CN112125982B - Application of Pleurotus tuber-regium sclerotium polysaccharide - Google Patents
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Abstract
The protective effect of Pleurotus tuber-Regium Scleropolysaccharide (PTRs) on acetaminophen (APAP) -induced C57BL/6 mouse acute liver injury was studied. The results show that: compared with the model group, the serum contents of glutamic-pyruvic transaminase and lactic dehydrogenase of mice of each dose group of PTRs are extremely obviously reduced, the contents of glutamic-oxaloacetic transaminase of medium and high dose groups are extremely obviously reduced, and the contents of alkaline phosphatase of high dose groups are extremely obviously reduced. The malondialdehyde content in the livers of the mice in the PTRs medium and high dose groups is extremely obviously reduced, and the superoxide dismutase content is extremely obviously increased; the content of glutathione peroxidase in the high-dose group is extremely obviously increased. The PTRs can improve mouse liver cell fatty lesion, necrosis and inflammatory infiltration, and obviously reduce liver cell damage. The PTRs are prompted to remarkably improve the oxidation resistance of the mice, and have an obvious protective effect on acute liver injury caused by APAP.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an application of Pleurotus tuber-regium sclerotium polysaccharide in preparation of a drug for preventing drug-induced liver injury.
Background
Pleurotus tuber-regium (Fr.) SingPleurotus tuber-regium) The pleurotus ostreatus, pleurotus poria cocos, poria cocos wolf and the like are mainly distributed in Yunnan province of China and countries such as Malaysia, Australia, Nigeria and Kenya, and are rare edible medicinal and edible fungi with seed entities and sclerotium. The Pleurotus tuber-regium fruiting body is rich in protein, and the sclerotium is mainly non-starch polysaccharide. Pleurotus tuber-regium is sweet and warm in nature, has effects of invigorating qi and replenishing blood, and can be used for treating headache, stomach ache, fever, asthma, smallpox and hypertension.
The liver, which is the main site for the metabolism of drugs in the body, is very susceptible to the drugs. It is reported that acetaminophen (APAP), carbon tetrachloride, thioacetamide, ethanol, D-galactose, diethylnitrosamine, dimethylnitrosamine and the like can promote the accumulation of active oxygen in liver cells, induce oxidative stress and cause liver injury, severe patients can cause liver failure and death of patients, and the screening of natural active ingredients with the effect of relieving liver injury has important application value. Research shows that different administration modes can obviously influence the drug effect and the metabolic mode of polysaccharide substances, and the intraperitoneal injection administration has more obvious effect than the intragastric administration. The protection effect of the mouse on acetaminophen-induced drug-induced liver injury is studied by adopting a mouse experiment of intraperitoneal injection of Pleurotus tuber-Regium Scleropolysaccharide (PTRs).
Disclosure of Invention
The invention aims to provide an application of Pleurotus tuber-regium sclerotium polysaccharide in preparing a medicine for preventing drug-induced liver injury.
In order to achieve the purpose, the invention adopts the following technical scheme:
the preparation method of the pleurotus tuber-regium sclerotium polysaccharide comprises the following steps:
drying Pleurotus tuber-regium sclerotium at 60 deg.C, pulverizing with pulverizer, and sieving with 60 mesh sieve. Weighing 1 kg of powder, adding 3 times of petroleum ether, refluxing for 2 h for degreasing, repeating for 2 times, filtering, drying filter residue at 60 ℃, and mixing the materials according to a material-liquid ratio of 1:20 g.mL-1Adding distilled water, extracting in boiling water bath for 2 times (each time for 2.5 hr), vacuum filtering the extractive solution, collecting supernatant, mixing supernatants, and concentrating under reduced pressure to 2L; adding 95% ethanol into the concentrated solution to make ethanol final concentration 80%, stirring, standing overnight at 4 deg.C, centrifuging 3600 g for 15 min, sequentially washing precipitate with acetone, diethyl ether and anhydrous ethanol, dissolving the precipitate in double distilled water, dialyzing with 8000 Da semipermeable membrane, and freeze drying to obtain powdery Pleurotus Tuber Regium scleropolysaccharide.
The invention has the advantages that: the PTRs prepared by the invention can improve mouse liver cell fatty lesion, necrosis and inflammatory infiltration, and obviously reduce liver cell damage. The PTRs are prompted to remarkably improve the oxidation resistance of the mice, and have an obvious protective effect on acute liver injury caused by APAP.
Drawings
FIG. 1 is a liquid chromatogram of PTRs and standard substance, wherein A is the liquid chromatogram of PTRs, and B is a mixed standard chromatogram of mannose, rhamnose, glucose and galactose.
FIG. 2 is a graph of the effect of PTRs on pathological changes in mouse liver tissue (200X); a: a normal control group; b: a model group; c: a positive control group; d: PTRs Low dose group (100 mg. kg)-1) (ii) a e: PTRs middle dose group (200 mg. kg)-1) (ii) a f: PTRs high dose group (400 mg kg)-1）。
Detailed Description
1. Materials and animals
Pleurotus tuber-regium (Fr.) SingP. tuber-regium) The dry sclerotium is purchased from Luo needle Zhen Ding Hu village in Linchuan region of Fuzhou city in Jiangxi province.
54 SPF-grade C57BL/6 mice, male, 6-8 weeks old, and 20-25 g in weight, provided by Jiangsu Seisakusho model biological research center, with production license numbers: SCXK (su) 2018-.
2. Primary reagents and instruments
Petroleum ether, phenol (west longa gmbh); absolute ethanol (Fuyu Fine chemical Co., Ltd., Tianjin); glucose, galactose, mannose, rhamnose, acetaminophen (APAP), N-acetylcysteine (NAC) (leaf Biotech, Inc., Shanghai); concentrated sulfuric acid (Shanghai Vocko Biotech Co., Ltd.); trifluoroacetic acid (shandongkun new materials science and technology limited); chloroform (Nanjing Runzi petrochemical Co., Ltd.); 1-phenyl-3-Methyl-5-pyrazolone solution (3-Methyl-1-phenyl-2-pyrazolin-5-one, PMP) (Wuxi market SpA chemical Co., Ltd.); alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), Lactate Dehydrogenase (LDH), alkaline phosphatase (AKP), Malondialdehyde (MDA), superoxide dismutase (SOD), glutathione-peroxidase (GSH-Px), Hematoxylin and Eosin (HE) staining kit (Nanjing institute of bioengineering).
DFT-200 high speed universal pulverizer (large mechanical Co., Ltd., forest, Wenling City); SF6-02B electric heating constant temperature blast drying oven (Hengfeng medical appliances, Inc. of yellow stone); AR223CN electronic balance (aohaus instruments ltd); MS105DU balance (mettler-toledo group, switzerland); WO-5L constant temperature water bath (Shanghai surge Instrument and Equipment Co., Ltd.); HDM-1000 thermostatic electric heating jacket (Changzhou Guohua electric appliances Co., Ltd.); an SHZ-DIII circulating water type vacuum pump (Shanghai Bangxi Instrument science and technology Co., Ltd.); DLSB-5L/20 cryogenic coolant circulation pump (Steud City Prov. instruments, Inc.); RE2000A rotary evaporator (shanghai yanglong biochemical instruments factory); LXJ-IIB Low speed high capacity Multi-tube centrifuge (Shanghai' an pavilion scientific Instrument factory); UV-1800 UV spectrophotometer (Shimadzu instruments, Japan); DM4000B research grade biomicroscopy (come, germany).
Example 1 preparation of aqueous extracts of Pleurotus Tuber Regium (PTRs), determination of the Total sugar content of the PTRs and analysis of monosaccharide composition
Drying Pleurotus tuber-regium sclerotium at 60 deg.C, pulverizing with pulverizer, and sieving with 60 mesh sieve. Weighing 1 kg of powder, adding 3 times volume of petroleum ether, refluxing for 2 h for degreasing, repeating for 2 times, filtering, drying the filter residue at 60 deg.C, and mixing the filtrate with the filtrate at a ratio of 1:20 (g.mL)-1) Adding distilled water, extracting in boiling water bath for 2 times (2.5 hr each time), vacuum filtering the extractive solution, collecting supernatant, mixing supernatants, and concentrating under reduced pressure to 2L. Adding 95% ethanol to the concentrated solution to make ethanol final concentration 80%, stirring, standing at 4 deg.C overnight, 3600gCentrifuging for 15 min, sequentially washing the precipitate with acetone, diethyl ether and anhydrous ethanol, dissolving the precipitate in double distilled water, dialyzing with 8000 Da semipermeable membrane, and freeze drying to obtain powdery Pleurotus Tuber Regium scleroglucan PTRs. The yield of PTRs is calculated as follows:
drawing standard curve and measuring total sugar content by phenol-sulfuric acid method, collecting PTRs 10 mg, adding ultrapure water to make 100 μ g/mL-1The solution was taken 1 mL and 1 mL5% phenol and 5 mL concentrated H were added sequentially2SO4Reacting in boiling water bath for 15 min, cooling, measuring absorbance at 490 nm, and calculatingTotal sugar content in PTRs.
The method comprises the steps of performing monosaccharide component analysis by using 1-phenyl-3-methyl-5-pyrazolone solution (PMP) derivatization and High Performance Liquid Chromatography (HPLC) (Chenfeng, Zhaowanling, Wanxing apparatus, and the like. the content of polysaccharide in Angelica keiskei roots is determined by a phenol-sulfuric acid method. chemical engineering management, 2019, 1(26): 11-13.). Precisely weighing 10 mg of PTRs, placing the PTRs into a test tube with a plug, and adding 2 mL of 2 mol.L-1Trifluoroacetic acid, sealing with a plug, hydrolyzing at 110 deg.C for 2 hr, cooling at room temperature, and adding 3 mol/L-1Adjusting the pH value to 7.0 by using sodium hydroxide, metering the volume to 5 mL by using distilled water, and precisely measuring 0.2 mL for PMP derivatization. HPLC chromatographic conditions were column: eclipse XDB-C18 (4.6 mm. times.250 mm, 5 μm), mobile phase: (A) acetonitrile, (B) 0.1 mol. L-1Ammonium acetate solution, 0-20 min (A: 17% -19%), 20-40 min (A: 19%); flow rate: 0.8 mLmin-1(ii) a Column temperature: 30 ℃; sample introduction amount: 15 mu L of the solution; detection wavelength: 250 nm. And comparing and analyzing the standard substance spectrogram to obtain the monosaccharide composition.
The results are shown in FIG. 1, in which FIG. 1A shows PTRs, and FIG. 1B shows four monosaccharide mixing standards of mannose, rhamnose, glucose and galactose. The yield of PTRs is (3.68 +/-0.03)%, and the total sugar content is (28.7 +/-0.3)%. The polysaccharides in PTRs are mainly composed of 4 monosaccharides, namely mannose, rhamnose, glucose and galactose, and the molar ratio of the four monosaccharides is 1:0.37:1.84:0.38 as calculated by standard yeast. The highest content of glucose is followed by mannose, galactose, rhamnose. The four monosaccharides were labeled as follows:
mannose standard koji: y =2434 x-993.35; r2=0.9952；
And (3) marking rhamnose: y =1617.6 x-605.35; r2=0.9999；
Marking glucose as yeast: y =1375.6 x-592.71; r2=0.9995；
And (3) galactose standard koji: y =779.48 x-271.83; r2=0.9997。
Example 2 mice grouping and dosing
The mice are raised in an SPF experimental animal house, the indoor temperature is 20-25 ℃, the relative humidity is 50-70%, and the noise is less than 60%dB, 12 hours each time, alternating light and shade every day, and adaptively feeding for one week. The 54 mice were randomly divided into 6 groups of 9 mice each, namely a normal control group, a model group, and a positive control group (NAC dose of 200 mg. kg)-1) Low, medium and high dose groups of PTRs (doses of 100, 200, 400 mg. kg, respectively)-1). Injecting PBS solution into abdominal cavity of normal control group and model group mice, and injecting 10 mg/mL into abdominal cavity of positive control group according to dosage-1NAC solution (prepared from PBS solution), low, medium and high PTRs dose components are intraperitoneally injected with 5, 10 and 20 mg/mL respectively-1PTRs solution (formulated from PBS solution) was administered for 6 d consecutively 1 time daily. After 12 h on day 6, fasting without water supply for 12 h, and performing final administration for 1 h, except for normal control group, performing intraperitoneal injection of PBS solution, and performing 400 mg/kg for the rest groups-1Dosage intraperitoneal injection of 20 mg/mL-1The following assays were performed on APAP solutions (prepared from PBS solution, heated in a 55 ℃ water bath until APAP was completely dissolved) after 6 h without water deprivation:
(1) mouse serum ALT, AST, LDH and AKP content determination: blood was collected from the eyeball and plasma was at 300 deg.CgSerum is taken after centrifugation for 15 min under the condition, and the ALT, AST, LDH and AKP content of the serum is determined according to the kit instruction. Alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), Lactate Dehydrogenase (LDH) and alkaline phosphatase (AKP) kits were purchased from Nanjing Biotech institute for Biotechnology engineering.
As can be seen from Table 1, compared with the normal control group, the contents of ALT, AST, LDH and AKP in the serum of the model group mice are remarkably increased, which indicates that APAP induces acute drug-induced liver injury of the mice and causes the related biochemical indexes in the serum to be remarkably changed. Compared with a model group, the ALT and LDH content in the serum of mice with low, medium and high PTRs is remarkably reduced; the AST content in the serum of the mice in the PTRs medium and high dose groups is remarkably reduced; the serum AKP content of the mice in the PTRs high-dose group is remarkably reduced, and the serum AKP content of the mice in the low-dose and medium-dose groups is remarkably reduced.
TABLE 1 ALT, AST, LDH and AKP content in mouse serum
Data are mean ± standard deviation (n = 9);##: compared with the normal control group,P<0.01；*: in comparison with the set of models,P<0.05，**：P<0.01
Values are means ± SD(n=9), # P<0.05，## P<0.01 vs control group；* P<0.05，** P<0.01 vs model group.
(2) measuring the MDA, SOD and GSH-Px contents of mouse liver: cutting the liver of the left lobe, accurately weighing 0.1 g of liver tissue with 5 mL of 4% more, adding 9 times volume of physiological saline, manually grinding on ice to homogenize the tissue, 300%gCentrifuging for 15 min, collecting supernatant, and detecting contents of MDA, SOD and GSH-Px according to kit specification, wherein the kit is purchased from Nanjing to build a bioengineering institute of Biotechnology.
As shown in Table 2, compared with the normal control group, the liver tissues of the mice in the model group have remarkably increased MDA content, remarkably reduced SOD content and remarkably reduced GSH-Px content, which indicates that the oxidation resistance of the mouse body is remarkably reduced. Compared with the model group, the MDA content in the liver of the mice in the PTRs medium and high dose groups is remarkably reduced, and the MDA content in the mouse in the low dose group is remarkably reduced; SOD content in the liver of the mice in the PTRs medium and high dose groups is remarkably increased, and SOD content in the mice in the low dose groups is remarkably increased; the GSH-Px content in the liver of the mice in the PTRs high-dose group is remarkably increased, and the GSH-Px content in the mice in the medium-dose group is remarkably increased. The PTRs are prompted to obviously improve the anti-oxidative stress capability of organisms.
TABLE 2 MDA, SOD and GSH-Px content in mouse liver
#: compared with the normal control group,P<0.05; other notes are in Table 1
(3) Mouse liver tissue staining section and observation: fixing polyformaldehyde, embedding the slices in paraffin, performing hematoxylin-eosin (HE) staining (Xiufei. different fixative solutions influence on HE staining of mouse liver paraffin slices. medicine front edge, 2013, 2(33): 116-.
As can be seen from FIG. 2, in comparison with the normal control group, the hepatic lobule structure of the model group mice is destroyed, and the degeneration, bleeding and necrosis of the liver cells occur near the central vein, accompanied by partial cell nucleus contraction and lysis, and partial inflammatory cell infiltration, which indicates that APAP induces the acute liver injury of the mice. Compared with the model group, the mouse liver lobule treated by the PTRs medium and high dose groups has complete structure, the liver chordae arrangement tends to be neat, the liver cell morphology is obviously improved, and the inflammatory infiltration and degenerative necrosis cells of cells near the central vein are obviously reduced, which indicates that the PTRs can improve the pathological change of the mouse liver tissue induced by APAP.
APAP can saturate glucuronic acid and sulfation metabolic pathways, a large amount of APAP is metabolized by a cytochrome P450 enzyme system to generate a toxic intermediate NAPHQ, GSH is rapidly exhausted, excessive active oxygen is generated, the scavenging capacity of antioxidant enzymes such as SOD and GSH-Px is exceeded, oxidative stress injury of the liver is induced, and the effects of increasing permeability and cell membrane damage of liver cell membranes, increasing intracellular MDA content and increasing ALT, AST, LDH and AKP content in blood are mainly shown. The research result shows that: the PTRs can obviously reduce the contents of ALT, AST, LDH and AKP in mouse serum, reduce the content of liver MDA, improve the contents of liver SOD and GSH-Px, improve the pathological change of liver tissues, have a certain protection effect on APAP-induced acute liver injury of mice, and are presumed to be related to the improvement of the anti-oxidative stress capability of organisms.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (1)
1. The application of Pleurotus tuber-regium sclerotium polysaccharide in preparing medicine for preventing acetaminophen drug-induced liver injury is characterized in that the preparation method of Pleurotus tuber regium sclerotium polysaccharide comprises the following steps:
drying sclerotium of Pleurotus tuber-regium at 60 deg.C, pulverizing with pulverizer, and sieving with 60 mesh sieve; weighing 1 kg of powder, adding 3 times of petroleum ether, refluxing for 2 h for degreasing, repeating for 2 times, filtering, drying filter residue at 60 ℃, and mixing the materials according to a material-liquid ratio of 1:20 g.mL-1Adding distilled water, extracting in boiling water bath for 2 times (each time for 2.5 hr), vacuum filtering the extractive solution, collecting supernatant, mixing supernatants, and concentrating under reduced pressure to 2L; adding 95% ethanol into the concentrated solution to make ethanol final concentration 80%, stirring thoroughly, standing overnight at 4 deg.C, centrifuging 3600 g for 15 min, and washing the precipitate with acetone, diethyl ether, and anhydrous ethanol sequentially; dissolving the precipitate in double distilled water, dialyzing with 8000 Da semipermeable membrane, and freeze drying to obtain powdered Pleurotus Tuber Regium scleropolysaccharide.
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| "虎奶多糖免疫功能实验研究";黄必胜等;《时珍国医国药》;20000820;第11卷(第8期);第685-686页 * |
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