Use of spondias mombin polysaccharides in the preparation of products for preventing or treating liver damage

Abstract

The application discloses application of sargent cypress seed polysaccharide in preparation of products for relieving or treating liver injury, and belongs to the field of biological medicine.The liver injury is acute liver injury induced by LPS / D-GalN.The application plays a protective role of sargent cypress seed polysaccharide on acute liver injury induced by LPS / D-GalN by reducing the content of AST and ALT in liver cells, improving the level of SOD, CAT and GSH oxidase in the cells, reducing the level of MDA in the cells, reducing the amount of inflammatory factors IL-6, TNF-alpha and NO.The application prevents and treats acute liver injury induced by LPS / D-GalN by pre-injection of sargent cypress seed polysaccharide into the abdominal cavity.The application proves the preventive or therapeutic effect of sargent cypress seed polysaccharide on drug-induced acute liver injury, and provides a certain theoretical basis for development and application of sargent cypress and research and development of liver-protecting products.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of fan palm seed polysaccharide in the preparation of products for the prevention or treatment of liver damage. Background Technology

[0002] The seed of the Chinese fan palm (Livistona chinensis R.Br.), belonging to the genus Livistona in the family Arecaceae, is widely used in traditional Chinese medicine for treating hepatitis and cancer. Currently, research on the seed mainly focuses on its chemical composition and anti-cancer effects, while research on its polysaccharide components is relatively limited. Existing studies have shown that the seed contains abundant polysaccharides with potential biological activity.

[0003] Polysaccharides, natural macromolecules derived from the cell membranes of higher plants, animals, and microbial cell walls, are fundamental building blocks of life and possess various biological activities such as anti-tumor, anti-inflammatory, antiviral, hypoglycemic, anti-aging, and immunomodulatory effects. Liviston's palm seeds, the seeds of the palm tree (Liviston's palm), are neutral in nature and have a bland, sweet, and astringent taste; they also possess anti-cancer properties. Clinically, Liviston's palm seeds and their compound preparations are used to treat esophageal cancer, choriocarcinoma, malignant hydatidiform mole, leukemia, and nasopharyngeal carcinoma. LPS induces the production of inflammatory cytokines, which lead to liver tissue damage. D-GalN, a specific hepatotoxic drug that inhibits macromolecule synthesis, enhances the toxic effects of LPS in the liver. LPS / D-GalN can induce acute liver injury, which is very similar to clinical hepatitis caused by endotoxemia and sepsis.

[0004] However, to date, there have been no studies on the effects of fan palm seed polysaccharides on acute liver injury. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes the application of *Ligustrum lucidum* seed polysaccharide in the preparation of products for the prevention or treatment of liver injury. Experiments have demonstrated that *Ligustrum lucidum* seed polysaccharide has significant preventive and protective effects against LPS / D-GalN-induced acute liver injury.

[0006] The present invention also provides a pharmaceutical composition for the prevention or treatment of liver injury.

[0007] The first aspect of the present invention provides the use of fan palm seed polysaccharide in the preparation of products for the prevention or treatment of liver damage.

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

[0009] This invention demonstrates that pre-intraperitoneal injection of *Ligustrum lucidum* seed polysaccharide, through the detection of intracellular AST and ALT levels, reduces pathological damage to liver tissue. Detection of intracellular SOD, CAT, GSH oxidase levels, and intracellular MDA levels confirms that *Ligustrum lucidum* seed polysaccharide has effective antioxidant effects on the liver, inhibiting cellular oxidative stress, relieving hepatotoxicity, and playing a hepatoprotective role. Furthermore, by detecting the levels of inflammatory factors IL-6, TNF-α, and NO, *Ligustrum lucidum* seed polysaccharide alleviates LPS / D-GalN-induced acute liver injury inflammation.

[0010] This invention demonstrates the preventive or therapeutic effects of fan palm seed polysaccharides on drug-induced acute liver injury, providing a theoretical basis for the development and application of fan palm seeds and the research and development of liver-protective products.

[0011] According to some embodiments of the present 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 fan palm seed polysaccharide is prepared by the following method:

[0014] The seeds of the fan palm were crushed into powder, and the residue was subjected to enzymatic hydrolysis, ultrasonic-assisted treatment, ethanol precipitation, and washing to obtain fan palm 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 present invention, the concentration of the ethanol is 95%.

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

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

[0019] According to some embodiments of the present invention, the enzyme used for enzymatic hydrolysis includes cellulase.

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

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

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

[0023] According to some embodiments of the present invention, the application of the fan palm seed polysaccharide in reducing serum ALT and AST levels.

[0024] According to some embodiments of the present invention, the application of the *Ligustrum lucidum* seed polysaccharide in enhancing the activity of antioxidant enzymes and reducing MDA levels.

[0025] According to some embodiments of the present invention, the application of the *Ligustrum lucidum* seed polysaccharide in reducing the levels of IL-6, TNF-α, and NO pro-inflammatory factors.

[0026] A second aspect of the present invention provides a pharmaceutical composition comprising the palm seed polysaccharide and / or pharmaceutically acceptable excipients thereof.

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

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

[0029] According to some embodiments of the present invention, the intraperitoneal injection time includes 3 to 5 days prior to the 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 *Ligustrum lucidum* polysaccharide is 7–28 mg / kg.

[0031] Preferably, when the drug is used, the dosage of the *Ligustrum lucidum* polysaccharide is 10–28 mg / kg.

[0032] More preferably, when the drug is used, the dosage of the *Ligustrum lucidum* polysaccharide is 28 mg / kg.

[0033] According to some embodiments of the present invention, the dosage is converted into an animal dosage based on the therapeutic dose of a unit drug in a living organism.

[0034] According to some embodiments of the present invention, the organism includes humans or other mammals.

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

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

[0037] According to some embodiments of the present invention, the pharmaceutical excipients comprise at least one of the following: solvent, propellant, solubilizer, cosolvent, emulsifier, colorant, binder, disintegrant, filler, lubricant, wetting agent, osmotic pressure regulator, stabilizer, flow aid, flavoring agent, preservative, suspending agent, diluent, coating material, fragrance, anti-adhesion agent, binding agent, penetration enhancer, pH adjuster, buffer, plasticizer, surfactant, foaming agent, defoamer, thickener, encapsulating agent, humectant, flocculant and anti-flocculation agent, filter aid, and release inhibitor.

[0038] Unless otherwise specified, the term "about" in this invention actually means that the error is allowed to be within ±2%, for example, about 100 is actually 100 ± 2% × 100.

[0039] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

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

[0041] Figure 1 This is a diagram illustrating the effect of *Ligustrum lucidum* seed polysaccharide on H&E staining results of mouse pathological sections according to an embodiment of the present invention.

[0042] Figure 2 This is a diagram illustrating the effect of *Ligustrum lucidum* seed polysaccharide on intracellular AST levels according to an embodiment of the present invention.

[0043] Figure 3 This is a diagram illustrating the effect of fan palm seed polysaccharide on intracellular ALT levels according to an embodiment of the present invention.

[0044] Figure 4 This is a diagram illustrating the effect of fan palm seed polysaccharide on intracellular CAT levels according to an embodiment of the present invention.

[0045] Figure 5 This is a diagram illustrating the effect of *Ligustrum lucidum* seed polysaccharide on intracellular GSH levels according to an embodiment of the present invention.

[0046] Figure 6 This is a diagram illustrating the effect of fan palm seed polysaccharide on intracellular SOD levels according to an embodiment of the present invention.

[0047] Figure 7 This is a diagram illustrating the effect of fan palm seed polysaccharide on intracellular MDA levels according to an embodiment of the present invention.

[0048] Figure 8 This is a diagram illustrating the effect of fan palm seed polysaccharide on intracellular IL-6 levels according to an embodiment of the present invention.

[0049] Figure 9This is a diagram illustrating the effect of fan palm seed polysaccharide on intracellular TNF-α levels according to an embodiment of the present invention.

[0050] Figure 10 This is a diagram illustrating the effect of fan palm seed polysaccharide on intracellular NO levels according to an embodiment of the present invention.

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

[0052] The following will describe the concept and technical effects of the present invention clearly and completely with reference to the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0053] In the description of this invention, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0054] The reagents and consumables used in this invention are all commercially available products that can be purchased on the market.

[0055] Example 1

[0056] This embodiment provides a method for preparing the *Ligustrum lucidum* seed polysaccharide of the present invention, specifically including the following steps:

[0057] Take 100g of dried fan palm seeds, pulverize them and pass them through a 60-mesh sieve. Add 10 times the volume of 95% ethanol and reflux twice, 2 hours each time. Pour off the supernatant. Place the residue in a 48℃ oven to dry and set aside. Add water to the dried residue at a material-to-liquid ratio of 1:20, enzymatically hydrolyze in a 50℃ water bath for 30 minutes, then inactivate at 80℃ for 10 minutes. Filter to obtain the extract. Sonicate the extract for 30 minutes, then concentrate the extract to 20% of its original volume. Add 4 times the volume of ethanol and let stand overnight at 4℃. Filter to obtain the residue. Wash the residue sequentially with anhydrous ethanol, ether, and acetone to obtain the precipitate. After drying, the crude polysaccharide is obtained.

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

[0059] A certain amount of polyamide was added to the deproteinized polysaccharide solution. The mixture was magnetically stirred at 50℃ for 30 minutes (300 rpm). After stirring, the solution was filtered through double-layered filter paper and centrifuged at 8000 rpm for 15 minutes to obtain the polysaccharide solution. The decolorized polysaccharide solution was then poured into a dialysis bag (molecular weight 8000–14000 Da), clamped at both ends, and placed in a beaker filled with water for dialysis. The water was changed every 12 hours, and dialysis was performed for 48 hours. After dialysis, the polysaccharide solution in the dialysis bag was poured out, pre-frozen at -20℃, and then freeze-dried in a vacuum freeze dryer to obtain total polysaccharides from *Ligustrum lucidum* seeds.

[0060] Determination of crude polysaccharide yield: The yield of polysaccharides from fan palm seeds was calculated using the following formula:

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

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

[0063] Infrared spectroscopy analysis:

[0064] FT-IR can simultaneously collect high-resolution data over a wide spectral range to obtain information on functional groups. Fourier transform infrared spectroscopy (FT-IR) analysis of total polysaccharides extracted from *Livistona chinensis* seeds using ultrasound-assisted enzyme extraction determined the functional group characteristics of the polysaccharides and provided a preliminary understanding of their chemical composition. The FT-IR spectral results of total polysaccharides from *Livistona chinensis* seeds are shown below. Figure 1As shown, the results indicate that polysaccharides generally exhibit similar characteristic absorption peaks. The absorption peak near 3386.29 cm⁻¹ is attributed to the stretching vibration of the OH group, a characteristic peak of carbohydrates. The peak at 2925.49 cm⁻¹ may be attributed to the CH stretching vibration. The peak at 1649.74 cm⁻¹ may be attributed to the C=O bound water peak. The peak at 1418.23 cm⁻¹ is likely the vibrational absorption peak of the OH group in the carboxyl group. The weak absorption peaks at 1076.21 cm⁻¹ and 1038.61 cm⁻¹ indicate the presence of COH and COC glycosidic bonds, confirming the presence of a pyranose ring in LCS. The small, weak peak in the 891.83 cm⁻¹ region indicates the presence of a β-glycosidic bond, while the absorption peak in the 572.10 cm⁻¹ region indicates the presence of an α-glycosidic bond.

[0065] Example 2

[0066] This embodiment provides an LPS / D-Gal-induced mouse model of acute liver injury.

[0067] Table 1. Reagent sources in the embodiments of the present invention

[0068]

[0069] Experimental materials:

[0070] Seventy-five male C57BL / 6J mice of SPF grade, weighing 20-25g, were used in the experiment. The mice were 6-8 weeks old and underwent an acclimatization diet for about one week.

[0071] Reagent preparation:

[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. Before injection into mice, further dilute with PBS to the required concentration. Inject into the peritoneal cavity in combination with D-GalN in a volume of 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. After sterilization by filtration using a 0.22 μm filter, store at -20°C. Before injection into mice, further dilute with PBS to the required concentration. The combined injection volume with LPS is 200 μL.

[0074] Drugs: The positive control drug silybin (25 mg / kg) was dissolved in DMSO and then in physiological saline. The polysaccharide LCP from the palm seed (7 mg / kg, 14 mg / kg, 28 mg / kg) was also dissolved in physiological saline.

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

[0076] SPF-grade male C57BL / 6J mice were randomly divided into two groups. The concentration of the positive drug group in the preliminary test was set at 25 mg / kg. The model group was induced by using different concentrations of LPS / D-GalN in combination. The blank control group was injected intraperitoneally with an equal volume of physiological saline. Blood was collected from the eyeballs at 6 h, 12 h, and 24 h, with 3 mice at each time point. Before blood collection, 5% aphthol was injected at 0.2 mL / 10 g. Liver function indicators were then measured using a biochemical analyzer. The model group was considered to have a significant increase in liver function enzyme index compared with the normal group (blank control group), thus determining the optimal modeling time and concentration.

[0077] The experimental group received intraperitoneal injection of the drug for 5 days, followed by intraperitoneal injection of LPS / D-GalN solution. (On day 5 after administration according to Table 2, the blank control group received an equal volume of physiological saline, and the positive control group and model group all received an equal volume of LPS / D-GalN intraperitoneally.) Blood was then collected from the orbital fossa at the time points specified above. The drugs were administered to groups according to the table below, with 5 mice in each group in the preliminary experiment, for a total of 35 mice.

[0078] Table 2 Grouping of Pre-experimental Models in Embodiments of the Invention

[0079]

[0080] At the designated time (approximately 5 hours), blood was collected from the mouse's eyeballs and the mouse was euthanized by dislocation of its neck. The liver was then separated for pathological tissue examination to determine the optimal concentration and time for modeling and the optimal drug concentration, thereby determining the best time for material collection from the mice and avoiding premature death.

[0081] Based on the preliminary experimental results, the modeling time, concentration, and drug dosage were determined, with 10 animals per group and a total of 40 animals.

[0082] Table 3 Optimal Grouping of Embodiments of the Invention

[0083]

[0084] At the designated time (approximately 5 hours), 5% aphthylamine was injected at 0.2 mL / 10 g. Blood was then collected from the mice's eyeballs, and the mice were euthanized by cervical dislocation. The livers were separated, with a portion used for histopathological examination and fixed with 4% paraformaldehyde; the remainder was frozen at -80°C or in liquid nitrogen for subsequent experimental testing.

[0085] Example 3

[0086] This embodiment provides the effects and results of LCP on liver tissue pathology.

[0087] Mouse liver tissue was fixed in 4% paraformaldehyde solution for 24 h, embedded in paraffin, and sectioned to a size of 5 μm. The tissue was then stained with hematoxylin and eosin (H&E), and pathological changes were observed under a light microscope. Hepatocytes from model group 2 after injury were treated with LCP from experimental group 3.

[0088] The Ctrl and LCP groups (experimental group 3) showed normal liver tissue morphology and cellular structure. The LPS / D-GalN group (model group 2) showed significant pathological changes in liver tissue, including disordered liver structure, lobular deformation, cell necrosis, saccular degeneration, and inflammatory cell infiltration. Compared with the LPS / D-GalN group, LCP pretreatment significantly improved liver injury induced by D-GalN / LPS, as shown in the results. Figure 1 As shown in the figure. The above results indicate that LCP has a protective effect against D-GalN / LPS-induced liver injury in mice.

[0089] Example 4

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

[0091] ALT and AST are widely used as biomarkers for assessing liver damage. Blood was collected from the ocular sacs of anesthetized mice, allowed to stand at room temperature (4°C), centrifuged at 1500 rpm for 15 minutes, and the supernatant serum was collected and stored at -80°C. AST and ALT were detected using a Nanjing Jiancheng reagent kit after appropriate dilution. Compared with the Ctrl group, the LPS / D-GalN group showed significantly elevated ALT and AST levels, indicating severe liver damage in these mice. In contrast, experimental group 3, pretreated with LCP, showed significantly decreased AST and ALT levels (p<0.0001). Figure 2 and Figure 3 As shown, this suggests that LCP has a protective effect against acute liver injury.

[0092] Example 5

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

[0094] Mouse liver tissue was removed from a -80°C freezer and homogenized using a homogenizer. The homogenate was centrifuged at 1200 rpm for 15 minutes at 4°C, and the supernatant was discarded. The antioxidant capacity of LCP was evaluated by detecting SOD, CAT, GSH, and MDA levels using a Nanjing Jiancheng reagent kit.

[0095] The LPS / D-GalN group significantly reduced the levels of CAT, GSH, and SOD (e.g., Figure 4 , Figure 5 and Figure 6 As shown), and increased the amount of MDA (e.g.) Figure 7As shown in the figure, this indicates that LPS / D-GalN induced oxidative stress in liver tissue. Compared with the LPS / D-GalN group, the LCP group significantly increased the levels of CAT, GSH, and SOD, and decreased the level of MDA. These results indicate that LCP has an effective antioxidant effect on the liver.

[0096] Example 6

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

[0098] Mouse serum was removed from -80°C, thawed, and then analyzed using an ELISA kit to detect IL-6, TNF-α, and NO. Pro-inflammatory factors IL-6, TNF-α, and NO are closely associated with LPS / D-GalN-induced acute liver injury and inflammation.

[0099] Compared to the Ctrl group, the LPS / D-GalN group showed a significant increase in IL-6, TNF-α, and NO levels. However, LCP treatment significantly reduced IL-6, TNF-α, and NO levels. Figure 8 , Figure 9 and Figure 10 As shown.

[0100] The results show that *Ligustrum lucidum* seed polysaccharide has a good effect in preventing or treating LPS / D-GalN-induced acute liver injury, and can reduce the levels of AST and ALT in hepatocytes, alleviating pathological damage to liver tissue; it increases the levels of SOD, CAT, and GSH oxidases in cells, while reducing the amount of MDA, indicating that *Ligustrum lucidum* seed polysaccharide has an effective antioxidant effect on the liver, inhibits cellular oxidative stress, relieves hepatotoxicity, and plays a hepatoprotective role; it reduces the levels of IL-6, TNF-α, and NO, indicating that *Ligustrum lucidum* seed polysaccharide alleviates the inflammation of LPS / D-GalN-induced acute liver injury.

[0101] This invention demonstrates that fan palm seed polysaccharide exerts a protective effect against LPS / D-GalN-induced acute liver injury by reducing ALT and AST, and alleviating oxidative stress and inflammatory factors. Therefore, fan palm seed polysaccharide has the potential to become a drug for the treatment of acute liver injury.

[0102] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. The application of *Livistona chinensis* seed polysaccharide in the preparation of drugs for the prevention or treatment of liver injury, characterized in that, The liver injury described was an acute liver injury induced by LPS / D-GalN. The fan palm seed polysaccharide was prepared by the following method: The seeds of the fan palm were crushed into powder, and after reflux with ethanol, the residue was enzymatically hydrolyzed, ultrasonically assisted, precipitated with ethanol, and washed sequentially to obtain fan palm polysaccharide. The concentration of the ethanol is 95%; The washing solution consists of anhydrous ethanol, diethyl ether, and acetone, washed sequentially.

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

3. The application according to claim 1, characterized in that, The application of the *Livistona chinensis* polysaccharide in enhancing the activity of antioxidant enzymes and reducing MDA levels.

4. The application according to claim 1, characterized in that, The application of the *Ligustrum lucidum* seed polysaccharide in reducing the levels of IL-6, TNF-α, and NO pro-inflammatory factors.

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