Preparation for preventing and treating liver damage and application thereof

Abstract

The present invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a preparation for preventing and treating liver damage and its application. In the preparation, the mass ratio of glucuronic acid, arabinose and codonopsis polysaccharide is 1-2:1-2:1-2. The preparation has an auxiliary protective effect on chemical liver damage, achieves excellent effects in alleviating the toxicity of sterigmatocystin and relieving liver damage, and has potential medicinal value.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a preparation for preventing and treating liver damage and application thereof. Background Art

[0002] Sterigmatocystin (STC) is one of the mycotoxins and is commonly found in grains and animal feed. It contains a bifuran ring of xanthone compounds, which are mainly produced by fungi such as Aspergillus versicolor and Aspergillus nidulans. STC can cause serious health problems and has multiple toxicities, including liver toxicity, gastrointestinal toxicity, genotoxicity and neurotoxicity, etc., which will cause serious health problems after entering the body. Moreover, since STC is a precursor of aflatoxin and has a structure similar to aflatoxin toxin, the toxicity it produces is second only to aflatoxin and has been classified as a carcinogen Class 2B by the International Agency for Research on Cancer IARC. At present, sterigmatocystin is widely present in nature and can contaminate a variety of food crops such as barley, wheat, peanuts, corn, soybeans and coffee beans, especially the pollution of feed forage such as peanuts, corn and wheat. In addition, the detection rate of STC in carpets and ceilings in humid living environments is also significantly increased.

[0003] The liver is the main target organ of STC toxicity. STC can induce liver damage in a variety of animals and cause liver oxidative stress, accompanied by increased levels of lipid peroxidation markers and decreased antioxidant enzyme activity. Therefore, it is urgent to find safe and effective methods to alleviate the damage caused by STC to the body. However, current research work is mostly focused on toxicological research and detection methods such as STC mutagenicity and carcinogenicity, while there are few reports on its prevention and control measures.

[0004] According to existing research, the liver is the main target organ of STC, and its liver toxicity to animals has been confirmed. For example, stericocyanate can induce liver damage in a variety of animals such as pigs, chickens, and rats. Its toxicity mechanisms include the induction of oxidative stress, mitochondrial dysfunction, apoptosis, cell cycle arrest, as well as changes in immune system function and activation of different signaling pathways. In addition, there is currently a lot of evidence to confirm the toxicity of STC to the human liver. Epidemiological analysis shows that STC is present at a high level in the blood and urine of patients with hepatocellular carcinoma (HCC), and there is a clear link between it and the risk of developing HCC. For example, a study that measured STC levels in blood and urine samples of patients with cirrhosis and HCC found that 26.2% of patients had high STC levels, with the STC levels in the blood and urine of patients with cirrhosis rising to 0.626 ng / ml and 1.053 ng / ml, respectively, and the STC levels in the blood and urine of patients with HCC rising to 2.02 ng / ml and 9.39 ng / ml, respectively, which were much higher than the blood STC (0.014 ng / ml) and urine STC (0.005 ng / ml) levels of the control group. It can be seen that inducing liver damage is one of the important ways in which STC exerts its toxic effects in the body. Therefore, finding components or preparations that alleviate STC-induced liver damage may be an effective strategy to alleviate the toxicity of STC to the body.

[0005] Codonopsis pilosula polysaccharides (CPP) is the main active ingredient of the edible and medicinal plant Codonopsis pilosula, which has antioxidant, immune-enhancing, anti-fatigue, anti-tumor and memory-improving effects. According to existing studies, CPP can alleviate liver damage and liver oxidative stress caused by BCG / lipopolysaccharide and high-fat diet.

[0006] Based on previous studies, the inventors speculated that CPP has the potential to alleviate STC-induced acute liver injury. However, to date, there have been no reports on the use of CPP to alleviate STC-induced acute liver injury and thereby alleviate the toxic effects of STC on the body, nor have there been any reports on how to compound CPP with other monosaccharides to further enhance its ability to alleviate STC-induced acute liver injury. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention aims to provide a preparation for preventing and treating liver damage and its application. By forming a composite preparation with codonopsis polysaccharide, glucuronic acid and arabinose, the level of codonopsis polysaccharide in alleviating liver damage is significantly improved.

[0008] To achieve the above object, in one aspect, the present invention provides a preparation for preventing and treating liver damage, the preparation comprising glucuronic acid, arabinose and codonopsis polysaccharide;

[0009] Among them, glucuronic acid, also known as glucuronic acid, is a uronic acid formed by oxidation of the C-6 hydroxyl group of glucose to a carboxyl group, which has the effects of protecting the liver and detoxifying.

[0010]

[0011] L-Arabinose has a blocking effect on the metabolic conversion of sucrose, and has application prospects in weight loss and diabetes control. The American Medical Association has listed arabinose as a nutritional supplement or over-the-counter drug for anti-obesity agents. The Japanese Ministry of Health, Labor and Welfare has listed arabinose as a special health food additive for regulating blood sugar in its list of specific health foods. As an emerging healthy sugar, arabinose is expected to be widely used in food.

[0012]

[0013] Furthermore, in the preparation, the mass ratio of glucuronic acid, arabinose and codonopsis polysaccharide is 1-2:1-2:1-2.

[0014] Furthermore, in the preparation, the mass ratio of glucuronic acid, arabinose and codonopsis polysaccharide is 1:1:1.

[0015] Furthermore, the preparation also includes auxiliary materials acceptable in medicine or feed.

[0016] In a second aspect, the present invention provides a use of the preparation described in the first aspect;

[0017] In a third aspect, the present invention provides a use of the preparation according to the first aspect in preparing a product for alleviating the toxicity of sterigmatocystin.

[0018] Furthermore, the product is a medicine or feed.

[0019] In a fourth aspect, the present invention provides a use of the preparation described in the first aspect in preparing a product for alleviating acute liver injury.

[0020] Furthermore, the product is a medicine or feed.

[0021] In a fifth aspect, the present invention provides a use of the preparation described in the first aspect in preparing a product having an auxiliary protective effect on chemical liver damage.

[0022] Furthermore, the product is a medicine or feed.

[0023] In a sixth aspect, the present invention provides a use of the preparation described in the first aspect in preparing a product for alleviating weight loss caused by acute liver injury.

[0024] Furthermore, the product is a medicine or feed.

[0025] In a seventh aspect, the present invention provides a use of the preparation described in the first aspect in preparing a product for regulating changes in component levels in serum caused by acute liver injury.

[0026] Furthermore, the components include one or more of aspartate aminotransferase, alanine aminotransferase, triglyceride or total cholesterol.

[0027] Furthermore, the change in the component level in the serum is an increase in the component concentration.

[0028] Furthermore, the adjustment is reduction.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] 1. Based on a cell model, the present invention analyzes the effects of different monosaccharide components in alleviating STC-induced liver damage, and uses glutathione (GSH) and the lipid synthesis gene SREBP1C as screening indicators, and finds that glucuronic acid and arabinose have the best alleviating effects.

[0031] 2. The present invention compounds glucuronic acid, arabinose and codonopsis polysaccharide to form a CGA complex, studies the mitigation effect of CGA on STC toxicity based on an in vitro cell model, and uses oxidative stress genes as evaluation indicators. It is found that the mitigation effect of CGA on STC toxicity is better than that of glucuronic acid, arabinose or codonopsis polysaccharide alone, that is, glucuronic acid, arabinose and codonopsis polysaccharide in the CGA complex have a synergistic effect in mitigating STC toxicity;

[0032] 3. The present invention evaluated the alleviating effect of CGA complex on acute liver injury based on an animal model, and found that the alleviating effect of CGA complex on STC-induced acute liver injury was better than that of single Codonopsis polysaccharide, glucuronic acid or arabinose, that is, glucuronic acid, arabinose and Codonopsis polysaccharide in the CGA complex had a synergistic effect in alleviating acute liver injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The figure shows the alleviating effect of different preparations on liver injury cells treated with STC. Among them, A is the GSH gene expression level, B is the SREBP1C gene expression level, C is the GSH gene expression level, and D is the SOD gene expression level.

[0034] Figure 2The figure shows the alleviating effect of different preparations on acute liver injury induced by STC in mice. A is the weight of mice, B is the serum TC level, C is the serum TG level, D is the serum ALT level, E is the serum AST level, F is the gene expression level of IL-6 in liver, G is the gene expression level of TNF-α in liver, H is the gene expression level of Bcl2 in liver, I is the gene expression level of Bax in liver, J is the gene expression level of SOD in liver, K is the gene expression level of GSH in liver, L is the NAS score of liver pathological injury, and M is the HE staining picture of liver pathological injury. DETAILED DESCRIPTION

[0035] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods are conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0036] 1. Experimental Methods

[0037] 1.1 Liver tissue staining and pathological scoring

[0038] Fresh livers were fixed in 4% tissue fixative overnight, dehydrated with gradient ethanol (50%, 75% and 95%) and embedded in paraffin. Tissue sections were dewaxed and hydrated and then stained with hematoxylin and eosin (H&E). Images were captured using an optical microscope and morphological examinations were performed to analyze the extent of acute liver injury.

[0039] The pathological feature of lobular inflammation in the liver was evaluated histopathologically and a comprehensive score was derived based on the NAS criteria to quantitatively assess the degree of acute liver injury. In the NAS scoring system, each pathological feature is scored on a scale of 0 to 3, with the following criteria: lobular inflammation, assessed based on the number of inflammatory foci per 200× field of view, scored as 0 (no inflammatory foci), 1 (<2 inflammatory foci), 2 (2-4 inflammatory foci), and 3 (>4 inflammatory foci)

[0040] 1.2 Determination of liver function indicators

[0041] Blood samples of mice were collected into anticoagulation tubes, left to stand at 4°C for 4h, centrifuged at 1000g for 15min at 4°C, and the upper serum was aliquoted and stored at -20°C. 50mg of liver tissue was accurately weighed in 500μL PBS, and 10% tissue homogenate (w / v) was prepared in an ice water bath, centrifuged at 2500rpm for 10min at 4°C, and the supernatant was aliquoted and stored at -20°C for further analysis. The activity of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was determined using commercially available kits.

[0042] 1.3 Determination of triglycerides and total cholesterol

[0043] Using the triglyceride and total cholesterol assay kit, take the prepared serum, mix it with the working solution in a 96-well plate, incubate at 37°C for 10 min, and measure the absorbance at 510 nm and 500 nm, respectively. According to the formula provided in the instructions, quantify the concentrations of triglyceride (TG) and total cholesterol (TC) in serum, and detect the corresponding contents in serum and liver homogenate.

[0044] 1.4 Analysis of related gene expression levels in liver

[0045] According to the RNA extraction protocol, total RNA was extracted from liver tissue using TRIzol reagent. RNase-free pipette tips and tubes were used during the experiment. The liver sample was ground into powder with a small amount of liquid nitrogen, resuspended in TRIzol and fully lysed, then chloroform was added and shaken vigorously to form an emulsion. The solution was allowed to stand at room temperature for 3 minutes and centrifuged at 12000g for 15 minutes at 4°C. The upper aqueous phase was transferred to a new centrifuge tube, gently mixed upside down with an equal volume of isopropanol, allowed to stand at room temperature for 10 minutes, and then centrifuged to form a gelatinous precipitate. The supernatant was discarded, and the RNA was washed three times with 75% ethanol, air-dried at room temperature to remove residual solvent, and redissolved with an appropriate amount of enzyme-free sterile water. The concentration and purity of the total RNA obtained were detected by NanoDrop One. The extracted RNA was stored at -80°C after aliquoting. The extracted RNA was reverse transcribed into cDNA and gDNA was removed using a PCR amplification instrument using the reverse transcription kit SweScript All-in-One RT SuperMix containing reaction buffer, primers and reverse transcriptase. According to the instructions of the kit 2×SYBR Green qPCR Master Mix, cDNA, upstream and downstream primers, 2×SYBR qPCR Mix and double distilled water were mixed in a flat-bottom 96-well plate and plated in a Bio-Rad IQ TM 5 system, and real-time fluorescence quantitative PCR amplification of cDNA was performed.

[0046] 2. Experimental results

[0047] Example 1 The alleviating effect of different monosaccharides on STC-treated liver damaged cells

[0048] After thawing and resuscitation, human normal hepatocyte LO-2 cells were cultured in DMEM medium containing 10% fetal bovine serum and placed in an incubator at 37°C and 5% CO2. When the cells proliferated to 75% to 85%, they were routinely digested, passaged and frozen. The experiment was conducted in the following groups to evaluate the mitigating effect of each monosaccharide on STC toxicity.

[0049] (1) Normal control group (CON group): cells in the normal group were cultured with complete medium;

[0050] (2) Model group (STC group): A liver injury cell model treated with STC was constructed. Under STC treatment, the expression level of the cell's antioxidant gene GSH was significantly downregulated, while the lipid synthesis gene SREBP1C was significantly upregulated. The cells in the model group were cultured in a medium containing 30 μmol / L STC;

[0051] (3) Glucuronic acid group (GlcA group): 30 μmol / L STC was added for culture, and 10 mmol / L glucuronic acid was added for intervention;

[0052] (4) Galacturonic acid group (GalA group): 30 μmol / L STC was added for culture, and 10 mmol / L galacturonic acid was added for intervention;

[0053] (5) Arabinose group (Ara group): 30 μmol / L STC was added for culture, and 10 mmol / L arabinose was added for intervention;

[0054] (6) Mannose group (Man group): 30 μmol / L STC was added for culture, and 10 mmol / L mannose was added for intervention;

[0055] (7) Glucosamine group (GlcN group): 30 μmol / L STC was added for culture, and 10 mmol / L glucosamine was added for intervention;

[0056] After 24 hours of intervention with each preparation, all cells were collected and resuspended with 1 mL of TRIzol for full lysis, then chloroform was added and shaken vigorously to form an emulsion. The solution was allowed to stand at room temperature for 3 minutes and centrifuged at 12,000 g for 15 minutes at 4 °C. The upper aqueous phase was transferred to a new centrifuge tube, gently mixed with an equal volume of isopropanol by inversion, allowed to stand at room temperature for 10 minutes, and then centrifuged to form a gelatinous precipitate. The supernatant was discarded, and the RNA was washed three times with 75% ethanol, air-dried at room temperature to remove the residual solvent, and redissolved with an appropriate amount of enzyme-free sterile water. The concentration and purity of the total RNA obtained were detected by NanoDrop One. The extracted RNA was stored at -80 °C after aliquoting. The extracted RNA was reverse transcribed into cDNA and gDNA was removed using a PCR amplification instrument using the reverse transcription kit SweScript All-in-OneRT SuperMix containing reaction buffer, primers, and reverse transcriptase. According to the instructions of the kit 2×SYBRGreen qPCR Master Mix, cDNA, upstream and downstream primers, 2×SYBR qPCR Mix and double distilled water were mixed in a flat-bottom 96-well plate and plated in a Bio-Rad IQ TM 5 system, and real-time fluorescence quantitative PCR amplification of cDNA was performed.

[0057] After treatment with each monosaccharide, it was found that glucuronic acid and arabinose could significantly alleviate STC-induced cellular oxidative stress and lipid metabolism abnormalities, which could significantly increase GSH ( Figure 1 A) Gene expression level, significantly reduced SREBP1C ( Figure 1 B) Gene expression level, thereby improving the antioxidant level of cells and maintaining normal lipid metabolism of cells.

[0058] Example 2 The alleviating effect of different preparations on STC-treated liver damaged cells

[0059] After thawing and resuscitation, human normal hepatocyte LO-2 cells were cultured in DMEM medium containing 10% fetal bovine serum and placed in an incubator at 37°C and 5% CO2. When the cells proliferated to 75% to 85%, they were routinely digested, passaged and frozen. The experiments were conducted in the following groups to evaluate the mitigating effect of each preparation on STC toxicity.

[0060] (1) Control group (CON group): The CON group was cultured with complete medium;

[0061] (2) Model group (STC group): The STC group was cultured with medium containing 30 μmol / L STC;

[0062] (3) Codonopsis pilosula polysaccharide group (CPP group): Codonopsis pilosula polysaccharide with a concentration of 10 mmol / L was prepared with normal saline, 30 μmol / L STC was added to the CPP group for culture, and 10 mmol / L Codonopsis pilosula polysaccharide was added for intervention;

[0063] (4) Glucuronic acid group (GlcA group): Glucuronic acid at a concentration of 10 mmol / L was prepared with normal saline, and 30 μmol / L STC was added to the GlcA group for culture, and 10 mmol / L glucuronic acid was added for intervention;

[0064] (5) Arabinose group (Ara group): 10 mmol / L arabinose was prepared with normal saline. 30 μmol / L STC was added to the Ara group for culture, and 10 mmol / L arabinose was added for intervention;

[0065] (6) Complex group (CGA group): CGA complex with a concentration of 10 mmol / L was prepared using normal saline. It was prepared with Codonopsis pilosula polysaccharide, glucuronic acid and arabinose in a ratio of 1:1:1. In the CGA group, 30 μmol / L STC was added for culture, and CGA complex with a concentration of 10 mmol / L was added for intervention.

[0066] After 24 hours of intervention with each preparation, all cells were collected and resuspended with 1 mL of TRIzol for full lysis, then chloroform was added and shaken vigorously to form an emulsion. The solution was allowed to stand at room temperature for 3 minutes and centrifuged at 12,000 g for 15 minutes at 4 °C. The upper aqueous phase was transferred to a new centrifuge tube, gently mixed with an equal volume of isopropanol by inversion, allowed to stand at room temperature for 10 minutes, and then centrifuged to form a gelatinous precipitate. The supernatant was discarded, and the RNA was washed three times with 75% ethanol, air-dried at room temperature to remove the residual solvent, and redissolved with an appropriate amount of enzyme-free sterile water. The concentration and purity of the total RNA obtained were detected by NanoDrop One. The extracted RNA was stored at -80 °C after aliquoting. The extracted RNA was reverse transcribed into cDNA and gDNA was removed using a PCR amplification instrument using the reverse transcription kit SweScript All-in-OneRT SuperMix containing reaction buffer, primers, and reverse transcriptase. According to the instructions of the kit 2×SYBRGreen qPCR Master Mix, cDNA, upstream and downstream primers, 2×SYBR qPCR Mix and double distilled water were mixed in a flat-bottom 96-well plate and plated in a Bio-Rad IQ TM 5 system, and real-time fluorescence quantitative PCR amplification of cDNA was performed.

[0067] The results showed that compared with the CPP group, GlcA group and Ara group, CGA could significantly increase the antioxidant gene GSH ( Figure 1 C) and SOD ( Figure 1 The expression level of CGA was significantly reduced, thereby alleviating STC-induced liver cell damage and achieving a detoxification effect. It can be seen that the components in the CGA complex have a synergistic effect in alleviating STC toxicity and can synergistically play an auxiliary protective role against chemical liver damage.

[0068] Example 3 Alleviating effects of different preparations on STC-induced acute liver injury in mice

[0069] Eight-week-old C57BL6 / J male mice were purchased and fed with normal chow, with free access to food and water. After a one-week adaptation period, the mice were randomly divided into 6 groups (as follows).

[0070] STC was used to construct an acute liver injury model in mice. Inflammation-related genes, oxidative stress-related genes and apoptosis-related genes were used as evaluation indicators to analyze the alleviating effects of CGA complex, Codonopsis pilosula polysaccharide, arabinose and glucuronic acid on the toxicity of STC.

[0071] (1) Control group (CON group): The CON group was gavaged with normal saline;

[0072] (2) Model group (STC group): The STC group was intragastrically administered with normal saline and 750 μg / kg body weight (BW) of STC;

[0073] (3) Codonopsis pilosula polysaccharide group (CPP group): The CPP group was intragastrically administered with 450 mg / kg Codonopsis pilosula polysaccharide (CPP) and 750 μg / kg BW STC;

[0074] (4) Glucuronic acid group (GlcA group): The GlcA group was intragastrically administered with 450 mg / kg glucuronic acid and 750 μg / kg BW of STC;

[0075] (5) Arabinose group (Ara group): The Ara group was intragastrically administered with 450 mg / kg arabinose and 750 μg / kg BW of STC;

[0076] (6) Complex group (CGA group): CPP, arabinose and glucuronic acid were combined in a ratio of 1:1:1 to form CGA complex. The CGA group was intragastrically administered with 450 mg / kg CGA complex and 750 μg / kg BW STC.

[0077] The oral dose for each group was 0.2 ml, and the oral administration lasted for 14 days. The body weight and food intake of mice in each group were recorded every day during the experiment, and the growth of the mice was observed. On the 15th day of the experiment, the mice were fasted for 12 hours, weighed and photographed after anesthesia, and about 500 μL-1 mL of blood was collected from the eye sockets and transferred to anticoagulant tubes. The mice were then killed and dissected, and the intact liver was collected. The body weights of mice and their livers, serum AST, ALT, TC and TG levels, and the expression levels of liver TNF-α, IL-6, GSH, SOD, Bax and Bcl2 genes were measured, and liver HE staining and NAS scores were performed to comprehensively evaluate the alleviating effects of each group on STC-induced acute liver injury.

[0078] The results showed that compared with the control group, CPP group, Glca group and Ara group, the CGA group could significantly increase the body weight of mice to the CON level ( Figure 2 A), significantly reduced serum TC ( Figure 2 B)TG( Figure 2 C) ALT( Figure 2 D) and AST( Figure 2 E) levels, and significantly reduced the liver inflammatory factor IL-6 ( Figure 2 F), TNF-α( Figure 2 G) and pro-apoptotic Bax ( Figure 2 I) and significantly increased the expression levels of genes that inhibit apoptosis, such as Bcl2 ( Figure 2 H) and antioxidant gene SOD ( Figure 2 J) and GSH ( Figure 2 K) expression level, significantly alleviated liver pathological damage ( Figure 2 L and Figure 2 M), thereby alleviating STC-induced acute liver injury, and further alleviating the toxicity of STC. It can be seen that the components in the CGA complex have a synergistic effect in alleviating liver injury.

[0079] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work shall all fall within the scope of protection of the present invention.

Claims

1. A preparation for preventing and treating liver damage, characterized in that: The preparation comprises glucuronic acid, arabinose and codonopsis pilosula polysaccharide, and the mass ratio of the glucuronic acid, arabinose and codonopsis pilosula polysaccharide is 1-2:1-2:1-2.

2. The preparation according to claim 1, characterized in that The mass ratio of glucuronic acid, arabinose and codonopsis pilosula polysaccharide is 1:1:

1.

3. The preparation according to claim 1 or 2, characterized in that The preparation also includes auxiliary materials acceptable in medicine or feed.

4. Use of the preparation according to any one of claims 1 to 3 in the preparation of a product for alleviating acute liver injury induced by sterigmatocystin.

5. Use of the preparation according to any one of claims 1 to 3 in the preparation of a product for alleviating weight loss caused by acute liver injury induced by sterigmatocystin.

6. Use of the preparation according to any one of claims 1 to 3 in the preparation of a product for regulating changes in serum component levels caused by acute liver injury induced by sterigmatocystin, wherein the components include one or more of aspartate aminotransferase, alanine aminotransferase, triglycerides or total cholesterol.

7. The use according to any one of claims 4 to 5, characterized in that: The product is a medicine or a feed.

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