Ribes nigrum extract and application thereof in preparation of liver injury medicine

Abstract

The invention belongs to the field of natural medicines and medical health care, and discloses a Ribes margaritae extract for improving liver injury, which is prepared by the following steps: by taking the overground part of Ribes margaritae as a raw material and water or 40-95% ethanol as an extraction solvent, carrying out heating reflux extraction, and centrifuging, concentrating and freeze-drying the extracting solution. Pharmacodynamic tests prove that the Ribes marginatum has a remarkable liver injury protection effect, can reverse serum liver function indexes and liver tissue oxidative stress indexes and improve the pathological state of liver tissues for cholestatic liver injury mice and alcoholic liver injury mice, and is low in effective dose and free of toxic and side effects. The invention also discloses an application of the Ribes margaritae extract in preparation of a medicine for improving liver injury or a health-care product with an auxiliary protection effect on liver injury. The liver injury is cholestatic liver injury and alcoholic liver injury.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, specifically to the field of natural medicine and medical health care, and relates to a Ribes cuneiformis extract and application thereof in preparing a liver injury drug, and specifically to a Ribes cuneiformis extract for improving liver injury and application thereof in preparing a drug for treating cholestatic liver injury and alcoholic liver injury. Background Art

[0002] Cirrhosis is a terminal disease that develops from various liver diseases and is one of the leading causes of death worldwide. Cirrhosis ranks as the 11th leading cause of death and the 15th leading source of disease burden worldwide, accounting for 2.2% of global deaths and 1.5% of disability-adjusted life years in 2016. In 2017, cirrhosis caused 1.32 million deaths, of which about two-thirds were men and one-third were women. Epidemiological surveys show that the absolute number of liver disease cases worldwide (covering all stages of disease severity) is estimated to be 1.5 billion, of which cholestatic liver diseases (such as primary cholangitis and primary sclerosing cholangitis) account for 1% and alcoholic liver disease accounts for 2%, which is an important cause of threat to the lives and health of people around the world.

[0003] Cholestasis is a pathological condition caused by intrahepatic or extrahepatic factors, characterized by obstruction of bile production, secretion and excretion, which results in bile not flowing normally into the duodenum and accumulating in the liver, thus triggering a series of pathological reactions. Extrahepatic cholestasis is usually due to bile duct obstruction, and common causes include bile duct stones, pancreatic cancer, biliary cancer, ampullary cancer and benign bile duct strictures. Intrahepatic cholestasis is caused by hepatocellular dysfunction or lesions or obstructions of capillary bile ducts, small bile ducts and interlobular bile ducts. Hepatobiliary diseases with cholestasis as the main manifestation due to various liver lesions are collectively referred to as cholestatic liver diseases. Clinical symptoms include jaundice, itchy skin, fatigue, steatorrhea, xanthomas and hepatic bone diseases. As the disease progresses, hyperbilirubinemia may occur, and in severe cases, cirrhosis, liver failure and ultimately life-threatening. In addition, because the key components of cholestatic liver disease are extensive oxidative stress and inflammatory responses, this can lead to more serious overall liver damage.

[0004] At present, the main treatments for cholestatic liver disease include drug therapy and liver transplantation. Ursodeoxycholic acid is the first choice drug approved by the FDA, but its efficacy is limited. As an alternative drug, obeticholic acid is used for patients with primary biliary cholangitis who do not respond to ursodeoxycholic acid, but obeticholic acid can cause side effects such as itching and abdominal pain. There is currently no specific treatment, and symptoms such as systemic itching, expensive medical expenses and a long course of the disease have placed a serious physical and mental burden on patients. Liver transplantation is the only effective treatment for end-stage cholestatic liver disease, but it may face problems such as bile duct obstruction, massive bleeding, immune rejection and cancer after surgery. Alcoholic liver disease refers to a liver disease caused by long-term and heavy drinking. In the early stage, it often manifests as alcoholic fatty liver, which then develops into alcoholic hepatitis, liver fibrosis and cirrhosis, and even liver failure. In recent years, with the improvement of people's living standards, the proportion of patients with alcoholic liver disease has continued to rise, seriously endangering people's health. Studies have found that alcoholic liver disease is mainly the result of the interaction of multiple factors such as inflammatory response, oxidative stress and enteric endotoxin induced directly or indirectly by ethanol and its metabolites. In particular, enteric endotoxemia caused by impaired intestinal barrier function and endotoxin-activated Kupffer cells play an important role in the occurrence and development of alcoholic liver disease. Although patients with early alcoholic liver disease can improve or even reverse their condition by quitting drinking, there is still no specific clinical drug for patients in the middle and late stages. For patients with advanced cirrhosis, the only effective treatment is liver transplantation, but the shortage of liver sources and the high cost of liver transplantation seriously restrict the development of surgery. Therefore, it is of great significance to find new clinically effective drugs with few side effects to treat cholestatic liver disease and alcoholic liver disease.

[0005] Ribes diacanthum Pall, whose official scientific name is Ribes linn., belongs to the genus Ribes Linn. of the Saxifragaceae family. The plant is mainly distributed in Inner Mongolia, northern Heilongjiang, eastern Jilin in China, as well as North Korea, northeastern Mongolia and Siberia in Russia. Ribes diacanthum Pall is widely used as a medicinal plant in folk medicine and has been documented to treat urinary system diseases. Summary of the invention

[0006] The purpose of the present invention is to solve the problems of poor therapeutic effect and toxic side effects of existing drugs for treating cholestasis and alcoholic liver disease, and to provide a Ribes cuneiformis extract for improving liver damage and a preparation method thereof.

[0007] In order to achieve the above object, the present invention adopts the following technical scheme:

[0008] The invention discloses a Ribes cuneiformis extract for improving liver damage. The extract is obtained by taking fresh Ribes cuneiformis as raw material, using water or 40% to 95% ethanol as extraction solvent, performing reflux extraction, and subjecting the extract to centrifugation, concentration and freeze drying.

[0009] Preferably, the Ribes cuneiformis extract is prepared by using the aerial part of Ribes cuneiformis as raw material, water as extraction solvent, performing reflux extraction, and centrifuging, concentrating and freeze-drying the extract.

[0010] Another object of the present invention is to provide a method for preparing the Ribes cuneiformis extract for improving liver damage, comprising the following steps:

[0011] Step 1: Selection of raw materials: using the aerial part of Ribes cuneiformis as raw material, grinding and sieving to obtain Ribes cuneiformis medicinal powder;

[0012] Step 2, extraction: using an extraction solvent to reflux extract the Ribes cuneiformis powder to obtain an extract;

[0013] Step 3, concentration: the extract is centrifuged to remove impurities, the supernatant is taken, the supernatant is concentrated under reduced pressure, the concentrate is stored at -20°C overnight, and freeze-dried to obtain the Ribes cuneiformis extract.

[0014] As an improvement, in step one, the above-ground parts of Ribes cuneiformis include fruits, stems and leaves.

[0015] As an improvement, in step one, the aerial parts of Ribes cuneiformis are powdered and passed through a No. 2 sieve.

[0016] As an improvement, in step 2, the extraction solvent is water, 40% ethanol, 70% ethanol or 95% ethanol.

[0017] Specifically, the water can be double distilled water.

[0018] As an improvement, in step 2, the solid-liquid ratio of the extraction solvent to Ribes cuneiformis is 8:1-12:1 g / mL, preferably 10:1 g / mL, where 10:1 g / mL means that 100 g of medicinal material is added with 1000 mL of extraction solvent for reflux extraction.

[0019] As an improvement, in step 2, the number of extractions is 2 to 4 times, preferably 2 times; the time of each extraction is 0.5 to 1.5 hours, preferably 1 hour; after each extraction, the extraction is filtered and the extracts are combined. Specifically, the filtration is performed using gauze.

[0020] As an improvement, in step three, the centrifugal speed is 8000 rpm, and the centrifugal time is 3 to 5 minutes.

[0021] As an improvement, in step three, the supernatant is filtered, and the obtained filtrate is concentrated under reduced pressure until the crude drug concentration of the concentrate (i.e., the amount of medicinal material added (g) / the volume of the medicinal solution (mL)) is 1 g / mL.

[0022] Specifically, the concentrated solution is stored in a -20°C refrigerator overnight.

[0023] As an improvement, in step three, the freeze-drying temperature is -35°C.

[0024] Specifically, the freeze drying is performed using a freeze dryer with a cold trap temperature of -35°C for one week.

[0025] After verification in a mouse model of cholestasis caused by short-term common bile duct ligation, the extract of Ribes cuneiformis has a significant effect on improving early cholestatic liver damage, can effectively improve the serum biochemical indexes and liver tissue pathological state of BDL mice, and increase the activity of superoxide dismutase (SOD) and glutathione (GSH). After verification in a mouse model of alcoholic liver disease caused by four weeks of 50% ethanol gavage, the extract of Ribes cuneiformis has a significant effect on improving alcoholic liver damage, can effectively improve the serum liver function indexes and liver tissue pathological state of mice with alcoholic liver disease, and increase GSH activity, and reduce the levels of MDA and triglycerides (TG). In short, pharmacodynamic experiments have shown that the extract of Ribes cuneiformis has a significant protective effect on liver damage, and can reverse serum liver function indexes, liver tissue oxidative stress indexes, and improve liver tissue pathological state for mice with cholestatic liver damage and mice with alcoholic liver damage, and the effective dose is low and has no toxic side effects. Therefore, the invention may become an alternative drug for the prevention and treatment of liver diseases.

[0026] Another object of the present invention is to provide the use of the Ribes cuneiformis extract in the preparation of a medicine for improving liver damage or a health product having an auxiliary protective effect on liver damage.

[0027] The liver damage is cholestatic liver damage and alcoholic liver damage.

[0028] Another object of the present invention is to provide the use of the Ribes cuneiformis extract in the preparation of a drug for treating cholestatic liver injury and alcoholic liver injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Flow chart for modeling and drug administration of experimental animals.

[0030] Figure 2 The statistical results of serum biochemical indices (ALT, AST, ALP, γ-GT, DBIL, TBA) and liver tissue biochemical indices (GSH, SOD) of mice in the sham operation group, common bile duct ligation model group, ursodeoxycholic acid group and each dose group of Ribes cuneiformis extract.

[0031] Figure 3 Representative results of H&E staining of liver tissues of mice in the sham operation group, common bile duct ligation model group, ursodeoxycholic acid group, and Ribes cuneiformis extract groups at different doses (scale bar 250 μm).

[0032] Figure 4 The liver tissue and pathological scores of mice in the sham operation group, common bile duct ligation model group, ursodeoxycholic acid group and Ribes cuneiformis extract groups at different doses were statistically analyzed.

[0033] Figure 5 The statistical results are of serum biochemical indices (ALT, AST) and liver tissue biochemical indices (GSH, TG, MDA) of mice in the blank control group, alcoholic liver disease model group and different dose groups of Ribes cuneiformis extract.

[0034] Figure 6 Representative results of H&E staining of liver tissues of mice in the blank control group, alcoholic liver disease model group, and different dose groups of Ribes cuneiformis extract (scale bar 250 μm).

[0035] Figure 7 Statistical analysis of liver tissue pathological scores of mice in the blank control group, alcoholic liver disease model group, and different dose groups of Ribes cuneiformis extract. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described in detail below through specific implementation methods, but the implementation methods of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following implementation methods are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; the materials, reagents, etc. used can all be obtained from commercial channels.

[0037] Example 1

[0038] A Ribes cuneiformis extract for improving liver damage, which is obtained by using Ribes cuneiformis as a raw material, extracting with water, and centrifuging, concentrating and freeze-drying the extract, and specifically comprising the following steps:

[0039] Step 1: Powder the above-ground part of Ribes truncatula and pass through a No. 2 sieve;

[0040] Step 2: Take 100 g of medicinal material powder, use double distilled water as the extraction solvent, heat and reflux extract twice, add 1000 mL of double distilled water each time, heat and reflux extract for 1 hour each time, filter with 8 layers of gauze after each heating and reflux extraction to obtain the extract, and combine the two extracts;

[0041] Step 3: Centrifuge the combined extracts (8000 rpm) for 3 minutes, take the supernatant, filter the supernatant under reduced pressure to obtain a filtrate, and concentrate the filtrate to 100 mL at 50°C using a rotary evaporator, i.e., the crude drug concentration is 1 g / mL; the obtained concentrate is stored in a -20°C refrigerator overnight, and then freeze-dried using a freeze dryer with a cold trap temperature of -35°C for one week.

[0042] Example 2

[0043] A Ribes cuneiformis extract for improving liver damage, which is obtained by extracting Ribes cuneiformis with 40% ethanol, centrifuging, concentrating and freeze-drying the extract, and comprising the following steps:

[0044] Step 1: Powder the above-ground part of Ribes truncatula and pass through a No. 2 sieve;

[0045] Step 2: Take 100g of medicinal material powder, add 1000mL of 40% ethanol, extract twice, heat and reflux for 1 hour each time, filter with 8 layers of gauze after each heating and reflux extraction, and combine the two extracts;

[0046] Step 3: Centrifuge the combined extracts (8000 rpm) for 3 minutes, take the supernatant, and filter the supernatant under reduced pressure to obtain a filtrate, which is concentrated to 100 mL at 50°C using a rotary evaporator; the concentrated solution is stored in a -20°C refrigerator overnight, and then freeze-dried using a freeze dryer with a cold trap temperature of -35°C for one week.

[0047] Example 3

[0048] A Ribes cuneiformis extract for improving liver damage is prepared from Ribes cuneiformis as a raw material, extracted with 70% ethanol, and the extract is centrifuged, concentrated, and freeze-dried, and comprises the following steps:

[0049] Step 1: Powder the above-ground part of Ribes truncatula and pass through a No. 2 sieve;

[0050] Step 2: Take 100g of medicinal material powder, add 1000mL of 70% ethanol, extract twice, heat and reflux for 1 hour each time, filter with 8 layers of gauze after each heating and reflux extraction, and combine the two extracts;

[0051] Step 3: Centrifuge the combined extracts (8000 rpm) for 3 minutes, take the supernatant, filter the supernatant under reduced pressure to obtain a filtrate, and concentrate the filtrate to 100 mL at 50°C using a rotary evaporator; store the concentrated solution in a -20°C refrigerator overnight, and then freeze-dry it in a freeze dryer with a cold trap temperature of -35°C for one week.

[0052] Example 4

[0053] A Ribes cuneiformis extract for improving liver damage, which is obtained by extracting Ribes cuneiformis with 95% ethanol, centrifuging, concentrating and freeze-drying the extract, and comprising the following steps:

[0054] Step 1: Powder the above-ground part of Ribes truncatula and pass through a No. 2 sieve;

[0055] Step 2: Take 100g of medicinal material powder, add 1000mL of 95% ethanol, extract twice, heat and reflux for 1 hour each time, filter with 8 layers of gauze after each heating and reflux extraction, and combine the two extracts;

[0056] Step 3: Centrifuge the combined extracts (8000 rpm / min) for 3 minutes, take the supernatant, filter the supernatant under reduced pressure to obtain a filtrate, and concentrate the filtrate to 100 mL at 50°C using a rotary evaporator; store the concentrate in a -20°C refrigerator overnight, and then freeze-dry it in a freeze dryer with a cold trap temperature of -35°C for one week.

[0057] Example 5

[0058] Pharmacodynamic evaluation of Ribes cuneiformis extract (RDP) in preventing and treating cholestasis induced by common bile duct ligation in mice.

[0059] 1. Experimental animal grouping and drug preparation

[0060] Thirty-six SPF male ICR mice (weight 24-26 g) were weighed after adaptive feeding for 1 week and randomly divided into 6 groups, 6 mice in each group:

[0061] (1) Sham operation group (Sham);

[0062] (2) common bile duct ligation model group (BDL);

[0063] (3) ursodeoxycholic acid group (positive drug, UDCA, dosage 15 mg / kg);

[0064] (4) low-dose group of Ribes cuneiformis extract (RDP, administration dose 5 mg / kg);

[0065] (5) medium-dose group of Ribes cuneiformis extract (RDP, administration dose 15 mg / kg);

[0066] (6) High-dose group of Ribes cuneiformis extract (RDP, dosage 45 mg / kg).

[0067] The extract of Ribes cuneiformis (Example 1) is easily soluble in water, and double distilled water is used to prepare the Ribes cuneiformis solutions with concentrations of 0.5 mg / mL, 1.5 mg / mL, and 4.5 mg / mL, respectively, which are used for administration to mice in the low, medium, and high dose groups of the Ribes cuneiformis extract; ursodeoxycholic acid is poorly soluble in water, and double distilled water containing 5% DMSO is used to prepare a 1.5 mg / mL ursodeoxycholic acid solution, which is used for administration to mice in the ursodeoxycholic acid group. The intragastric administration volume of mice in the ursodeoxycholic acid group and each dose group of the Ribes cuneiformis extract is 10 mL / kg; all groups of mice receive an equal volume (0.1 mL / 10 g) of intragastric administration.

[0068] 2. Preparation and administration of cholestatic liver injury model

[0069] Experimental animal modeling and drug administration (Experimetal design, Drug administration) specific operations such as Figure 1 .

[0070] Days 1-3: Every day, the ursodeoxycholic acid group and each dosage group of Ribes cuneiformis extract (Example 1) were given preventive oral administration; the sham operation group and the common bile duct ligation model group were given oral administration of double distilled water.

[0071] Day 4: After fasting for 12 hours, mice were subjected to common bile duct ligation. The specific steps are as follows:

[0072] Mice were anesthetized by intraperitoneal injection of pentobarbital (dose 70 mg / kg) and then laparotomy was performed. A short incision of no more than 2 cm was made below the xiphoid process of the sternum, and the abdomen was opened and the liver hilum was exposed. The common bile duct was separated and ligated at both ends with 2-0 surgical sutures, without removing the middle bile duct. The abdominal cavity was then quickly closed and the wound was sutured with 4-0 surgical sutures.

[0073] The mice in the sham operation group underwent the above steps, except that the common bile duct was separated but not ligated.

[0074] The ligation time of each mouse was accurately recorded so that blood and liver tissue samples could be collected on time 48 hours after the operation.

[0075] After surgery, 10% sodium ampicillin solution was applied to the wound to avoid infection, and the mice were placed under a red warm lamp to maintain body temperature to promote anesthesia and postoperative recovery. On the day of surgery, after the mice woke up, the ursodeoxycholic acid group and Ribes cuneiformis extract groups were given drugs according to the dose, and the sham operation group and the common bile duct ligation model group were given double distilled water by gavage.

[0076] Day 5: Observe the wound healing of mice, and apply 10% sodium ampicillin solution to the wound to avoid infection. Within 24 hours after common bile duct ligation, the ursodeoxycholic acid group and Ribes cuneiformis extract groups continued to be administrated according to the dose, and the sham operation group and common bile duct ligation model group were gavaged with double distilled water.

[0077] Day 6: Observe the wound healing of mice, and apply 10% sodium ampicillin solution to the wound to avoid infection. Within 48 hours after common bile duct ligation, the ursodeoxycholic acid group and Ribes cuneiformis extract groups continued to be administrated according to the dose, and the sham operation group and common bile duct ligation model group were gavaged with double distilled water.

[0078] After the last medication, samples were collected 48 hours after surgery strictly according to the recorded time. No food or water was allowed for 12 hours before sample collection.

[0079] 3. Collection of serum and tissue samples

[0080] The mice were fasted but not watered for 12 hours before sampling. The eyeballs of the mice were removed to collect blood. After standing for 1 hour, the blood was centrifuged at 3000rpm for 15 minutes, and the serum was collected and packaged. The blood was stored in a -80℃ refrigerator away from light until it was tested. The mice were dissected, the liver was removed, the water was absorbed with filter paper, and the liver was weighed and cut into two parts. One part was fixed with 4% paraformaldehyde, and the rest of the samples were stored in a -80℃ refrigerator until it was used.

[0081] 4. Serum biochemical index detection

[0082] Serum biochemical indices, including the enzyme activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (γ-GT), and the contents of direct bilirubin (DBIL) and total bile acid (TBA), were detected by an automatic biochemical analyzer according to the instructions of the kit.

[0083] Compared with the sham operation group, the enzyme activities of ALT, AST, ALP, γ-GT, and the contents of DBIL and TBA in the serum of mice in the common bile duct ligation model group were significantly increased (P<0.01), indicating that 48 hours after the common bile duct ligation, extremely serious cholestatic liver damage had been caused. Compared with the common bile duct ligation model group, the enzyme activities of ALT, AST, ALP, γ-GT, and the contents of DBIL and TBA in the serum of mice in the various dose groups of Ribes cuneiformis extract and ursodeoxycholic acid groups were significantly decreased (P<0.05, P<0.01), indicating that Ribes cuneiformis extract and ursodeoxycholic acid can effectively protect the liver damage of mice with cholestasis, and the effect of Ribes cuneiformis extract in the medium and high dose groups was more obvious.

[0084] Biochemical indices of liver tissue, including superoxide dismutase (SOD) and glutathione (GSH), were detected according to the kit instructions.

[0085] The results are as follows Figure 2 As shown. Compared with the sham operation group, the activities of SOD and GSH in the liver of mice in the common bile duct ligation model group were significantly reduced (P<0.01). Compared with the common bile duct ligation model group, the activities of SOD and GSH in the liver of mice in each dose group of Ribes cuneiformis extract were significantly increased (P<0.05, P<0.01). The results showed that Ribes cuneiformis extract can improve the antioxidant capacity of the liver of mice with cholestatic liver injury and reduce the damage of oxidative stress to the liver.

[0086] 5. Observation of liver tissue pathology

[0087] Liver pathology was scored, with a pathology score of 0-4, and the pathology score was determined (Sharma S, et al. Pediatr Surg Int. 2011; 27(5): 451-461. doi: 10.1007 / s00383-010-2845-5). Figure 3 and Figure 4 As shown. In the blank control group, the hepatocyte morphology was intact, the cell nucleus was clearly visible, and the cell morphology was normal. Compared with the blank control group, the model group showed inflammatory cell infiltration, hepatocyte degeneration, and regional necrosis. Compared with the model group, the Ribes cuneiformis extract group could improve the liver tissue pathological damage caused by common bile duct ligation. In the low-dose group, inflammatory cells still aggregated in the portal area, and the necrotic site was regionally scattered. In the medium and high-dose groups, the liver necrosis area was significantly reduced, and the hepatocytes were neatly arranged with intact morphology. This indicates that Ribes cuneiformis extract and ursodeoxycholic acid can significantly reverse the liver tissue pathological damage in mice with cholestasis, and the effect of Ribes cuneiformis extract in the medium and high dose groups is the most significant.

[0088] Example 6

[0089] Pharmacodynamic evaluation of Ribes cuneiformis extract in preventing and treating alcoholic liver injury

[0090] 1. Experimental animals and groups

[0091] 50 SPF male ICR mice (weighing 24-26g) were weighed and randomly divided into 5 groups after adaptive feeding for 1 week, with 10 mice in each group:

[0092] (1) Blank control group (Control);

[0093] (2) alcoholic liver disease model group (ALD);

[0094] (3) low-dose group of Ribes cuneiformis extract (administered dose, 5 mg / kg);

[0095] (4) medium-dose group of Ribes cuneiformis extract (administered dose, 15 mg / kg);

[0096] (5) High-dose group of Ribes cuneiformis extract (administered dose, 45 mg / kg).

[0097] 2. Preparation and administration of alcoholic liver injury model

[0098] The preparation of the Ribes cuneiformis extract solution (Example 1) used in the low, medium and high dose groups of the Ribes cuneiformis extract is the same as that in Example 5.

[0099] Every morning, the extract of Ribes cuneiformis (Example 1) was administered by gavage according to the set dose, and the blank control group and the alcoholic liver disease model group were given an equal volume of double distilled water; every afternoon, except for the blank control group, which was given an equal volume of double distilled water, the mice in the other groups were gavaged with 50% ethanol (absolute ethanol was diluted with double distilled water to 50% ethanol, and the gavage volume for mice was 12-14 mL / kg) for 4 consecutive weeks. During the modeling period, the weight of the mice was weighed and recorded every week. After the end of the fourth week of administration, the weight of the mice was weighed and recorded; the mice were fasted for 12 hours but not water, and the eyeballs of the mice were removed for blood collection. After standing for 1 hour, they were centrifuged at 3000 rpm for 15 minutes, and the serum was collected, packaged, and placed in a -80°C refrigerator to avoid light for testing. The mice were dissected, the liver was removed, the water was absorbed by filter paper, and it was cut into 2 parts after weighing. One part was fixed with 4% paraformaldehyde, and the remaining samples were stored in a -80°C refrigerator for standby use.

[0100] The results are as follows Figure 5 As shown. Compared with the blank control group, the serum ALT and AST activities of mice in the model group were significantly increased (P<0.01), indicating that long-term drinking can lead to abnormal liver function. Compared with the model group, the serum ALT and AST activities of mice in the high-dose group of Ribes cuneiformis extract were significantly reduced (P<0.05). It shows that Ribes cuneiformis extract may alleviate the damage to liver function caused by long-term drinking. Compared with the blank control group, the GSH activity in the liver of mice in the model group was significantly reduced (P<0.01), and the MDA and TG contents were significantly increased (P<0.01). Compared with the model group, the GSH activity in the liver of mice in the Ribes cuneiformis extract group was significantly increased (P<0.05, P<0.01), and the MDA and TG contents were significantly decreased (P<0.01). It shows that Ribes cuneiformis extract can improve the antioxidant capacity of the liver of mice with alcoholic liver injury, reduce the level of lipid peroxidation, and reduce the damage of oxidative stress to the liver.

[0101] Liver pathology was scored according to Sharma S, et al. Pediatr Surg Int. 2011; 27(5): 451-461. (doi: 10.1007 / s00383-010-2845-5), with a pathology score of 0-4, and the pathology score was determined. Figure 6 and Figure 7As shown. The blank control group showed clear liver lobules, neatly arranged liver cells and complete structure. Compared with the blank control group, the basic structure of liver cells in the model group disappeared, showing diffuse swelling of liver cells, loose and translucent cytoplasm, and the cytoplasm of some liver cells was almost completely transparent and balloon-like, and the liver sinusoids were narrowed and almost disappeared. Compared with the model group, the liver cells of the mice in the low-dose group of Ribes cuneiformis showed slight fatty degeneration, and the liver tissue damage of the mice in the medium and high-dose groups of Ribes cuneiformis was significantly reduced, and the morphological structure was close to that of the normal group. The results showed that the extract of Ribes cuneiformis can effectively protect the liver tissue pathological damage of mice with alcoholic liver disease model, and the effect was more obvious in the medium and high-dose groups.

[0102] The above-mentioned pharmacodynamic tests have proved that Ribes cuneiformis has a significant protective effect against liver damage. It can reverse serum liver function indicators and liver tissue oxidative stress indicators in mice with cholestatic liver damage and alcoholic liver damage, and improve the pathological state of liver tissue. The effective dose is low and there are no toxic side effects. Therefore, Ribes cuneiformis extract may become an alternative drug for the prevention and treatment of liver diseases.

Claims

1. A Ribes cuneiformis extract for improving liver damage, characterized in that: The invention takes the aerial part of Ribes cuneiformis as raw material, uses water or 40% to 95% ethanol as extraction solvent, performs heating reflux extraction, and obtains the extract after centrifugation, concentration and freeze-drying.

2. The Ribes cuneiformis extract according to claim 1, characterized in that: The invention is prepared by taking the aerial part of Ribes cuneiformis as raw material and water as extraction solvent, performing heating reflux extraction, and subjecting the extract to centrifugation, concentration and freeze-drying.

3. A method for preparing the Ribes cuneiformis extract for improving liver damage according to claim 1, characterized in that: The steps include: Step 1: Selection of raw materials: using the aerial part of Ribes cuneiformis as raw material, grinding and sieving to obtain Ribes cuneiformis medicinal powder; Step 2, extraction: using an extraction solvent to reflux extract the Ribes cuneiformis powder to obtain an extract; Step 3, concentration: centrifuge the extract, take the supernatant, concentrate the supernatant under reduced pressure, store the concentrate at -20°C overnight, and freeze-dry to obtain the Ribes cuneiformis extract.

4. The method for preparing the Ribes cuneiformis extract for improving liver damage according to claim 3, characterized in that: In step 2, the extraction solvent is water, 40% ethanol, 70% ethanol or 95% ethanol.

5. The method for preparing the Ribes cuneiformis extract for improving liver damage according to claim 4, characterized in that: In step 2, the ratio of the extraction solvent to the solid liquid of Ribes cuneiformis is 8:1 to 12:1 g / mL.

6. The method for preparing the Ribes cuneiformis extract for improving liver damage according to claim 3 or 4, characterized in that: The solid-liquid ratio of the extraction solvent to Ribes cuneiformis is 10:1 g / mL.

7. The method for preparing the Ribes cuneiformis extract for improving liver damage according to claim 3, characterized in that: In step 2, the extraction is performed 2 to 4 times; the time for each extraction is 0.5 to 1.5 hours; after each extraction, the extraction is filtered and the extracts are combined.

8. The method for preparing the Ribes cuneiformis extract for improving liver damage according to claim 5, characterized in that: In step 3, the centrifugal speed is 8000 rpm, and the centrifugal time is 3 to 5 minutes; The supernatant was filtered, and the obtained filtrate was concentrated under reduced pressure until the crude drug concentration of the concentrate was 1 g / mL.

9. Use of the Ribes cuneiformis extract according to claim 1 or 2 in the preparation of a medicament for improving liver damage or a health product having an auxiliary protective effect on liver damage.

10. Use of the Ribes cuneiformis extract according to claim 1 or 2 in the preparation of a drug for treating cholestatic liver injury and alcoholic liver injury.

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