阅读说明：本技术 肉苁蓉总苷及松果菊苷在制备用于预防和治疗酒精性肝损伤伴肠道损伤的药物中的应用 (Application of herba cistanches Deserticolae total glycosides and echinacoside in preparing medicine for preventing and treating alcoholic liver injury and intestinal injury ) 是由 姜勇 屠鹏飞 孙嘉旭 王富江 逯颖媛 曾克武 于 2021-07-16 设计创作，主要内容包括：本发明提供了肉苁蓉总苷及松果菊苷在制备用于预防和治疗酒精性肝损伤伴肠道损伤和调节肠道菌群的药物中的应用。药理实验结果表明,在酒精性肝损伤小鼠模型中,肉苁蓉总苷及松果菊苷能够减轻小鼠肝脏组织形态损伤,降低小鼠肝脏的脂质沉积,降低小鼠肝脏谷丙转氨酶(ALT)和谷草转氨酶(AST)含量,减轻小鼠肝脏的氧化损伤,减少内毒素等有害物质对肝脏的损害,同时还能够减轻小鼠小肠组织形态损伤,改善肠壁绒毛完整性,以及调节肠道菌群紊乱。由此可见,本发明具有开发成一种能够同时保护酒精性肝损伤和酒精性肠道损伤的药物的前景,有重要的临床意义和商业价值。(The invention provides application of total cistanchis glycosides and echinacoside in preparing a medicament for preventing and treating alcoholic liver injury with intestinal tract injury and adjusting intestinal flora. Pharmacological experiment results show that in an alcoholic liver injury mouse model, total cistanchis glycosides and echinacoside can relieve morphological injury of mouse liver tissues, reduce lipid deposition of mouse liver, reduce contents of alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) in mouse liver, relieve oxidative injury of mouse liver, reduce damage of harmful substances such as endotoxin to liver, relieve morphological injury of mouse small intestine tissues, improve intestinal villus integrity and regulate intestinal flora disorder. Therefore, the invention has the prospect of developing a medicament capable of simultaneously protecting alcoholic liver injury and alcoholic intestinal injury, and has important clinical significance and commercial value.)

1. Application of herba cistanches Deserticolae total glycosides in preparing medicine for preventing and treating alcoholic liver injury with intestinal canal injury and regulating intestinal canal flora is provided.

2. Use according to claim 1, characterized in that: the total cistanchis glycosides are extracted from cistanche deserticola (Cistancheseriola) or Cistabutea tubulosa (Cistanchutlla).

3. Use according to claim 1 or claim 2, characterized in that: the extraction method of the total cistanchis glycosides comprises the following steps:

extracting herba cistanches 5kg with 8 times of water under reflux for 2 hr for three times, and mixing filtrates; concentrating the filtrate under reduced pressure to a certain volume, adding 95% ethanol until the alcohol content reaches 60%, and standing overnight; filtering, concentrating the filtrate under reduced pressure, purifying with macroporous resin, sequentially eluting with water and ethanol of different concentrations, collecting 40% ethanol eluate, concentrating under reduced pressure, and drying to obtain herba cistanches Deserticolae total glycosides.

4. Use according to any one of claims 1 to 3, characterized in that: the total cistanches glycoside comprises one or more of echinacoside, tubular flower glycoside A, verbascoside, isoverbascoside, 2' -acetyl verbascoside or 8-epibrucic acid, and the content of each component is 8-20%, 0.2-1.0%, 2-10%, 1-10% and 1-10% by weight percentage respectively.

5. Application of echinacoside in preparing medicine for preventing and treating alcoholic liver injury with intestinal canal injury and regulating intestinal canal flora is provided.

6. Use according to any one of claims 1 to 5, characterized in that: in an alcoholic liver injury mouse model, the total cistanchis glycosides and the echinacoside can relieve the morphological injury of mouse liver tissues, reduce the lipid deposition of the mouse liver, reduce the contents of glutamic-pyruvic transaminase (ALT) and glutamic-oxaloacetic transaminase (AST) in the mouse liver, relieve the oxidative injury of the mouse liver, reduce the damage of harmful substances such as endotoxin and the like to the liver, and simultaneously can relieve the morphological injury of the mouse small intestine tissues, improve the intestinal villus integrity and regulate the intestinal flora disorder.

7. A pharmaceutical preparation for preventing and treating alcoholic liver injury with intestinal injury and regulating intestinal flora is characterized in that: the pharmaceutical formulation comprising the medicament of any one of claims 1-6, and a pharmaceutically acceptable carrier.

8. The pharmaceutical formulation of claim 7, wherein: the medicinal preparation also comprises other medicines for treating alcoholic liver injury, wherein the other medicines for treating alcoholic liver injury are one or more of schisandra chinensis, puerarin, silymarin, reductive glutathione, glutamine or vitamin D.

9. The pharmaceutical formulation of claim 8, wherein: the other drug for treating alcoholic liver injury is silymarin, and the weight ratio of the drug in any one of claims 1-6 to the silymarin is 5: 1-1: 5.

10. the pharmaceutical formulation according to any one of claims 7-9, characterized in that: the pharmaceutical preparation is an oral dosage form, and preferably, the oral dosage form is a capsule, a tablet, a granule or an oral liquid.

Technical Field

The invention belongs to the field of medicines, relates to a new application of cistanche deserticola extract, and particularly relates to an application of total cistanche deserticola glycosides and echinacoside in preparing medicines for preventing and treating alcoholic liver injury with intestinal tract injury and regulating intestinal flora.

Background

Alcohol consumption is a factor in a variety of diseases and conditions, and most alcoholics experience tissue damage or organ dysfunction. 20% -30% of heavy alcoholics suffer from alcoholic fatty hepatitis and alcoholic liver diseases such as liver cirrhosis. Chronic drinking can have a devastating effect on growth factors, cytokines and immune function, damaging body organs through inflammatory reactions. It has been reported that the gastrointestinal tract may be an important source of inflammation for alcohol-mediated organ damage.

The alcoholic liver disease is liver injury caused by long-term large-scale drinking, and the long-term large-scale drinking is a single pathogenic factor of the disease, but the pathogenesis of the alcoholic liver disease is quite complex, and the pathogenic factor comprises the metabolic toxicity effect of alcohol, oxidative stress, endotoxemia, cell factor, immunoregulation and the like. The accumulation of alcohol and metabolites releases a large amount of inflammatory mediators, which can destroy the tight connection of intestinal mucosa epithelial cells, increase the permeability of intestinal mucosa epithelial cells to endotoxin, and damage the liver by excessive endotoxin through blood circulation. In addition, the liver and the intestinal tract are physiologically connected via the hepatic portal vein, and chronic alcohol intake not only causes the permeability of intestinal mucosa to be increased, but also causes the disorder of intestinal flora (see Nie jiao, etc., "the effects of ECM, MMP-1 and TIMP in intestinal mucosa injury accompanied by alcoholic liver disease", Shandong medicine, Vol. 52, No. 48, 2012, pages 1-3). It can be seen that alcoholic liver injury and alcoholic intestinal injury are closely related, but currently, studies on alcoholic injury are mainly focused on alcoholic liver disease, and studies on other aspects such as alcoholic intestinal injury are often neglected.

Cistanche deserticola is a perennial medicinal plant of cistanche genus of Orobanchaceae family, has extremely high medicinal value and is good reputation of desert ginseng. In China, cistanche deserticola is mainly distributed in areas of Sinkiang and inner Mongolia, and Gansu and Ningxia are also distributed. There are 4 varieties of 1 cistanche plants in China, namely cistanche deserticola C.deserves Y.C.Ma, cistanche salsa C.salsa (C.A.Mey.) G.Beck, cistanche baillosa C.salsa var.biflora P.F.Tu et Z.C.Lou, cistanche tubulosa C.tubulosa (Schenk) R.Wight and cistanche salsa C.sinensis G.Beck (Tupengfeng et al, modern Chinese medicine 2015,17(4):297 + 301). Wherein, desert cistanche and cistanche tubulosa are officially approved as the basic plants of the traditional Chinese medicine cistanche and are recorded in the Chinese pharmacopoeia. Research shows that the polysaccharide extract and the phenylethanoid glycoside of cistanche deserticola can improve the recovery of serum and liver indexes of an alcohol-induced liver injury model mouse, improve the survival of HepG2 cells, and relieve fatty microvesicles and necrotic cells in liver tissues of a model animal, so that the polysaccharide extract and the phenylethanoid glycoside of cistanche deserticola have obvious protective effect on alcohol-induced chronic liver injury. Further research has shown that the polysaccharide-rich cistanche deserticola extract can reduce inflammatory mucosal hyperplasia and helicobacter pylori infection in the intestines of mice by activating immune system, and has the effect of preventing colorectal cancer and intestinal inflammation (see, margue et al, "cistanche deserticola research progress and industrialization status", Shandong agricultural science, vol. 52, 12 th, 2020, p. 133-140).

Echinacoside (ECH) is a natural compound extracted from cistanche deserticola and is white crystalline powder. Echinacoside has many biological effects, such as neuroprotective effect, liver protecting effect, anti-tumor, anti-apoptosis, anti-aging, immunoregulation and reproduction promoting effect, and also has effects of lowering blood sugar, reducing blood lipid, promoting bone formation, resisting pulmonary hypertension and preventing atherosclerosis. Previously, studies have shown that Echinacoside can relieve alcohol-induced oxidative stress and liver cirrhosis (see, ZhiTao, et al, "ecological improvements of alcohol-induced oxidative stress and hepatic by infection SREBP1c/FASN pathway via PPAR α," Food and Chemical society, Vol 148,2021,111956). In addition, studies have reported that the extract of cistanche tubulosa rich in Echinacoside can reduce Dextran Sodium sulfate Induced Colitis in Mice (see YaminJia, et al, "examination of electrolyte Sulphate Sodium-Induced Colitis in Mice by Echinacoside-Enriched extract of Cistauthitus tubulosa", PHYTOTHERAPY RESEARCH, Vol 28,2014, 110-) 119).

From the above review of the prior art, the research on the treatment of alcoholic liver diseases by using total cistanchis glycosides and echinacoside is only on the liver of a single target organ, but does not relate to the research on other organ injuries related to alcoholic liver injury (particularly, alcoholic intestinal injury closely related to the alcoholic liver injury). In addition, the research on the treatment of intestinal diseases by total cistanchis glycosides and echinacoside mainly focuses on immune-related inflammation models, and the pathophysiological mechanism of enteritis simulated by the models is completely different from that of alcoholic intestinal injury. Therefore, at present, the research on the prevention and treatment of alcoholic liver injury with intestinal injury by using total cistanchis glycosides and echinacoside at the same time is not available, and the invention just fills the technical gap. The research result of the invention has the prospect of developing a medicament capable of simultaneously protecting alcoholic liver injury and alcoholic intestinal injury and adjusting intestinal flora, and has important clinical significance and commercial value.

Disclosure of Invention

The invention aims to solve the defects of the prior art, realize the development and utilization of natural products by adopting a modern pharmaceutical research method, and provide the application of total cistanchis glycosides and echinacoside in preparing medicines for preventing and treating alcoholic liver injury with intestinal injury and regulating intestinal flora by combining a large amount of pharmacodynamics experiment screening.

In order to achieve the purpose of the invention, the invention adopts the following technical scheme:

in a first aspect, the invention provides application of total cistanchis glycosides in preparing a medicament for preventing and treating alcoholic liver injury with intestinal tract injury and regulating intestinal flora.

Alternatively, in the above use, the total cistanchis glycosides are extracted from cistanche deserticola (Cistanchesericola) or cistanche tubulosa (Cistanchebusosa).

Alternatively, in the above use, the extraction method of total cistanchis glycosides comprises the following steps:

extracting herba cistanches 5kg with 8 times of water under reflux for 2 hr for three times, and mixing filtrates. Concentrating the filtrate under reduced pressure to a certain volume, adding 95% ethanol until the alcohol content reaches 60%, and standing overnight; filtering, concentrating the filtrate under reduced pressure, purifying with macroporous resin, sequentially eluting with water and ethanol of different concentrations, collecting 40% ethanol eluate, concentrating under reduced pressure, and drying to obtain herba cistanches Deserticolae total glycosides.

Optionally, in the above application, the total cistanches glycosides comprise one or more of echinacoside, tubular flower glycoside A, verbascoside, isoverbascoside, 2' -acetyl verbascoside or 8-epinux vomica acid, and the content of each component is 8-20%, 0.2-1.0%, 2-10%, 1-10% and 1-10% by weight respectively.

In a second aspect, the invention provides the use of echinacoside in the manufacture of a medicament for the prevention and treatment of alcoholic liver injury with intestinal injury and for the modulation of intestinal flora.

Alternatively, in the above uses, in an alcoholic liver injury mouse model, the total cistanchis glycosides and echinacoside can reduce morphological injury of mouse liver tissue, reduce lipid deposition of mouse liver, reduce contents of glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST) in mouse liver, reduce oxidative injury of mouse liver, reduce damage of harmful substances such as endotoxin to liver, and simultaneously can reduce morphological injury of mouse small intestine tissue, improve intestinal villus integrity and regulate intestinal flora disorder.

In a third aspect, the invention provides a pharmaceutical formulation for the prevention and treatment of alcoholic liver injury with intestinal injury and for modulating the intestinal flora, the pharmaceutical formulation comprising a medicament as described in the first and second aspects above, and a pharmaceutically acceptable carrier.

Optionally, in the above pharmaceutical preparation, the pharmaceutical preparation further comprises other clinically commonly used drugs for treating alcoholic liver injury. Such as, but not limited to, schisandra, puerarin, silymarin, reduced glutathione, glutamine, vitamin D, or the like.

Optionally, in the above pharmaceutical preparation, the other drug for treating alcoholic liver injury is silymarin, and the weight ratio of the drug of the first aspect or the second aspect to silymarin is 5: 1-1: 5.

preferably, the drug of the first or second aspect is echinacoside, the other drug for treating alcoholic liver injury is silymarin, and the weight ratio of echinacoside to silymarin is 4: 1.

alternatively, in the above pharmaceutical formulation, the pharmaceutical formulation is an oral dosage form.

Alternatively, in the above pharmaceutical preparation, the oral dosage form is a capsule, a tablet, a granule or an oral liquid. Preferably tablets or capsules.

The pharmaceutically acceptable carrier refers to conventional pharmaceutical carriers in the field of pharmaceutical preparations, and is selected from one or more of fillers, binders, disintegrants, lubricants, suspending agents, wetting agents, pigments, flavoring agents, solvents and surfactants.

Fillers of the present invention include, but are not limited to, starch, microcrystalline cellulose, sucrose, dextrin, lactose, powdered sugar, glucose, and the like; such lubricants include, but are not limited to, magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, poloxamers, and the like; such binders include, but are not limited to, water, ethanol, starch slurry, syrup, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, sodium alginate, polyvinylpyrrolidone, and the like; such disintegrants include, but are not limited to, starch effervescent mixtures, i.e., sodium bicarbonate and citric acid, tartaric acid, low substituted hydroxypropylcellulose, and the like; suspending agents include, but are not limited to, polysaccharides such as acacia gum, agar, alginic acid, cellulose ethers, carboxymethyl chitin ester, and the like; including but not limited to water, balanced salt solutions, and the like.

The pharmaceutical composition can be prepared into various solid oral preparations, liquid oral preparations and the like. The pharmaceutically acceptable oral solid preparation comprises the following components: common tablet, dispersible tablet, enteric coated tablet, granule, capsule, dripping pill, powder, etc., and oral liquid preparation includes oral liquid, emulsion, etc. The various dosage forms can be prepared according to the conventional process in the field of pharmaceutical preparation.

The total cistanchis glycosides and echinacoside used in the invention can be extracted and separated from the cistanches by the extraction and separation method of the invention or other biological extraction methods reported in the literature, and can also be purchased from commercial products.

In the above-mentioned medical applications, the administration time, administration frequency and administration frequency of total cistanchis glycosides and echinacoside, etc. are determined according to the specific diagnosis result of the disease state, which is within the skill of those skilled in the art.

In order to better understand the essence of the present invention, the following detailed description section uses pharmacodynamic experiments and results to further illustrate the new use of total cistanchis glycosides and echinacoside in the pharmaceutical field.

Compared with the prior art, the invention has the following beneficial effects:

the invention combines the advantages of our country in the aspect of natural product research, screens out the new application of the total cistanchis glycosides and the echinacoside in the aspects of preventing and treating alcoholic liver injury with intestinal tract injury and regulating intestinal flora. In an alcoholic liver injury mouse model and an alcoholic liver injury mouse model, the total cistanchis glycosides and the echinacoside can relieve the morphological injury of mouse liver tissues, reduce the lipid deposition of the mouse liver, reduce the contents of glutamic-pyruvic transaminase (ALT) and glutamic-oxaloacetic transaminase (AST) in the mouse liver, relieve the oxidative injury of the mouse liver, reduce the damage of harmful substances such as endotoxin and the like to the liver, and simultaneously can also relieve the morphological injury of the mouse small intestine tissues, improve the intestinal wall villus integrity and regulate the intestinal flora disorder.

It is worth mentioning that after the research on the effects of the echinacoside and other clinically used drugs for treating alcoholic liver injury in combination for preventing and treating alcoholic liver injury with intestinal injury and regulating intestinal flora, the inventor finds that the echinacoside and silymarin have obvious synergistic effect in preventing and treating alcoholic liver injury with intestinal injury, and particularly the synergistic effect of the echinacoside and the silymarin is optimal under the condition of specific weight ratio, so that the administration dosage of the echinacoside and the silymarin can be remarkably reduced in application, and the related toxic and side effects are reduced.

Therefore, the invention has the prospect of developing a medicament capable of simultaneously protecting alcoholic liver injury and alcoholic intestinal injury, and has important clinical significance and commercial value.

Drawings

FIG. 1: TGs (400mg/kg) can improve the morphological damage of liver tissue of mice with alcoholic liver injury. n is 5 and the scale bar is 100 μm.

FIG. 2: TGs inhibit lipid deposition in the liver of alcoholic liver injury mice. A: oil red O staining, B: and (5) carrying out statistics on lipid deposition data. In comparison with the NOR,^*P<0.05, comparing with MOD,^#P<0.05, n-5, scale bar 100 μm.

FIG. 3: TGs can reduce ALT and AST content in liver of mice with alcoholic liver injury. In comparison with the NOR,^*P<0.05, comparing with MOD,^#P<0.05，n＝5。

FIG. 4: TGs can improve the tissue morphology of small intestine of mice with alcoholic liver injury. n is 5 and the scale bar is 100 μm.

FIG. 5: TGs can reduce the expression of PV1 protein in the intestinal wall of mice with alcoholic liver injury. A: immunofluorescence staining of PV1 protein, B: quantitative statistics of PV1 protein expression, compared to NOR,^*P<0.01, compared to MOD,^#P<0.05, n is 3, and the scale bar is 100 μm.

FIG. 6: TGs can remarkably improve the content of ET, DAO, D-LA and LPS in the serum of mice with alcoholic liver injury. In comparison with the NOR,^*P<0.05; in comparison with the MOD, the results of,^#P<0.05，n＝5。

FIG. 7: TGs can enhance CAT, GSH-Px and SOD activity of liver tissue of mice with alcoholic liver injury and reduce MDA content. In comparison with the NOR,^*P<0.05; in comparison with the MOD, the results of,^#P<0.05，n＝5。

FIG. 8: TGs activate the Nrf-2/Keap-1 signal pathway of liver tissue of mice with alcoholic liver injury. A: immunofluorescence staining and nuclear entry rate statistics of Nrf-2 protein, wherein the scale bar is 50 μm, B: and (3) carrying out immunofluorescence staining and expression quantitative statistics on the Keap-1 protein, wherein the scale bar is 100 mu m. In comparison with the NOR,^*P<0.01, compared to MOD,^#P<0.05，n＝5。

FIG. 9: the ECH can improve the morphological damage of liver tissues of mice with alcoholic liver injury. n is 5 and the scale bar is 100 μm.

FIG. 10: the ECH can reduce the liver lipid deposition of the mice with alcoholic liver injury. n is 5 and the scale bar is 200 μm.

FIG. 11: the ECH can reduce the ALT and AST content of the liver of the mouse with alcoholic liver injury. In comparison with the NOR,^*P<0.05, comparing with MOD,^#P<0.05，n＝5。

FIG. 12: ECH can enhance the activities of CAT, GSH-Px and SOD in liver tissues of mice with alcoholic liver injury and reduce the content of MDA. In comparison with the NOR,^*P<0.05; in comparison with the MOD, the results of,^#P<0.05，n＝5。

FIG. 13: the ECH can improve the tissue morphology of the small intestine of the mice with alcoholic liver injury. n is 5 and the scale bar is 100 μm.

FIG. 14: ECH improves intestinal wall villus integrity.

FIG. 15: ECH can reduce the expression of PV1 protein on the intestinal wall of mice with alcoholic liver injury. A to F are respectively: NOR, MOD, ECH50, ECH100, ECH200 group, compared to NOR,^*P<0.01, compared to MOD,^#P<0.05, n is 3, and the scale bar is 100 μm.

Detailed Description

The invention is further illustrated with reference to specific examples. It should be understood that the specific embodiments described herein are illustrative only and are not limiting upon the scope of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products which are not known to manufacturers and are available from normal sources.

The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.

Unless otherwise indicated, all percentages and parts referred to in the present invention are percentages and parts by weight.

Example 1: method for extracting total glucosides of cistanche deserticola, total polysaccharides of cistanche deserticola and total oligosaccharides of cistanche deserticola

Extracting herba cistanches 5kg with 8 times of water under reflux for 2 hr for three times, and mixing filtrates. Concentrating the filtrate under reduced pressure to a certain volume, adding 95% ethanol until the alcohol content reaches 60%, precipitating at low temperature for 12h, filtering, and precipitating to obtain total polysaccharides of herba cistanches Deserticolae; standing overnight; filtering, concentrating the filtrate under reduced pressure, purifying with macroporous resin, and sequentially eluting with water and ethanol of different concentrations. Collecting water eluate, concentrating under reduced pressure to obtain herba cistanches Deserticolae total oligosaccharide; collecting 40% ethanol eluate, concentrating under reduced pressure, and drying to obtain herba cistanches Deserticolae total glycosides.

Total cistanchis glycosides mainly contain phenylethanoid glycosides and iridoid glycosides. Measuring the contents of echinacoside, tubular flower glycoside A, verbascoside, isoverbascoside, 2' -acetyl verbascoside, and 8-epibrucic acid by high performance liquid chromatography, respectively 16.31%, 0.41%, 4.17%, 2.27%, 1.20%, and 2.78%; the content of total phenylethanoid glycosides and total iridoid glycosides are determined by ultraviolet spectrophotometry with echinacoside and 8-epibrucic acid as reference. The results show that the product contains 43.21% of total phenylethanoid glycosides calculated as echinacoside and 32.18% of total iridoid glycosides calculated as 8-epibrucic acid, calculated as dry product.

Example 2: pharmacodynamic study of Total cistanchis glycosides and Echinacoside for protecting alcoholic hepatic injury with intestinal injury 2.1 pharmacodynamic study result of Total cistanchis glycosides for protecting alcoholic hepatic injury with intestinal injury

30 male healthy Kunming mice were randomly divided into six groups, namely a normal group (NOR), a model group (MOD), a cistanche total glycosides group (TGs, 400mg/kg), a cistanche polysaccharides group (PSs, 400mg/kg) and a cistanche oligosaccharides group (OSs, 400mg/kg), and 10 mice were each group. Establishing alcoholic liver injury model, molding for 7 days, respectively administering physiological saline and medicine, and continuously administering for 14 days.

HE staining (fig. 1) showed that the liver tissue cells of NOR group were normal in morphology, well-aligned and intact; MOD group structure disorder, irregular cell arrangement, loose cell gaps and unclear cell boundary; after TGs intervention, the cell morphology change is obviously better than that of MOD group, and the cell number is more than that of MOD group, which shows that the total cistanchis glycosides have liver protection function.

Oil red O staining (fig. 2) showed that the NOR group had a small amount of lipid deposition, while the MOD group had a significant increase in lipid deposition; compared with MOD group, the lipid deposition is obviously reduced after TGs intervention, which shows that total cistanchis glycosides can reduce the lipid deposition in liver of mice with alcoholic liver injury.

Biochemical marker detection (fig. 3) showed a significant increase in the levels of ALT and AST in the MOD group compared to the NOR group; compared with MOD group, the ALT and AST levels of TGs group are obviously reduced; shows that the total cistanchis glycosides inhibit liver injury and have liver protection effect.

The small intestine HE staining result (figure 4) shows that NOR group small intestine histiocyte morphology is normal, villi are arranged regularly, intestinal wall is uniform, cell structure is complete, and cell gap is compact; MOD group has disordered structure, irregularly arranged villi, broken shape and thinned intestinal wall; after TGs intervention, the cell morphology is obviously changed, the intestinal wall is thickened, and the total cistanchis glycosides have a protective effect on the integrity of the intestinal wall and villi.

Plasma membrane associated protein 1(PV1) is an integral membrane protein associated with the endothelial diaphragm and serves as a marker for the breakdown of the intestinal vascular barrier. As seen from the immunofluorescence staining results (fig. 5), the expression level of PV1 protein was significantly increased in the small intestine wall in MOD group compared to NOR group; compared with MOD group, the expression level of PV1 protein in TGs group is reduced obviously, which shows that total cistanchis glycosides can promote the repair of small intestine wall.

Alcohol can destroy the integrity of the intestinal tract, so that bacterial metabolites in the intestinal tract enter the systemic circulation and reach the liver through the portal vein, and further liver injury is aggravated. FIG. 6 shows that total cistanchis glycosides can reduce the content of endotoxin, lipopolysaccharide, diamine oxidase and D-lactic acid (D-LA) in blood by reducing the permeability of alcohol in the intestinal tract, and prevent harmful substances from entering the liver through the portal vein, thereby protecting the liver.

The oxidative stress factor detection result (figure 7) of the sample shows that compared with the NOR group, the MOD group has obviously reduced activities of superoxide dismutase (SOD), Catalase (CAT) and glutathione peroxidase (GSH-Px), and the MDA content is obviously increased; after TGs intervention, the activities of SOD, CAT and GSH-Px are obviously improved, and the content of MDA is obviously reduced.

Under the condition of oxidative stress, the Kel ch-like epichlorohydrin related protein 1/nuclear factor E2 related factor 2(Keap-1/Nrf-2) compound can participate in the transcriptional activation of various antioxidant genes, thereby providing a cytoprotective effect. As seen from the immunofluorescent staining results (fig. 8), Nrf-2 nuclear entry rate in liver of MOD group was significantly decreased compared to NOR group; the Keap-1 protein expression level is obviously increased; compared with the MOD group, the Nrf-2 nuclear entry rate of the TGs group is obviously increased, and the Keap-1 protein expression level is obviously reduced.

The results show that the total cistanchis glycosides can increase the activities of antioxidant enzymes SOD, CAT and GSH-Px by activating a Keap-1/Nrf-2 signal channel to play a role in liver protection.

2.2 pharmacodynamic study result of Echinacoside for protecting alcoholic liver injury with intestinal injury

60 male Kunming mice were randomly divided into a normal group (NOR), a model group (MOD), a bifendate positive group (BIF, 100mg/kg), an echinacoside low dose group (ECH 50mg/kg), an echinacoside medium dose group (ECH 100mg/kg), and an echinacoside high dose group (ECH 200 mg/kg). Molding by adopting a stomach-filling red star Erguotou wine mode, performing stomach filling and administration on the physiological saline and each medicine respectively 7 days after molding, and taking materials after continuous administration for 14 days.

The results of HE staining (FIG. 9) show that the NOR group of liver tissue cells have normal morphology, regular cell arrangement, uniform staining, complete cell structure and compact cell gaps; MOD group structure disorder, irregular cell arrangement, loose cell gaps and unclear cell boundary; after ECH intervention, the change of cell morphology of each group is obviously better than that of the model group, and the number of cells is more than that of the model group, which indicates that echinacoside has liver protection effect.

The results of oil red O staining (fig. 10) showed that the NOR group had a small amount of lipid deposition, while the MOD group had a significant increase in lipid deposition; compared with MOD group, the lipid deposition is obviously reduced after ECH (50mg/kg, 100mg/kg and 200mg/kg) intervention; the echinacoside can reduce the liver lipid deposition of mice with alcoholic liver injury.

The results of biochemical assays showed (FIG. 11) that ALT and AST levels were significantly increased in the MOD group compared to the NOR group; compared with MOD group, ALT and AST levels in ECH group are obviously reduced and are dose-dependent, which shows that echinacoside can inhibit alcoholic liver injury and has liver protection effect.

The detection result of oxidative stress factors (figure 12) shows that compared with the NOR group, the activities of SOD, CAT and GSH-Px in the MOD group are obviously reduced, the MDA content is obviously increased, and compared with the MOD group, the activities of SOD, CAT and GSH-Px are obviously increased after ECH (50 mu mol/L, 100 mu mol/L and 200 mu mol/L) intervenes, and the MDA content is obviously reduced, which indicates that the echinacoside has obvious antioxidation.

The small intestine tissue is shown after HE staining (figure 13), the NOR group small intestine tissue has normal cell shape, uniform villi arrangement, uniform intestinal wall, complete cell structure and compact cell gap; MOD group has disordered structure, irregularly arranged villi, broken shape and thinned intestinal wall; the cell morphology was significantly altered following ECH (50mg/kg, 100mg/kg and 200mg/kg) intervention, thickening the intestinal wall, indicating that echinacoside can improve the integrity of the intestinal wall and villi.

The electron microscope results show (fig. 14), NOR group small intestine villi are arranged neatly, no fracture, and villi gap is dense; the MOD group fluff is irregularly arranged and broken, and fluff gaps are increased; the shapes of villi of various groups are obviously changed after ECH (Ech drying), the villi are arranged regularly, the gaps become small, and the echinacoside is proved to be capable of improving the integrity of intestinal villi.

As seen from the immunofluorescence staining results (fig. 15), the expression level of PV1 protein was significantly increased in the small intestine wall in MOD group compared to NOR group; compared with MOD group, the PV1 protein expression level of each ECH group is obviously reduced, which shows that echinacoside can promote the repair of small intestine wall.

The sequencing result of the intestinal flora shows that the intestinal flora structure in the mice with alcoholic liver injury is obviously changed, so that the abundance of actinomycetes and bacilli such as bacteroidetes, actinomycetes, bacilli, micrococcaceae, rikenella, rossia and the like is reduced, the abundance of firmicutes, ruminomycetes, butyrospirillum and the like is increased, and the administration of echinacoside can reverse the changes, which indicates that the echinacoside can regulate the intestinal flora disorder.

2.3 pharmacodynamic preliminary evaluation result of echinacoside and other clinically common medicines for treating alcoholic liver injury for preventing and treating alcoholic liver injury with intestinal injury

An extension experiment is also carried out on the basis of the experiments of the parts 2.1 and 2.2, and the drug effect of preventing and treating alcoholic liver injury with intestinal injury after the echinacoside is combined with other clinically common drugs for treating the alcoholic liver injury is preliminarily investigated.

120 male Kunming mice were randomly divided into a normal group (NOR), a model group (MOD), an echinacoside low dose group (ECH 50mg/kg), an echinacoside high dose group (ECH 100mg/kg), a silymarin low dose group (SLM 12.5mg/kg), a silymarin medium dose group (SLM 25mg/kg), a silymarin high dose group (SLM 50mg/kg), a combined 1 group (ECH 100mg/kg + SLM 25mg/kg), a combined 2 group (ECH 50mg/kg + 12.5mg/kg), a combined 3 group (ECH 50mg/kg + SLM 50mg/kg), a combined 4 group (ECH 100mg/kg + puerarin 25mg/kg) and a combined 5 group (ECH 100mg/kg + glutathione reducing 25 mg/kg). Molding by adopting a stomach-filling red star Erguotou wine mode, performing stomach filling and administration on the physiological saline and each medicine respectively 7 days after molding, and taking materials after continuous administration for 14 days.

Detecting the apoptosis inhibition condition of the intestinal epithelial cells: and detecting the apoptosis condition of the intestinal epithelial cells of the mice by using a TUNEL apoptosis detection kit. The method comprises the following operation steps: dewaxing, digesting with protease for 20min, cleaning, adding TUNEL reaction mixture, sealing at 37 deg.C for 30min, adding substrate for color development, counterstaining with hematoxylin, dehydrating, and sealing. Each group was randomly selected from 10 sections, each section was selected from 10 representative high power fields (x400), the number of TUNEL-stained positive cells among 500 cells was counted, and the apoptosis inhibition index represented by 1- (apoptotic cell number/total cell number) x 100%.

The synergy index is judged by adopting a gold positive mean q value method, and the q value is obtained by the following formula: q ═ P_A+B/(P_A+P_B-P_A×P_B). In the formula P_A、P_BAnd P_A+BRespectively the treatment rates of the A medicine group, the B medicine group and the combination of the two medicines. q is less than 1, which indicates that the two medicines generate antagonism after being used together; q is more than 1, which indicates that the two drugs produce synergistic effect after being combined together, and q is 1, which indicates that the two drugs produce additive effect after being combined together.

The specific results are as follows:

by one-way anova, # # indicates P <0.01 compared to the blank; and indicate P <0.05 and P <0.01 compared to the model group.

As can be seen from the results in the above table, the combination of echinacoside and silymarin examined in the above experiments all showed significant synergy, and the weight ratio of echinacoside to silymarin was 4: 1 is optimal.

In conclusion, pharmacological experimental results show that, in an alcoholic liver injury mouse model and an alcoholic liver injury mouse model, the total cistanchis glycosides and the echinacoside can reduce the morphological injury of the liver tissue of the mouse, reduce the lipid deposition of the liver of the mouse, reduce the contents of alanine Aminotransferase (ALT) and glutamic-oxaloacetic transaminase (AST) in the liver of the mouse, reduce the oxidative injury of the liver of the mouse, reduce the damage of harmful substances such as endotoxin and the like to the liver, simultaneously can also reduce the morphological injury of the small intestine tissue of the mouse, improve the villus integrity of the intestinal wall and regulate the disorder of the intestinal flora.

It is worth mentioning that after the research on the effect of the echinacoside and other clinically used drugs for treating alcoholic liver injury in the aspects of preventing and treating alcoholic liver injury with intestinal injury, the inventor finds that the echinacoside and silymarin have obvious synergistic effect in the aspects of preventing and treating alcoholic liver injury with intestinal injury, and particularly the synergistic effect of the echinacoside and the silymarin is optimal under the condition of a specific weight ratio, so that the administration dosage of the echinacoside and the silymarin can be obviously reduced in application, and the related toxic and side effects are reduced.

Therefore, the invention has the prospect of developing a medicament capable of simultaneously protecting alcoholic liver injury and alcoholic intestinal injury and regulating intestinal flora, and has important clinical significance and commercial value.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.