Application of livistona chinensis seed polysaccharide in preparation of product for preventing or treating liver injury

Abstract

The invention discloses application of livistona chinensis seed polysaccharide in preparation of a product for relieving or treating liver injury, and belongs to the field of biological medicine, and the liver injury is LPS / D-GalN induced acute liver injury. According to the application disclosed by the invention, by reducing the contents of AST and ALT in liver cells, improving the levels of SOD, CAT and GSH oxidase in the cells, reducing the level of MDA in the cells and reducing the amount of inflammatory factors IL-6, TNF-alpha and NO, the protective effect of the livistona chinensis seed polysaccharide on LPS / D-GalN induced acute liver injury is exerted. The livistona chinensis seed polysaccharide is injected into the intraperitoneal cavity in advance to prevent and treat the acute liver injury induced by LPS / D-GalN. The invention proves the prevention or treatment effect of the livistona chinensis seed polysaccharide in drug-induced acute liver injury, and provides a certain theoretical basis for the development and application of livistona chinensis seeds and the research and development of liver protection products.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to the application of Livistona sylvestris polysaccharide in preparing products for preventing or treating liver damage. Background Art

[0002] The traditional Chinese medicine Livistona chinensis R.Br. belongs to the palm family Livistona genus and is widely used in folk medicine to treat hepatitis and cancer. At present, the research on Livistona chinensis R.Br. mainly focuses on its chemical composition and anti-cancer effect, while the research on its polysaccharide components is relatively small. Studies have shown that Livistona chinensis R.Br. is rich in polysaccharides, which have potential biological activity.

[0003] Polysaccharides, as natural macromolecules from higher plants, animal cell membranes, and microbial cell walls, are one of the basic substances that constitute life and have a variety of biological activities such as anti-tumor, anti-inflammatory, antiviral, hypoglycemic, anti-aging, and immunomodulatory. Livistona seeds are the seeds of the palm plant Livistona, which are bland, sweet, and astringent, and have anti-cancer effects. Livistona seeds and their compound preparations are clinically used to treat esophageal cancer, choriocarcinoma, malignant hydatidiform mole, leukemia, nasopharyngeal carcinoma, etc. LPS induces the production of inflammatory cytokines, which cause liver tissue damage. D-GalN is a specific hepatotoxic drug that inhibits the synthesis of macromolecules, which enhances the toxic effect of LPS in the liver. LPS / D-GalN can induce acute liver damage, which is very similar to clinical hepatitis caused by endotoxemia and sepsis.

[0004] However, to date, there has been no study on the effect of palmetto seed polysaccharides in acute liver injury. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes the use of Livistona polysaccharide in the preparation of products for preventing or treating liver damage. Experiments have shown that Livistona polysaccharide has obvious preventive and protective effects on acute liver damage induced by LPS / D-GalN.

[0006] The present invention also provides a pharmaceutical composition for preventing or treating liver damage.

[0007] The first aspect of the present invention provides the use of Livistona chinensis polysaccharide in preparing a product for preventing or treating liver damage.

[0008] According to the first aspect of the present invention, there are at least the following beneficial effects:

[0009] The present invention pre-injects the palmetto seed polysaccharide intraperitoneally, and detects the intracellular AST and ALT contents, which proves that the pathological damage of liver tissue is alleviated; the intracellular SOD, CAT, GSH oxidase levels and the intracellular MDA level are detected, which confirms that the palmetto seed polysaccharide has an effective antioxidant effect on the liver, inhibits cell oxidative stress, relieves liver cell toxicity, and plays a role in liver protection; and the palmetto seed polysaccharide alleviates the acute liver injury inflammation induced by LPS / D-GalN by detecting the amounts of inflammatory factors IL-6, TNF-α and NO.

[0010] The invention proves the preventive or therapeutic effect of Livistona chinensis polysaccharide on drug-induced acute liver injury, and provides a certain theoretical basis for the development and application of Livistona chinensis and the research and development of liver protection products.

[0011] According to some embodiments of the invention, the liver injury is acute liver injury.

[0012] According to some embodiments of the present invention, the drug-induced acute liver injury is LPS / D-GalN-induced acute liver injury.

[0013] According to some embodiments of the present invention, the palm seed polysaccharide is prepared by the following method:

[0014] The seeds of Livistona chinensis were crushed into powder, and the residues were enzymatically hydrolyzed, ultrasonically assisted, ethanol precipitated and washed in sequence to obtain Livistona chinensis polysaccharide.

[0015] According to some embodiments of the present invention, the palm seeds are crushed and passed through a 50-70 mesh sieve.

[0016] According to some embodiments of the invention, the concentration of ethanol is 95%.

[0017] According to some embodiments of the present invention, the mass volume ratio of the palmetto seeds to ethanol is 1:(5-10).

[0018] According to some embodiments of the present invention, during the water bath enzymolysis, the solid-liquid ratio of the medicinal residue to water is 1:(10-20).

[0019] According to some embodiments of the invention, the enzymatic hydrolysis enzyme comprises cellulase.

[0020] According to some embodiments of the present invention, the enzymatic hydrolysis time is 30 to 40 minutes, and the enzymatic hydrolysis temperature is 40 to 50°C.

[0021] According to some embodiments of the present invention, the ultrasound-assisted treatment is performed for 30 to 40 minutes.

[0022] According to some embodiments of the present invention, the washing solution comprises anhydrous ethanol, ether and acetone in sequence.

[0023] According to some embodiments of the present invention, the palmetto seed polysaccharide is used to reduce the levels of ALT and AST in serum.

[0024] According to some embodiments of the present invention, the use of Livistona sylvestris seed polysaccharide in enhancing the activity of antioxidant enzymes and reducing MDA levels.

[0025] According to some embodiments of the present invention, the palmetto seed polysaccharide is used to reduce the levels of IL-6, TNF-α and NO pro-inflammatory factors.

[0026] The second aspect of the present invention provides a pharmaceutical composition, which comprises the Livistona palm seed polysaccharide and / or its pharmaceutically acceptable excipients.

[0027] According to some embodiments of the present invention, the administration method of the Livistona palm seed polysaccharide in an animal is intraperitoneal injection.

[0028] According to some embodiments of the present invention, the intraperitoneal injection time includes prior LPS / D-GalN acute liver injury model injection.

[0029] According to some embodiments of the present invention, the intraperitoneal injection time includes 3 to 5 days of pre-LPS / D-GalN acute liver injury model.

[0030] According to some embodiments of the present invention, when the drug is used, the dosage of the palmetto seed polysaccharide is 7 to 28 mg / kg.

[0031] Preferably, when the drug is used, the dosage of the palmetto seed polysaccharide is 10 to 28 mg / kg.

[0032] More preferably, when the drug is used, the dosage of the Livistona palm seed polysaccharide is 28 mg / kg.

[0033] According to some embodiments of the present invention, the dosage is converted into an in vivo dosage of an animal according to the dosage of a unit drug for treating a biological body.

[0034] According to some embodiments of the invention, the organism comprises a human or other mammal.

[0035] According to some embodiments of the present invention, the drug can be prepared into a clinically acceptable drug preparation according to conventional preparation methods of traditional Chinese medicine.

[0036] According to some embodiments of the present invention, the dosage form of the pharmaceutical preparation is at least one of tablets, granules, oral liquid, pills, capsules and injections.

[0037] According to some embodiments of the present invention, the pharmaceutical excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, diluents, coating materials, fragrances, anti-adhesive agents, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, flocculants and deflocculating agents, filter aids and release retardants.

[0038] If there is no special explanation, the actual meaning of “about” in the present invention is that the error is allowed to be within the range of ±2%, for example, about 100 is actually 100±2%×100.

[0039] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0041] Figure 1 This is a graph showing the effect of Livistona chinensis polysaccharide on H&E pathological staining results of mouse pathological sections according to an embodiment of the present invention;

[0042] Figure 2 This is a graph showing the effect of palmetto seed polysaccharide on intracellular AST levels in an embodiment of the present invention;

[0043] Figure 3 This is a graph showing the effect of palmetto seed polysaccharide on intracellular ALT levels in an example of the present invention;

[0044] Figure 4 This is a graph showing the effect of palmetto seed polysaccharide on intracellular CAT levels in an embodiment of the present invention;

[0045] Figure 5 This is a graph showing the effect of palmetto seed polysaccharide on intracellular GSH levels in an embodiment of the present invention;

[0046] Figure 6 This is a graph showing the effect of palmetto seed polysaccharide on intracellular SOD levels in an embodiment of the present invention;

[0047] Figure 7 This is a graph showing the effect of palmetto seed polysaccharide on intracellular MDA levels in an embodiment of the present invention;

[0048] Figure 8 This is a graph showing the effect of palmetto seed polysaccharide on intracellular IL-6 levels in an example of the present invention;

[0049] Fig. 9This is a graph showing the effect of palmetto seed polysaccharide on intracellular TNF-α levels in an embodiment of the present invention;

[0050] Fig.10 This is a graph showing the effect of palmetto seed polysaccharide on intracellular NO levels in an embodiment of the present invention;

[0051] In the figure, the data are expressed as mean ± SD; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, &p<0.05, &&p<0.01, &&&&p<0.0001 compared with the LPS / D-GalN model group. DETAILED DESCRIPTION

[0052] The following will clearly and completely describe the concept of the present invention and the technical effects produced in combination with the embodiments, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by technicians in this field without creative work are all within the scope of protection of the present invention. The test methods used in the embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can all be reagents and materials obtained from commercial channels.

[0053] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0054] The reagents and consumables used in the present invention are all common commercial products and can be purchased in the market.

[0055] Example 1

[0056] This embodiment provides a method for preparing the polysaccharide of Livistona chinensis seeds of the present invention, which specifically comprises the following steps:

[0057] Take 100g of dried palm seed sample, crush it through a 60-mesh sieve, add 10 times the volume of 95% ethanol, reflux twice, each time for 2 hours, and pour out the upper liquid. Place the residue in a 48℃ oven to dry for use. Add water to the dried residue at a material-liquid ratio of 1:20, enzymolyze in a 50℃ water bath for 30 minutes, then inactivate at 80℃ for 10 minutes, filter to obtain an extract, ultrasonically assist the extract for 30 minutes, then concentrate the extract to 20% of the original volume, add 4 times the amount of ethanol and let it stand at 4℃ overnight, filter to obtain a residue, wash the residue with anhydrous ethanol, ether and acetone in turn, and obtain a precipitate that is a crude polysaccharide after drying.

[0058] The extracted crude polysaccharide was prepared into a 1 mg / mL polysaccharide concentrate, and an organic solvent (chloroform: n-butanol = 5:1) was added according to 1 / 5 of the volume of the concentrate. The mixture was stirred on a magnetic stirrer for 20 min (900 r / min), and centrifuged (6000 r / min, 3 min). The supernatant was retained and the precipitate was removed, and the previous steps were repeated until there was no protein precipitate.

[0059] Take a certain amount of polyamide and add it to the deproteinized polysaccharide solution, the temperature is 50℃, magnetic stirring is carried out for 30min (300r / min), after stirring, double-layer filter paper is used for suction filtration, and centrifugation is carried out at 8000r / min for 15min to obtain a polysaccharide solution. Dialysis and freeze-drying: Pour the decolorized polysaccharide solution into a dialysis bag (molecular weight is 8000-14000Da), clamp the two ends with dialysis bag clamps, and put it into a beaker filled with water for dialysis. Change the water every 12h for 48h. After the dialysis, pour out the polysaccharide solution in the dialysis bag, put it into a -20℃ refrigerator for pre-freezing, and then put it into a vacuum freeze dryer for freeze-drying, and finally freeze-dry to obtain the total polysaccharide of Livistona chinensis.

[0060] Determination of crude polysaccharide yield: The yield of polysaccharide from Livistona chinensis seeds was calculated according to the following formula:

[0061] Polysaccharide yield (%) = (crude polysaccharide mass / sample mass) × 100%;

[0062] According to formula (1), the yield of polysaccharide extracted by ultrasound-assisted enzyme method was 6.9%; the total polysaccharide content extracted by ultrasound-assisted enzyme method was 62.17%.

[0063] Infrared spectroscopy analysis:

[0064] FT-IR can collect high-resolution data simultaneously over a wide spectral range to obtain information about functional groups. The total polysaccharides of Livistona seeds extracted by ultrasound-assisted enzyme extraction were analyzed by Fourier transform infrared spectroscopy (FT-IR) to determine the functional group characteristics of the polysaccharides and gain a preliminary understanding of their chemical composition. Figure 1As shown, the results show that polysaccharides generally have similar characteristic absorption peaks. The absorption peak near 3386.29cm-1 is the stretching vibration absorption peak of OH, which is a characteristic peak of sugars. 2925.49cm-1 may be attributed to CH stretching vibration. 1649.74cm-1 may be attributed to the C=O bound water peak. 1418.23cm-1 may be the vibration absorption peak of OH in the carboxyl group. The weak absorption peaks at 1076.21cm-1 and 1038.61cm-1 are COH and COC glycosidic bonds, proving that LCS contains pyranose rings. The small weak peak in the 891.83cm-1 area indicates the presence of β-glycosidic bonds, and the absorption peak in the 572.10cm-1 area indicates the presence of α-type glycosidic bonds.

[0065] Example 2

[0066] This example provides an LPS / D-Gal-induced acute liver injury model in mice

[0067] Table 1 Sources of reagents in the embodiments of the present invention

[0068]

[0069] Experimental Materials:

[0070] 75 SPF male C57BL / 6J mice, weighing 20-25 g, were used. The experimental mice were 6-8 weeks old and were subjected to adaptive feeding for about one week.

[0071] Preparation of reagents:

[0072] LPS: Weigh an appropriate amount of LPS powder and dissolve it in PBS buffer to a concentration of 1 mg / mL. Store at -20°C and further dilute it with PBS to the required concentration before injection into mice. Combined with D-GalN, the intraperitoneal volume is 200 L.

[0073] D-GalN: Weigh an appropriate amount of D-GalN powder and dissolve it in PBS buffer to a concentration of 100 mg / mL. Filter and sterilize using a 0.22 μm filter and store at -20°C. Further dilute with PBS to the required concentration before injection into mice. The volume for co-injection with LPS is 200 μL.

[0074] Drugs: The positive drug Silybin (25 mg / kg) was dissolved with DMSO and then dissolved with normal saline. The palmetto seed polysaccharide LCP (7 mg / kg, 14 mg / kg, 28 mg / kg) was also dissolved with normal saline.

[0075] LPS / D-Gal-induced acute liver injury model in mice:

[0076] SPF-level male C57BL / 6J mice were randomly divided into groups. The concentration of the preliminary positive drug group was set at 25 mg / kg. The model group was modeled with different concentrations of LPS / D-GalN. The blank control group was intraperitoneally injected with an equal volume of normal saline. Eye blood was collected every 6 h, 12 h, and 24 h, with 3 mice at each time point. Before blood collection, 5% Avertin was injected at 0.2 mL / 10 g, and liver function indicators were determined using a biochemical analyzer. Compared with the normal group (blank control group), the model group had a significant increase in liver function enzyme index, thereby determining the optimal modeling time and modeling concentration.

[0077] The experimental group was treated with drugs by intraperitoneal injection for 5 days, and then intraperitoneally injected with LPS / D-GalN solution (after the 5th day of administration according to Table 2, the blank control group was intraperitoneally injected with an equal volume of saline, and the positive drug group and the model group were intraperitoneally injected with an equal volume of LPS / D-GalN), and then orbital blood was collected according to the above-set time points. The grouping and drug administration were carried out according to the table below, and there were 5 mice in each group in the preliminary experiment, for a total of 35 mice.

[0078] Table 2 Preliminary experimental model grouping of the embodiment of the present invention

[0079]

[0080] At the designated time (about 5 hours), blood was drawn from the mouse's eyeballs and the mouse was killed by cervical dislocation. The liver was separated and pathological tissue testing was performed to determine the optimal modeling concentration and time and the optimal dosing concentration, thereby determining the best time to collect samples from the mice to avoid premature death of the mice.

[0081] According to the preliminary experimental results, the modeling time, concentration, and drug dosage were determined, and each group was set to 10 mice, for a total of 40 mice.

[0082] Table 3 Optimal grouping of the embodiment of the present invention

[0083]

[0084] At the designated time (about 5 hours), 5% Avertin was injected at 0.2 mL / 10 g, and then blood was collected from the mice's eyes and they were killed by cervical dislocation. The liver was separated, and one part was used for histopathological detection and fixed with 4% paraformaldehyde; the other part was frozen at -80°C or in liquid nitrogen for subsequent experimental detection.

[0085] Example 3

[0086] This example provides the effect of LCP on liver tissue pathology and results

[0087] Mouse liver tissue was fixed in 4% paraformaldehyde solution for 24 hours, embedded in paraffin, and sliced ​​into 5 μm sections. Then it was stained with hematoxylin and eosin (H&E), and the pathological changes of liver tissue were observed under an optical microscope. The damaged liver cells of model group 2 were treated with LCP of experimental group 3.

[0088] Normal liver tissue morphology and cell structure can be seen in the Ctrl and LCP groups (experimental group 3). The LPS / D-GalN group (model group 2) showed obvious pathological changes in liver tissue, disordered liver structure, deformed liver lobules, cell necrosis, spherical degeneration, and inflammatory cell infiltration. Compared with the LPS / D-GalN group, LCP pretreatment significantly improved the liver damage caused by D-GalN / LPS. Figure 1 The above results showed that LCP had a protective effect on D-GalN / LPS-induced liver injury in mice.

[0089] Example 4

[0090] This example provides the effects of LCP on serum AST and ALT and the results

[0091] ALT and AST are widely used as biomarkers for assessing liver damage. The mice were anesthetized and blood was collected from the eyeballs. The blood was kept at room temperature at 4°C and centrifuged at 1500 rpm for 15 minutes to collect the upper serum, which was then stored at -80°C. AST and ALT were detected using the Nanjing Jiancheng kit after appropriate dilution. Compared with Ctrl, the ALT and AST levels in the LPS / D-GalN group were significantly increased, indicating that these mice had severe liver damage, while the AST and ALT levels in the experimental group 3 pretreated with LCP were significantly reduced (p<0.0001). Figure 2 and Figure 3 This suggests that LCP has a protective effect against acute liver injury.

[0092] Example 5

[0093] This example provides the effects of LCP on liver SOD, CAT, GSH and MDA levels and results

[0094] The mouse liver tissue was taken out of the -80℃ refrigerator, ground using a grinder, and the liver tissue homogenate was centrifuged at 4℃, 1200 rpm for 15 minutes, and then the tissue homogenate supernatant was removed. The Nanjing Jiancheng kit was used to detect the levels of SOD, CAT, GSH and MDA to evaluate the antioxidant capacity of LCP.

[0095] The LPS / D-GalN group significantly decreased the levels of CAT, GSH and SOD (e.g. Figure 4 , Figure 5 and Figure 6 ), and increased the amount of MDA (as shown Figure 7The results showed that LPS / D-GalN caused oxidative stress in liver tissue. The LCP group significantly increased the levels of CAT, GSH and SOD, and reduced the level of MDA compared with the LPS / D-GalN group. The above results show that LCP has an effective antioxidant effect on the liver.

[0096] Example 6

[0097] This example provides the effects of LCP on inflammatory factors IL-6, TNF-α and NO and the results

[0098] The mouse serum was taken out from -80℃ and thawed and then tested with ELISA kits for IL-6, TNF-α, and NO. Proinflammatory factors IL-6, TNF-α, and NO are closely related to acute liver injury inflammation induced by LPS / D-GalN.

[0099] Compared with the Ctrl group, the LPS / D-GalN group significantly increased the levels of IL-6, TNF-α, and NO. However, LCP treatment significantly reduced the levels of IL-6, TNF-α, and NO. Figure 8 , Fig. 9 and Fig.10 shown.

[0100] According to the above results, it can be seen that Livistona polysaccharide has a good effect in preventing or treating acute injury induced by LPS / D-GalN, and can reduce the levels of AST and ALT in hepatocytes and alleviate liver tissue pathological damage; it increases the levels of intracellular SOD, CAT, and GSH oxidase, and reduces the amount of MDA, indicating that Livistona polysaccharide has an effective antioxidant effect on the liver, inhibits cellular oxidative stress, relieves hepatocyte toxicity, and plays a role in liver protection; it reduces the amount of IL-6, TNF-α and NO, indicating that Livistona polysaccharide alleviates LPS / D-GalN-induced acute liver injury inflammation.

[0101] The present invention proves that the polysaccharide from Livistona chinensis seeds plays a protective role against acute liver injury induced by LPS / D-GalN by reducing ALT and AST and alleviating oxidative stress and inflammatory factors. Therefore, the polysaccharide from Livistona chinensis seeds may become a potential drug for treating acute liver injury.

[0102] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Application of Livistona palm seed polysaccharide in the preparation of products for preventing or treating liver damage.

2. The use according to claim 1, characterized in that: The liver injury is acute liver injury induced by LPS / D-GalN.

3. The use according to claim 1, characterized in that: The palm seed polysaccharide is prepared by the following method: The seeds of Livistona chinensis were crushed into powder, and the residues were enzymatically hydrolyzed, ultrasonically assisted, ethanol precipitated and washed in sequence to obtain Livistona chinensis polysaccharide.

4. The use according to claim 1, characterized in that: The application of the palm seed polysaccharide in reducing the levels of ALT and AST in serum.

5. The use according to claim 1, characterized in that: The invention discloses an application of the palm seed polysaccharide in enhancing the activity of antioxidant enzymes and reducing the MDA level.

6. The use according to claim 1, characterized in that: The invention discloses an application of the palm seed polysaccharide in reducing the levels of IL-6, TNF-α and NO pro-inflammatory factors.

7. A pharmaceutical composition, characterized in that The raw materials for preparing the pharmaceutical composition include the palmetto seed polysaccharide and / or pharmaceutically acceptable excipients thereof.

8. The pharmaceutical composition according to claim 7, characterized in that The administration method of the palm seed polysaccharide in animals is intraperitoneal injection.

9. The pharmaceutical composition according to claim 8, characterized in that The intraperitoneal injection time includes the prior LPS / D-GalN acute liver injury model injection.

10. The pharmaceutical composition according to claim 7, characterized in that When the drug is used, the dosage of the Livistona syringae polysaccharide is 7-28 mg / kg; preferably, the dosage of the Livistona syringae polysaccharide is 28 mg / kg.

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