Application of dalbergia odorifera leaf extract in medicine for improving non-alcoholic steatohepatitis

Abstract

The invention provides an application of a dalbergia odorifera leaf extract in a medicine for improving non-alcoholic steatohepatitis, and relates to the field of biological medicines. Experiments show that the dalbergia odorifera leaf extract can reduce the levels of AST and ALT, and reduce the levels of IL-6, TNF-alpha and IL-1beta; the symptoms of basic structure damage, fatty degeneration, balloon-like degeneration and inflammatory cell infiltration of hepatocytes can be relieved; red lipid droplets in the liver can be reduced, namely the dalbergia odorifera leaf extract can improve the non-alcoholic fatty liver hepatitis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of Dalbergia odorifera T. Chen leaf extract in drugs for improving non-alcoholic steatohepatitis. Background Art

[0002] The occurrence of non-alcoholic steatohepatitis (NASH) will gradually exacerbate liver fibrosis, and even lead to liver cirrhosis, thereby increasing the risks of liver failure and hepatocellular carcinoma.

[0003] NASH is a chronic liver disease associated with liver metabolic disorders and inflammation. The clinical manifestations of this disease are liver inflammation, steatosis (more than 5% of hepatocytes), and hepatocyte damage (ballooning degeneration); the levels of liver function indicators such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) increase; tumor necrosis factor- α (TNF- α ) and inflammatory cytokine indicators such as interleukin 6 (IL-6) increase.

[0004] At present, the main methods for preventing and treating NASH include adjusting the diet structure, controlling body weight, anti-inflammatory lipid regulation, and reducing enzymes to protect the liver, etc. The clinical drug treatment of NASH not only has unsatisfactory treatment effects, but also has serious adverse reactions. Therefore, seeking safer and more effective drugs for improving NASH has been the long-term focus of the world's pharmaceutical research field.

[0005] Dalbergia odorifera T. Chen Dalbergia odorifera is an evergreen semi-deciduous tree of the genus Dalbergia in the legume family and is the source plant of the traditional Chinese medicine Dalbergiae Lignum. Modern research shows that: Dalbergia odorifera T. Chen leaves have pharmacological effects such as antioxidant, cardiovascular protection, anti-inflammatory, anti-allergic, sedative, and antiplatelet aggregation. At present, the pharmaceutical research of Dalbergia odorifera T. Chen mainly focuses on its heartwood, and the comprehensive utilization research of Dalbergia odorifera T. Chen leaves has not attracted enough attention. The present invention provides an effective way for the comprehensive utilization of forestry waste - Dalbergia odorifera T. Chen leaves by determining that the Dalbergia odorifera T. Chen leaf extract has good activity in improving NASH. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide the application of Dalbergia odorifera T. Chen leaf extract in drugs for improving non-alcoholic steatohepatitis.

[0007] To achieve the above purpose, the present invention provides the application of Dalbergia odorifera T. Chen leaf extract in drugs for improving non-alcoholic steatohepatitis.

[0008] Furthermore, the preparation steps of the Dalbergia odorifera T. Chen leaf extract include: Mixing Dalbergia odorifera T. Chen leaves with an extraction solvent to form a mixture; At a preset temperature, the mixture is subjected to extraction treatment and filtration treatment by a preset extraction method to obtain a filtrate; The filtrate is subjected to reduced pressure concentration treatment and drying treatment to obtain an extract of Dalbergia odorifera T. Chen leaves.

[0009] Furthermore, the extraction solvent is water, methanol or ethanol with a concentration of 20% - 90%.

[0010] Furthermore, the preset temperature is 60°C - 100°C, and the preset extraction method is heat reflux extraction, warm maceration extraction or ultrasonic extraction.

[0011] Furthermore, the number of extraction treatments is 1 - 3 times, and the extraction time is 1h - 5h.

[0012] Furthermore, the mixing ratio of the Dalbergia odorifera T. Chen leaves to the extraction solvent is 1:1 - 1:20.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: Experiments show that the extract of Dalbergia odorifera T. Chen leaves can reduce the levels of AST and ALT, and reduce the levels of IL-6, TNF- α and IL-1 β levels; it can alleviate the destruction of the basic structure of hepatocytes, symptoms of fatty degeneration, ballooning degeneration and inflammatory cell infiltration; it can reduce the red lipid droplets in the liver, that is, the extract of Dalbergia odorifera T. Chen leaves can improve NASH. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a test result diagram of liver function index levels in the application of the extract of Dalbergia odorifera T. Chen leaves in the drug for improving non-alcoholic steatohepatitis in the embodiments of the present invention. Among them, A is the detection result diagram of AST content, and B is the detection result diagram of ALT content; Figure 2 It is a test result diagram of inflammatory cytokine index levels in the application of the extract of Dalbergia odorifera T. Chen leaves in the drug for improving non-alcoholic steatohepatitis in the embodiments of the present invention. Among them, A is the detection result diagram of IL-1β content, B is the detection result diagram of IL-6 content, and C is the detection result diagram of TNF-α content; Figure 3 It is a microscopic examination diagram of HE staining in the application of the extract of Dalbergia odorifera T. Chen leaves in the drug for improving non-alcoholic steatohepatitis in the embodiments of the present invention; Figure 4 It is a NASH score result diagram after HE staining in the application of the extract of Dalbergia odorifera T. Chen leaves in the drug for improving non-alcoholic steatohepatitis in the embodiments of the present invention; Figure 5 It is a microscopic examination diagram of oil red O staining in the application of the extract of Dalbergia odorifera T. Chen leaves in the drug for improving non-alcoholic steatohepatitis in the embodiments of the present invention; The following specific embodiments will further illustrate the present invention in conjunction with the above-mentioned drawings. Specific Embodiments

[0015] For the convenience of understanding the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. Several embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the disclosure of the present invention more thorough and comprehensive.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs. The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0017] Please refer to Figure 1 , the embodiment of the present invention provides the application of Dalbergia odorifera T. Chen leaf extract in drugs for improving non-alcoholic fatty liver hepatitis.

[0018] The preparation steps of the Dalbergia odorifera T. Chen leaf extract include: Mix Dalbergia odorifera T. Chen leaves with an extraction solvent to form a mixture; Preferably, the extraction solvent is water, methanol or ethanol with a concentration of 20% - 90%, and the mixing ratio of the Dalbergia odorifera T. Chen leaves to the extraction solvent is 1:1 - 1:20. In this embodiment, the extraction solvent is ethanol, and the mixing ratio of the Dalbergia odorifera T. Chen leaves to the extraction solvent is 1:5.

[0019] At a preset temperature, perform extraction treatment and filtration treatment on the mixture by a preset extraction method to obtain a filtrate; Preferably, the preset temperature is 60°C - 100°C, the preset extraction method is hot reflux extraction, warm soaking extraction or ultrasonic extraction, the number of extraction treatments is 1 - 3 times, and the extraction treatment time is 1h - 5h. In this embodiment, the preset temperature is 100°C, the preset extraction method is hot reflux extraction, the number of extraction treatments is 2 times, and the extraction treatment time is 3h.

[0020] Perform decompression concentration treatment and drying treatment on the filtrate to obtain Dalbergia odorifera T. Chen leaf extract.

[0021] To obtain the pharmacological effects of the Dalbergia odorifera T. Chen leaf extract, a pharmacological experiment is conducted on it: Animal grouping, model establishment and drug administration: After 7 days of adaptive feeding with normal diet, SD rats were randomly divided into a normal group, a model group, a low-dose Dalbergia odorifera T. Chen leaf group (400 mg·kg -1 ), a medium-dose Dalbergia odorifera T. Chen leaf group (800 mg·kg -1 ), and a high-dose Dalbergia odorifera T. Chen leaf group (1600 mg·kg -1 ), with 8 rats in each group.

[0022] The normal group was given basal diet, and the other groups were continuously given a high-sugar and high-fat diet (D12492, ResearchDiets, USA) for 12 weeks. During the experiment, the rats had free access to food and water to establish a NASH rat model.

[0023] After successful modeling, the Dalbergia odorifera T. Chen leaf treatment groups were given the corresponding drugs by gavage every day, and the gavage dose was 10 mL·kg -1 body weight, once a day for 12 consecutive weeks. The normal group and the model group were given the corresponding volume of distilled water.

[0024] Determination of liver function index levels: After 12 consecutive weeks of drug administration, blood was collected from the orbital cavity, centrifuged at 3500 r·min -1 for 10 min, and the serum was taken. The contents of AST and ALT in the rat serum were detected using a kit from Nanjing Jiancheng. Please refer to Figure 1 , after the rats were fed a high-fat diet for 12 weeks, compared with the normal group, the levels of serum AST and ALT in the model group were significantly increased ( P <0.01), indicating that the NASH rat model was successfully established. Compared with the model group, the levels of serum AST and ALT in each drug administration group were significantly decreased ( P <0.05, P <0.01). The results showed that the extract of Dalbergia odorifera T. Chen leaves had good activity in improving NASH.

[0025] Determination of inflammatory cytokine index levels: After 12 consecutive weeks of drug administration, the rat livers were dissected for homogenization, centrifuged at 3000 r·min -1 for 10 min, and the supernatant was taken. The contents of TNF- α , IL-6 and IL- β in the rat liver were detected using an Elisa kit from Nanjing Jiancheng. Please refer to Figure 2 , after the rats were fed a high-fat diet for 12 weeks, compared with the normal group, the levels of IL-1 β , IL-6 and TNF- α in the model group liver were significantly increased ( P <0.01), indicating that the NASH rat model was successfully established. Compared with the model group, the levels of IL-6 and TNF- αThe levels were significantly reduced ( P <0.05, P <0.01); IL-1 in the liver of rats in the high-dose group and the medium-dose group β Levels were significantly reduced ( P <0.05, P <0.01). The results showed that the extract of Dalbergia odorifera leaves had good activity in improving NASH.

[0026] HE staining: The drug was given continuously for 12 weeks, and the rat liver was dissected after blood was collected from the orbit. The blood in the tissue was rinsed with normal saline, and an appropriate amount of liver tissue was fixed in 4% paraformaldehyde solution, dehydrated, embedded in paraffin, and made into paraffin sections (4μm). After staining with hematoxylin-eosin method, the tissue was washed with gradient alcohol, transparentized with xylene, and sealed with neutral gum. The pathological changes of the liver were observed under a microscope, and the NASH activity score was statistically evaluated: hepatocyte fatty degeneration 0-3 points (hepatocyte fatty degeneration less than 5% is 0 points; 5%-33% is 1 point; 34%-66% is 2 points; greater than 66% is 3 points), ballooning degeneration 0-2 points (hepatocyte ballooning degeneration: no is 0 points; rare is 1 point; frequent is 2 points), inflammation 0-3 points (no intralobular inflammation is 0 points when counting necrotic foci under a 20x microscope; less than 2 is 1 point; 2-4 is 2 points; greater than 4 is 3 points), and the total score is 8 points. Please refer to Figure 3 and Figure 4 , the normal rat liver tissue structure is normal, the central veins are evenly distributed, the size of liver cells is relatively consistent, and the cytoplasm is uniform. Compared with the normal group, the basic structure of liver cells in the model group was destroyed, the area of ​​lipid accumulation increased, severe fatty degeneration occurred, the cytoplasm was crowded with vacuoles, inflammatory cell infiltration symptoms, and ballooning were visible. Compared with the model group, each drug-treated group can alleviate the destruction of the basic structure of liver cells, fatty degeneration, ballooning and inflammatory cell infiltration symptoms. NASH score showed that compared with the normal group, the liver NASH score of the model group was significantly increased ( P <0.01), indicating that the NASH rat model was successfully established. Compared with the model group, the liver NASH scores of rats in the high-dose group and the medium-dose group were significantly reduced ( P <0.01). The results confirmed that the extract of Dalbergia odorifera leaves has good biological activity in improving NASH.

[0027] Oil Red O staining: The drug was administered for 12 consecutive weeks, and the rat liver was dissected after blood was collected from the orbit. The rat liver tissue was embedded in OCT (optimal cutting temperature) fixative and stored in a -80°C refrigerator. Subsequently, the tissue was made into frozen sections and fixed with tissue fixative for 15 minutes, then stained with Oil Red O and observed under an optical microscope. Please refer to Figure 5, in the blank group, there were basically no large red lipid droplets in the liver tissue. In the model group, there were a large number of fused red lipid droplets in the liver of rats, and the area and color depth of the red lipid droplets both increased, indicating obvious lipid accumulation in the liver. Compared with the model group, the red lipid droplets in the livers of rats in each drug administration group were significantly reduced, and the area and color depth of the red lipid droplets both decreased. This result further confirmed that the extract of Dalbergia odorifera leaves has good biological activity in improving NASH.

[0028] Based on the above experimental results, the extract of Dalbergia odorifera leaves can reduce the levels of AST and ALT, and reduce the levels of IL-6, TNF- α and IL-1 β ; it can alleviate the damage of the basic structure of hepatocytes, symptoms of fatty degeneration, ballooning degeneration and inflammatory cell infiltration; it can reduce the red lipid droplets in the liver, that is, the extract of Dalbergia odorifera leaves can improve NASH.

[0029] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0030] The above embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but should not be construed as limiting the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims

1. Application of a Dalbergia odorifera leaf extract in a drug for improving non-alcoholic fatty hepatitis.

2. The use according to claim 1, characterized in that: The preparation steps of the Dalbergia odorifera leaf extract include: mixing the Dalbergia odorifera leaves with an extraction solvent to form a mixture; Performing extraction and filtering on the mixture at a preset temperature using a preset extraction method to obtain a filtrate; The filtrate is subjected to reduced pressure concentration and drying to obtain the Dalbergia odorifera leaf extract.

3. The use according to claim 2, characterized in that: The extraction solvent is water, 20% to 90% methanol or ethanol.

4. The use according to claim 2, characterized in that: The preset temperature is 60° C. to 100° C., and the preset extraction method is hot reflux extraction, warm immersion extraction or ultrasonic extraction.

5. The use according to claim 2, characterized in that: The number of extraction treatments is 1 to 3 times, and the time of the extraction treatment is 1 hour to 5 hours.

6. The use according to claim 2, characterized in that: The mixing ratio of the Dalbergia odorifera leaves and the extraction solvent is 1:1-1:20.

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