CN107929308B - Application of panax japonicus polysaccharide in preparation of medicine for preventing and/or treating liver cancer - Google Patents

Abstract

The invention belongs to the technical field of medical application of plant polysaccharide, and particularly discloses application of panax japonicus polysaccharide in preparation of a medicine for preventing and/or treating liver cancer. The application discovers for the first time that PSPJ (30-100mg/kg) has obvious inhibition effect on the in vivo growth of tumor cells of a mouse with liver cancer H22 tumor (the tumor inhibition rates are 41.7 percent and 54.3 percent respectively). Moreover, PSPJ has no obvious toxic effect on the general physical condition, the blood conventional index, and the liver and kidney functions of the liver cancer H22 tumor-bearing mice. The application also finds that the PSPJ has definite prevention and protection effects on the acute liver injury of mice induced by LPS/D-GalN, and the action mechanism of the PSPJ can prevent and prevent the liver injury, improve the function of the damaged liver and finally prevent the possibility of converting the liver injury into liver cancer by inhibiting the occurrence of oxidative stress reaction and inflammatory reaction.

Description

Application of panax japonicus polysaccharide in preparation of medicine for preventing and/or treating liver cancer

Technical Field

The invention relates to the technical field of medical application of plant polysaccharide, in particular to application of panax japonicus polysaccharide in preparing a medicament for preventing and/or treating liver cancer.

Background

Liver cancer is a malignant tumor seriously harming human health, and has high morbidity and high mortality. The pathogenesis of liver cancer is very complex, wherein chronic persistent inflammation and the deletion of cancer suppressor genes are important factors for liver cancer. At present, the mechanism of regulating liver cancer cell proliferation and apoptosis by most cancer suppressor genes is clearly researched. In view of the fact that some cancer suppressor genes are also expressed in immune cells, the tumor suppressor genes have dual functions of suppressing inflammation and suppressing cancer in immune cells and tumor cells, and further exert the effect of efficiently blocking the occurrence and development of inflammation canceration (particularly inflammation-related liver cancer) by targeting common signal molecules in the immune cells and the tumor cells. Therefore, the application mainly researches that the panax japonicus polysaccharide inhibits hepatocellular carcinoma through anti-inflammatory and immune activation, and provides theoretical basis for clinical application of the panax japonicus polysaccharide serving as an immune adjuvant in treating malignant diseases.

Related reports are not found in the prior art, and the application fills the blank.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide the application of panax japonicus polysaccharide in preparing the medicine for preventing and/or treating liver cancer.

Compared with the prior art, the invention has the following advantages and effects:

1. the method for preparing the panax japonicus polysaccharide (PSPJ) is simple, convenient and feasible, and has strong controllability.

2. The panax japonicus polysaccharide obtained by the method has high purity and high yield.

3. The application discovers that PSPJ has dose-dependent prevention and protection effects on mice acute liver injury induced by LPS/D-GalN for the first time. The action mechanism can inhibit the occurrence of oxidative stress reaction and inflammatory reaction, prevent and prevent liver injury, improve the function of damaged liver and finally prevent the transformation of inflammation to liver cancer.

4. The application discovers for the first time that PSPJ (30-100mg/kg) has a remarkable inhibiting effect (the tumor inhibition rates are 41.7% and 54.3% respectively) on the in-vivo growth of tumor cells of a mouse with liver cancer H22 tumor, and meanwhile, PSPJ has no obvious toxic effect on the general physical condition, the blood conventional index and the liver and kidney functions of the mouse with H22 tumor.

Drawings

FIG. 1 is the elution graph of crude polysaccharide from the Sephadex G-75 column chromatography of example 1.

FIG. 2 is an HPLC chart of the panax japonicus polysaccharide after column chromatography in example 1.

Chromatographic conditions are as follows: the column was Shodex Ohpak SB-804 HQ (8X 300mm), a differential photodetector, 0.1M NaCl as a mobile phase, a flow rate of 0.5mL/min, a column temperature of 35 ℃ and a sample introduction amount of 20. mu.L.

FIG. 3 is a survival diagram of the effect of Panax japonicus polysaccharides on mice with acute liver injury.

FIG. 4 morphological and histopathological changes of panax japonicus polysaccharide on liver injury of mice.

Detailed Description

The applicant will now further describe the technical solution of the present invention in detail with reference to specific examples. It should be understood that the following should not be construed as limiting the scope of the claims of the present invention in any way.

Example 1

Preparing panax japonicus polysaccharide:

the raw material Panax japonicus medicinal material is dried rhizome of Panax japonicus (Panax japonica C.A.Mey.) belonging to Araliaceae and genus Panax, is a family Panax japonicus, and is produced from Yingxiang Cheng-mu Yan-Xiang-Mi dam of Xuan En county of the Anshi soil family Miao nationality.

The method comprises the following steps:

taking dry panax japonicus medicinal material powder, adding 95% ethanol (V/V, the percentage concentration of the ethanol in the specification refers to volume percentage concentration, and is not described in detail later), refluxing for 3h at 60 ℃, and degreasing;

adding 80% ethanol, refluxing at 60 deg.C for 2 hr, and removing monosaccharide and oligosaccharide;

drying the residue in oven, extracting under reflux with purified water as solvent for 3 times (2 hr each time), mixing filtrates, concentrating in rotary evaporator to obtain fluid extract, adding ethanol, adjusting ethanol concentration to 80%, standing overnight at 4 deg.C, filtering the next day, precipitating, and oven drying to obtain rhizoma Panacis Japonici crude product;

dissolving the obtained crude product of panax japonicus with warm water (60 ℃), removing protein, dialyzing in running water for 48h, and freeze-vacuum drying the dialyzate to obtain crude polysaccharide of panax japonicus.

Purifying the crude rhizoma Panacis Japonici polysaccharide with DEAE-Sepharose FF column for 3 times; and then separating and purifying by a Sephadex G-75 column to obtain a main peak, dialyzing and desalting the peak eluent, and drying to obtain panax japonicus polysaccharide, PSPJ for short, wherein the related data are shown in table 1.1.

TABLE 1.1

Through detection, the obtained panax japonicus polysaccharide mainly comprises glucose, galacturonic acid, galactose, glucuronic acid, rhamnose, arabinose, xylose and the like, and the mass fraction is shown in table 1.2.

TABLE 1.2 Mass fraction table of monosaccharide components in Panax japonicus polysaccharides

The elution curve of crude rhizoma Panacis Japonici polysaccharide after DEAE-Sepharose FF and Sephadex G-75 column chromatography is shown in FIG. 1. Further, the purified panax japonicus polysaccharide was confirmed to be a homogeneous polysaccharide by HPLC chromatography, as shown in fig. 2.

Example 2

anti-H22 liver cancer cell activity and general toxicity study of panax japonicus polysaccharide prepared in example 1

2.1 materials of the experiment

2.1.1 animals and cell lines

Kunming species mice: the male and female halves of the Chinese medicament are respectively provided with the weight of 18-22 g, and the license number is as follows: SCXK (jae): 2008-0003.

The H22 cell line was gifted by Chen Luo-Yi teacher at the college of medicine of the university of the southern China and stored in a refrigerator at-80 ℃. Centrifuging during recovery, removing DMSO from the frozen stock solution, and diluting with normal saline to 1 ANG 10⁶Each dose is 0.2mL, inoculated in the abdominal cavity of a mouse, passaged after seven days, ascites of a third generation H22 ascites tumor mouse is taken, diluted by physiological saline according to a ratio of 1:10, and 0.2mL of each dose is inoculated in the subcutaneous part of the right forelimb armpit of the mouse, so as to establish an H22 solid tumor model.

2.1.2 Experimental reagents

Rhizoma Panacis Japonici polysaccharide: example 1 was prepared;

sodium chloride injection: wuhan Binshu Bihe pharmaceutical industry, batch number: 20150927.

heparin sodium: BIOSHARP Biotech, Inc., lot 201609;

ethylenediaminetetraacetic acid (EDTA), national drug group chemical agents ltd, lot No.: f20100108;

5-Fluorouracil (5-FU) Shanghai Crystal pure reagent, Ltd., batch No.: 20141108.

2.2 Experimental methods

2.2.1 methods for establishing and administering solid tumor model

60 Kunming mice are taken, and an H22 solid tumor model is established. After 2 days, the weight was measured and randomly divided into 12 individuals (male and female halves). A normal group (Non-tumor), a model group (salt), a positive control group (5-FU 10mg/kg) and a panax japonicus polysaccharide administration group (30, 100mg/kg) are set. Normal and model groups were given saline in the same manner as above. After the last administration, the mice are killed by cervical dislocation without water supply for more than 12 hours, and the axillary solid tumors are dissected and weighed and recorded. Other tissues and organs are quickly taken out and weighed and then stored for later use. The inhibition rate was calculated according to the following formula: tumor inhibition ratio (%) (average tumor weight in placebo-average tumor weight in administered group)/average tumor weight in placebo × 100. Organ index is calculated according to the following formula: organ index 10 × organ weight (mg)/mouse body weight (g)

2.2.2 measurement of Biochemical indicators of blood

A part of the blood of the mouse collected by the method of taking the blood from the venous plexus at the bottom of the frame is anticoagulated by EDTA and is used for detecting four blood conventional indexes, namely White Blood Cells (WBC), Red Blood Cells (RBC), Hemoglobin (HGB) and Platelets (PLT). The other part is anticoagulated by heparin, centrifuged for 10min at 3000r/min, and tested by serum for the following liver and kidney function indexes: transaminase (ALT, AST), urea nitrogen (BUN), Uric Acid (UA), and Creatinine (CRE).

2.2.3 data analysis

Results are expressed as Mean ± standard deviation (Mean ± SD). All statistical analyses were performed using the Orgin-7.5 software for t-test analysis, with P <0.05 indicating significant differences; p <0.01 indicates a very significant difference.

2.3 results and analysis

2.3.1 evaluation of inhibitory Effect of Panax japonicus polysaccharides on H22 hepatocellular carcinoma

To evaluate the inhibitory effect of panax japonicus polysaccharide on H22 hepatocellular carcinoma, a H22-bearing mouse solid tumor model was established and administered at the same dose, as shown in table 2.1, with the average tumor weight of 1.27g in the blank group. In the PSPJ (30 and 100mg/kg) groups, the mean tumor weights were reduced to 0.74g and 0.58g, respectively. The mean tumor weights were significantly different in the PSPJ-administered groups compared to the negative control group. Tumor inhibition (61.4%) after 5-FU treatment, but the mean body weight of this group of tumor-bearing mice also dropped sharply and one mouse died at the end of the study. In contrast, the PSPJ (100mg/kg) had a tumor suppression rate of 54.3%, had little effect on the body weight of tumor-bearing mice, and no mortality occurred in the mice of the administered group.

TABLE 2.1 inhibition of tumor growth in H22 tumor-bearing mice by Panax japonicus polysaccharides (Mean. + -. SE, n ═ 12)

Compared to the blank group:^#p<0.01；^btumor inhibitory rate.

2.3.2 evaluation of toxicity of Panax japonicus polysaccharides on H22 tumor-bearing mice

To assess the toxic effects of PSPJ on normal hepatocytes, serum parameters of liver function, i.e., ALT, AST activity, were measured. 5-FU treatment significantly increased serum levels of ALT and AST, but PSPJ had no effect on this (Table 2.2). To investigate the toxic effects of PSPJ on host kidney, we evaluated their serum kidney function markers, including BUN, UA and CRE. As shown in table 2.3, the positive drug group serum BUN and CRE were elevated and the PSPJ administered group remained nearly unchanged. At the same time, we also examined the effect of PSPJ on the concentration of blood WBC, RBC, HGB and PLT. The 5-FU group reduced WBC content in the blood compared to the blank group, while PSPJ did not have much influence on it, and the amount of RBC, HGB and PLT remained unchanged in all experimental groups (table 2.4).

TABLE 2.2 influence of Panax japonicus polysaccharides on liver function in tumor-bearing mice (Mean. + -. SE, n. 12)

Compared to the blank group:^#p<0.01.

TABLE 2.3 influence of Panax japonicus polysaccharides on renal function in tumor-bearing mice (Mean. + -. SE, n. 12)

Compared to the blank group:^#p<0.01

TABLE 2.4 influence of Panax japonicus polysaccharides on blood of tumor-bearing mice (Mean. + -. SE, n. 12)

Compared to the blank group:^#p<0.01.

2.4 discussion

In this embodiment, by establishing an H22 tumor-bearing mouse model, the body weight change, survival days, tumor weight, tumor suppression rate, blood routine, liver and kidney function, and other indicators of the mouse are observed. The result shows that the panax japonicus polysaccharide has a certain inhibition effect on hepatocellular carcinoma of H22 tumor-bearing mice and has no toxic effect. By recording the weight change of the mice before and after the experiment, the weight of the experimental mice is not abnormal after the panax japonicus polysaccharide is administrated, and the mice do not die before the experiment is finished. In the experimental process, observation shows that the diet, activity, hair and excrement of the mice in the panax japonicus administration group are normal and the drug poisoning phenomenon does not occur, while the mice in the 5-FU group are thinned, hairy and even dead. The tumor inhibition rates are calculated to show that the tumor inhibition rates after the panax japonicus polysaccharide is administrated are 41.7% and 54.3%, and a good tumor inhibition effect is shown.

Alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) are common indicators of liver function. The liver is the largest detoxifying organ of the human body, and the level of the liver and the liver is directly related to the liver function. After the panax japonicus polysaccharide is administrated, the liver function of an experimental mouse is not obviously abnormal, which indicates that the panax japonicus polysaccharide has no drug-induced toxicity. Urea Nitrogen (BUN), Uric Acid (UA) and Creatinine (CRE) are three most commonly used indicators of renal function. The kidney is the largest excretory organ of the human body, and the excretion does not affect the abnormal function of the kidney after the panax japonicus polysaccharide is administered. By detecting blood indexes, the panax japonicus polysaccharide has a trend of improving leucocytes, erythrocytes and blood platelets, and compared with a positive medicine, all blood indexes are obviously increased. The research results jointly prove that the panax japonicus polysaccharide has the capacity of inhibiting the growth of tumors with low toxicity and high efficiency in vivo.

Example 3

The protection effect of the panax japonicus polysaccharide prepared in the example 1 on the LPS/D-GalN induced acute liver injury of mice is as follows:

LPS/D-GalN is adopted to induce mice acute liver injury as a model, the prevention and protection effects of panax japonicus polysaccharide on acute liver injury are researched, and the mechanism is tried to be discussed.

3.1 laboratory animals and reagents

3.1.1 Experimental animals

48 SPF male Kunming mice with the body mass of 18-22 g are purchased from the research center of experimental animals in Hubei province, and the production license number of the experimental animals is as follows: SCXK (jaw) 2015-0018.

3.1.2 reagents

Rhizoma Panacis Japonici polysaccharide: example 1 was prepared; lipopolysaccharide (LPS) was purchased from Biosharp; galactosamine (D-GalN) was purchased from Tokyo Chemical Industry; the kit for measuring the content of alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), reduced Glutathione (GSH), superoxide dismutase (SOD) and Malondialdehyde (MDA) is purchased from Nanjing institute of biological research; the kit for measuring the contents of tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta) and interleukin-6 (IL-6) is purchased from Shanghai leaf Biotech company.

3.2 methods

3.2.1 group administration and animal modeling

The 48 Kunming mice were randomly divided into 4 groups, namely a normal control group, a model group and a PSPJ administration group, and were subjected to intragastric administration (the normal control group and the model group were administered with an equal volume of physiological saline, and the administration group was administered with 50mg/kg and 150mg/kg of PSPJ, respectively) 1 time per day for 7 days. After 2h of the last administration, LPS (8. mu.g/kg) and D-GalN (800mg/kg) were intraperitoneally injected into mice of each group except the normal control group to prepare an acute liver injury model.

3.2.2 sample Collection and processing

The death of the mice was observed and recorded within 24h, and blood was taken from the eyeball immediately before the death of the experimental mice, and serum was isolated. Immediately dissecting a mouse after blood collection, taking liver tissue small blocks with the same size at the same parts of the right lobe of the liver of the mouse, fixing the liver tissue small blocks in 10% neutral formaldehyde, observing histopathological changes of the liver by HE staining, and storing the rest livers at-80 ℃ for biochemical analysis.

3.2.3 correlation index detection

The death of each group of mice was recorded at different time points over 24h and survival curves were plotted. The liver was taken, photographed and weighed, and the liver index was calculated. Detecting the levels of ALT, AST, GSH, SOD and MDA in the serum of the mice according to the kit instruction; TNF-alpha, IL-1 beta and IL-6 levels in mouse liver homogenates were determined by ELISA.

3.2.4 data analysis

The experimental data are expressed as Mean ± standard deviation (Mean ± SE) and analyzed using Sigma-plot10.0 statistical software. And performing mean comparison analysis on two groups of samples by adopting a two-tailed t test, and distinguishing whether the multiple groups have statistical significance or not by adopting one-factor variance analysis, wherein if P is less than 0.05, the difference is proved to have statistical significance.

3.3 results and analysis

3.3.1 Effect of Panax japonicus polysaccharides on survival Rate of mice with acute liver injury

The acute liver injury of mice induced by LPS/D-GalN can cause a large amount of death of model mice in a short time (6h), and the prevention and protection effects of the drug on liver injury can be better evaluated by observing the survival time and survival rate of the mice after modeling. The experimental results show that: the mice in the model group begin to die about 4 hours after modeling, the time of death peak is about 5 hours, and the survival rate is 16.7% after 24 hours. In the administration group, the death peak time of the PSPJ low-dose group mice is about 8h, and the survival rate of 24h is 50%; the peak time of death of the PSPJ high dose group mice was close to 10h, and the 24h survival rate was 4.5 times higher than that of the model group, which was increased by 25% compared to the PSPJ low dose group (fig. 3). The result shows that the PSPJ has prevention and protection effects on mice acute liver injury induced by LPS/D-GalN and has better dose dependence. We then performed follow-up observations on survival time of surviving mice, and the results showed that: the ultimate survival time of the model group mice is 2 days, while the ultimate survival time of the PSPJ high dose group can reach even 7 days.

3.3.2 Effect of Panax japonicus polysaccharides on liver morphology and pathology of mice with acute liver injury

Liver appearance and pathological morphology are the most intuitive indicators for evaluating liver damage and the prevention and protection effects of drugs on damage. The liver of the normal control group mouse is light in appearance, smooth in edge and tough in quality; in the aspect of liver histopathology morphology, the cells are uniform in size, orderly in arrangement and complete in structure, and the cell nucleus is located in the center of the liver cells. The appearance color of the liver of the model group is dark red, and the liver has obvious swelling and blood stasis; pathological damage of liver tissue is serious, liver cells are degenerated, small vacuoles are filled in cytoplasm, and obvious liver bleeding and inflammatory cell infiltration are generated. After PSPJ administration, the appearance and the color of the liver of the mouse are obviously improved, pathological examination shows that the liver cell gap of the mouse in the PSPJ administration group is reduced, and the congestion and inflammatory cell infiltration conditions of the high-dose group are obviously improved and are close to normal. Indicating that PSPJ dose-dependently attenuated LPS/D-GalN-induced changes in liver appearance and microscopic morphology (experimental results are shown in FIG. 4).

3.3.3 Effect of Panax japonicus polysaccharides on ALT, AST and liver index levels in liver-injured mice

The liver index of the mouse indirectly reflects the degree of liver damage, and ALT and AST in the serum of the mouse can directly reflect the liver function of the mouse. After stimulation by LPS/D-GalN, the levels of ALT and AST in the serum of a model group mouse are respectively increased by 4.1 times and 3.1 times, and the liver index of the mouse is also significantly increased (^##P<0.01), indicating that LPS/D-GalN causes liver damage, manifested as edema of liver tissue and abnormal liver function. Compared with the model group, the serum levels of ALT and AST and the liver index of the mice in the PSPJ administration group are reduced to different degrees, wherein all indexes in the high dose group are reduced by more than 40 percent (the experimental results are shown in the table 3.1). The PSPJ can improve the liver function abnormality and the liver edema of the LPS/D-GalN induced liver injury mice dose-dependently.

TABLE 3.1 influence of Panax japonicus polysaccharides on liver function index and liver index in liver-injured mice (Mean + -SE, n ═ 12)

Comparison with Normal group^#P<0.05，^##P<0.01; comparison with model group^*P<0.05，^**P<0.01, the same as below.

3.3.4 Effect of Panax japonicus polysaccharides on liver injury mouse MDA, SOD and GSH levels

The antioxidant factors SOD, GSH and the oxidative stress marker MDA reflect the oxidative stress state of the organism. When LPS/D-GalN induces liver damage of mice, SOD and GSH activity in liver tissues are reduced, and MDA content is increased. In the model group, the levels of GSH and SOD in liver tissue were reduced by 3.2 times and 1.0 times respectively compared with the normal group, while the MDA level was increased by 0.97 times, and there was a significant difference (A)^##P<0.01), indicating that LPS/D-GalN induces mouse liver damage to be related to oxidative stress. The liver tissues of the mice in the PSPJ-administered group showed increased levels of GSH and SOD and decreased levels of MDA, compared to the model group, with significant changes in GSH and MDA in the high dose group (. about.p)<0.01) (results of the experiment are shown in table 3.2). The PSPJ can improve the oxidative stress state of LPS/D-GalN induced liver injury of mice.

TABLE 3.2 influence of Panax japonicus polysaccharides on MDA, SOD and GSH levels in liver-injured mice (Mean + -SE, n ═ 12)

Comparison with Normal group^#P<0.05，^##P<0.01; comparison with model group^*P<0.05，^**P<0.01, the same as below.

3.3.5 Effect of Panax japonicus polysaccharides on liver tissue inflammation mediators TNF-alpha, IL-1 beta and IL-6 of liver-injured mice

The degree of acute liver injury of the mouse has obvious positive correlation with the concentration levels of inflammatory mediators TNF-alpha, IL-1 beta and IL-6 in vivo. Compared with the normal group, the liver tissue concentrations of TNF-alpha, IL-1 beta and IL-6 in the model group mice are all obviously increased (^##P<0.01), the difference between the front and the back is about 10 times. Shows that the experiment mice induce serious liver injury after being stimulated by LPS/D-GalN, and promote a large amount of inflammatory factorsReleasing and also indicating that the molding of the experiment is successful. Compared with the model group, the concentrations of TNF-alpha, IL-1 beta and IL-6 in the damaged liver tissue are respectively reduced by 0.44 times, 0.62 times and 0.31 times after the PSPJ treatment, and the dose-effect relationship is better (the result is shown in a table 3.3). The PSPJ can prevent the release of inflammatory factors of mouse liver tissues and inhibit inflammatory response of the mouse liver tissues so as to achieve the effects of preventing and protecting the liver damage of mice induced by LPS/D-GalN.

TABLE 3.3 Effect of Panax japonicus polysaccharides on TNF-. alpha.IL-1. beta.and IL-6 in liver tissues of liver-injured mice (Mean. + -. SE, n. 12)

Comparison with Normal group^#P<0.05，^##P<0.01; comparison with model group^*P<0.05，^**P<0.01, the same as below.

3.4 discussion

The experimental result shows that the PSPJ can prolong the survival rate and survival time of experimental mice and improve the appearance and tissue morphology of livers; compared with the model group, after PSPJ administration, the serum AST, ALT and liver index of the mice are all significantly reduced (^**P<0.01), the levels of SOD and GSH in liver homogenate are increased, and the contents of MDA, TNF-alpha, IL-1 beta and IL-6 are all significantly reduced (^**P<0.01)。

Intraperitoneal injection of LPS and D-GalN is a convenient method for constructing a mouse acute liver injury model. LPS is a major component of endotoxin secreted by gram-negative bacteria, and it stimulates immune cells including macrophages to release inflammatory factors, thereby allowing hepatocytes to undergo apoptosis and necrosis. D-GalN can inhibit the synthesis of biomacromolecules represented by RNA and proteins by consuming uridine triphosphate in the liver, causing an inflammatory response in the liver and diffuse necrosis of liver cells. Under the synergistic action of LPS and D-GalN, the liver cells of experimental animals die in a large amount in a short time, and the physiological functions of the liver are seriously damaged. In the experiment, the survival rate of the model group mice in 24h is only 16.7%, the survival rate of the PSPJ group mice in 24h is obviously improved, the damage degree of liver cells is obviously reduced, and the contents of ALT and AST in serum are obviously lower than those of the model group. The experimental facts show that the PSPJ has the effects of protecting liver cells and resisting liver injury.

Liver damage caused by chemical drugs is generally closely related to changes in the oxidative stress status of the body. This will further cause a decrease in the levels of SOD and GSH in the liver and an increase in the level of MDA. Similarly, we found that in liver tissue of model mice, levels of SOD and GSH were decreased and MDA content was increased. The results of these experiments suggest that hepatic oxidative stress plays an important role in the LPS/D-GalN combination induced liver injury. Compared with the model group, the PSPJ high-dose group has the advantages that the activities of SOD and GSH are respectively improved by 4.02 times and 1.58 times, MDA is reduced by 1.65 times, and the difference has statistical significance. The PSPJ is shown to improve the oxidative stress condition of the damaged liver of the mouse.

TNF-alpha, IL-1 beta and IL-6 are inflammatory factors secreted by liver macrophages. Inflammatory factors such as TNF-alpha, IL-1 beta, IL-6 and the like act on a hepatocyte surface receptor to cause hepatocyte necrosis, and activate an intracellular NF-kB signal channel to further increase the release amount thereof in a positive feedback manner, so that hepatocyte damage is aggravated. We found that under the combined action of LPS/D-GalN, the contents of TNF-alpha, IL-1 beta and IL-6 in liver tissues are obviously increased, and the phenomenon can be effectively inhibited by PSPJ, and the inhibition of liver inflammatory reaction plays an important role in the hepatocyte protection activity of PSPJ.

In conclusion, the research results of this example preliminarily demonstrate that PSPJ has clear prevention and protection effects on acute liver injury induced by LPS/D-GalN in mice, and the action mechanism may be inhibition of oxidative stress and inflammatory reaction, so as to prevent and prevent liver injury, improve the function of damaged liver, and finally prevent the conversion of liver inflammation to liver cancer.

Claims (1)

1. Application of rhizoma Panacis Japonici polysaccharide in preparing medicine for treating H22 hepatocarcinoma;

the preparation method of the panax japonicus polysaccharide comprises the following steps:

taking dry rhizoma Panacis Japonici powder, adding 95V/V% ethanol, refluxing at 60 deg.C for 3 hr, and defatting;

adding 80V/V% ethanol, refluxing at 60 deg.C for 2 hr, and removing monosaccharide and oligosaccharide;

drying the residue in an oven, extracting under reflux with purified water as solvent for 3 times (2 hr each time), mixing filtrates, concentrating in a rotary evaporator to obtain fluid extract, adding ethanol, adjusting ethanol concentration to 80V/V%, standing overnight at 4 deg.C, filtering the next day, precipitating, and oven drying to obtain crude rhizoma Panacis Japonici product;

dissolving the obtained crude product of panax japonicus with 60 deg.C warm water, removing protein, dialyzing in running water for 48 hr, and freeze-vacuum drying the dialyzate to obtain crude polysaccharide of panax japonicus;

purifying the crude rhizoma Panacis Japonici polysaccharide with DEAE-Sepharose FF column for 3 times; and separating and purifying by a Sephadex G-75 column to obtain a main peak, dialyzing and desalting the peak eluent, and drying to obtain the panax japonicus polysaccharide.

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