Application of moringa oleifera leaf ethanol extract in improvement of metabolism-related fatty liver diseases

Abstract

The invention belongs to the field of food nutritional supplements and medicines, and particularly relates to application of a horseradish tree leaf ethanol extract in the aspect of improving metabolism-related fatty liver diseases. The invention aims to provide the moringa oleifera leaf ethanol extract, and the moringa oleifera leaf ethanol extract can effectively reduce triglyceride and free fatty acid levels in the liver of a mouse and reduce lipid accumulation in the liver, so that the moringa oleifera leaf ethanol extract has a remarkable effect in the aspect of treatment of metabolism-related fatty liver diseases. The invention particularly emphasizes the application of the moringa oleifera leaf ethanol extract in the aspects of reducing mouse liver weight, free fatty acid, triglyceride and liver staining. In addition, the composition is a natural product extracted from moringa oleifera leaves with homology of medicine and food, and is safe and reliable to take, simple in preparation method and low in cost.

Description

Technical Field

[0001] The invention relates to an ethanol extract of Moringa oleifera leaves, and belongs to the field of food nutritional supplements and medicines. More specifically, it relates to an application of the ethanol extract of Moringa oleifera leaves in improving metabolic associated fatty liver disease (MASLD). Background Art

[0002] Metabolic-associated fatty liver disease (MASLD) is a metabolic liver disease closely related to obesity. Its pathological characteristics are excessive accumulation of lipids in the liver, which may gradually progress to non-alcoholic steatohepatitis (NASH), liver fibrosis, cirrhosis and even hepatocellular carcinoma. Obesity is not only an important risk factor for MASLD, but also a risk factor for a variety of non-communicable diseases such as type 2 diabetes, cardiovascular disease and certain types of cancer. At present, the drugs and clinical strategies for the treatment of obesity and MASLD mainly include lifestyle intervention and drug therapy, such as dietary management, increased physical activity, and the use of drugs such as ω-3 fatty acids, glucagon (GCG) / glucagon-like peptide-1 (GLP-1) dual receptor agonists.

[0003] In the current medical and research fields, although drug therapy and lifestyle intervention have achieved certain results, these treatments are often accompanied by side effects and dependence. In contrast, edible and medicinal extracts have shown unique advantages in the treatment of obesity and metabolic associated fatty liver disease (MASLD). Flavonoids, saponins, polysaccharides and alkaloid compounds in edible and medicinal substances such as hawthorn, sea buckthorn and ginseng play an important role. They can promote intestinal peristalsis and activate the AMPK signaling pathway, thereby reducing fat intake and accelerating fat metabolism. These substances have the characteristics of both food and medicine, are highly safe and have few side effects. They are suitable for long-term consumption and preventive applications, and provide a better choice for the treatment of related diseases. Moringa leaves ( Moringa oleifera Lam. ) As a traditional medicinal plant, it has many health benefits such as antioxidant, anti-inflammatory and metabolic regulation. Its medicinal and edible properties make it both a cure and a nutritional supplement, which is crucial in the comprehensive management of MASLD. At the same time, Moringa leaves are widely available and low in cost, which enhances its appeal in MASLD treatment options.

[0004] Although the bioactivity of Moringa leaves has been widely studied, its potential in the treatment of MASLD has not been fully explored, especially in the overall bioactivity of ethanol extracts, for which there are currently no relevant patents. Overall, Moringa leaves are ideal for developing new strategies for the treatment of MASLD, which brings new perspectives for the treatment of MASLD and creates conditions for in-depth research on its active ingredients, which is expected to discover more potential natural product ingredients, provide patients with a comprehensive solution, and lay the foundation for single compound research. Summary of the invention

[0005] The purpose of the present invention is to provide an ethanol extract of Moringa oleifera leaves, which can effectively reduce the weight of mice, reduce the levels of triglycerides and free fatty acids in mice, and reduce liver lipid accumulation, thereby showing significant effects in the treatment of metabolic-related fatty liver. The present invention particularly emphasizes the application of the ethanol extract of Moringa oleifera leaves in liver weight, free fatty acids, triglycerides, and liver staining.

[0006] An object of the present invention is to provide a method for preparing the Moringa leaf ethanol extract.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions: S1, drying the Moringa leaf material and crushing it into powder; S2. Cover the powder with 95% ethanol, soak for seven days, select the supernatant for filtration, and repeat three times; S3. Concentrate the filtrate under vacuum at 45°C to obtain an extract.

[0008] Furthermore, an application of an ethanol extract of Moringa leaves is characterized in that the extract is used to treat or prevent metabolic-related fatty liver disease and its related symptoms caused by a high-fat and high-cholesterol diet.

[0009] Furthermore, the pharmaceutical composition of the Moringa leaf ethanol extract is characterized in that the composition contains the Moringa leaf ethanol extract as an active ingredient.

[0010] In addition, the present invention also provides a specific application method of the Moringa leaf ethanol extract, which specifically comprises the following steps: S1. Purchase 40 8-week-old male C57BL / 6J mice weighing 18-20 g at the specific pathogen-free (SPF) level; S2. After one week of feeding at constant temperature and humidity, the rats were divided into five groups (8 in each group) according to their body weight: normal control group, high-fat and high-cholesterol model group, low-dose Moringa leaf ethanol extract intervention group, high-dose Moringa leaf ethanol extract intervention group, and positive drug rosiglitazone intervention group; S3. Except for the normal control group, the other groups were given a high-fat diet and intragastrically administered drugs at 10 a.m. every day for 60 consecutive days. S4, after step S3, the drug was stopped for 24 hours, blood was collected under anesthesia, the mice were killed by cervical dislocation and autopsied, the weight of the liver of the mice was recorded, and the lipid accumulation was observed; S5, weighing a certain weight of the mouse liver tissue collected in step S5, making paraffin sections, staining with hematoxylin-eosin (HE) and oil red O, respectively, and observing the pathological sections; S6. After step S5, serum of mice is collected to measure the content of triglyceride and free fatty acid in the mice.

[0011] Furthermore, in step S2, the extract was administered at a dosage of 200 mg / kg for the low-dose group, 400 mg / kg for the high-dose group, and 10 mg / kg for the positive drug group.

[0012] Furthermore, in step S3, the high-fat and high-cholesterol feed formula contains 60% fat and 1.2% cholesterol.

[0013] The present invention has the following advantages: In the first aspect of the present invention, the ethanol extract of Moringa oleifera leaves can significantly reduce the liver weight of mice and reduce the lipid accumulation in the liver of mice, and the significant difference is very obvious.

[0014] In a second aspect of the present invention, the ethanol extract of Moringa leaves can significantly reduce the content of triglycerides and free fatty acids in mice, and can effectively improve metabolism-related fatty liver disease.

[0015] The third aspect of the present invention provides a medicinal and edible extract which is of natural origin, highly safe and has few side effects.

[0016] A fourth aspect of the present invention provides a use of an ethanol extract of Moringa leaves in a preparation for preventing metabolic-related fatty liver disease and related diseases caused by a high-fat, high-cholesterol diet.

[0017] A fifth aspect of the present invention provides a preparation for preventing and improving metabolism-related fatty liver disease, wherein the preparation comprises an ethanol extract of Moringa leaves.

[0018] A sixth aspect of the present invention is a preparation for preventing hyperlipidemia-related diseases, the preparation comprising Moringa leaf ethanol extract.

[0019] In the seventh aspect of the present invention, Moringa leaves are a new resource food, and the extract thereof has little toxic side effects, is low in price, is easy to take, and can be used for a long time. It is expected to become a relatively safe and reliable drug that can be taken for a long time to improve metabolism-related fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a statistical graph of mouse liver weight data after 8 weeks of drug administration in the present invention.

[0021] Figure 2 These are actual photos of the mouse liver after 8 weeks of drug administration in the examples of the present invention, as well as photos of the changes in liver tissue lipid levels and histopathology observed by hematoxylin / eosin staining and Oil Red O staining under an optical microscope.

[0022] Figure 3This is a graph showing changes in triglyceride content in mouse liver after 8 weeks of drug administration in accordance with an embodiment of the present invention.

[0023] Figure 4 This is a graph showing changes in free fatty acid content in the liver of mice after 8 weeks of drug administration in accordance with an embodiment of the present invention. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0025] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0026] The preparation of the Moringa leaf ethanol extract comprises the following steps: S1, weigh 2 kg of dried Moringa leaves; S2, crushing the Moringa leaves in step S1 into fine powder using a crusher; S3, put the Moringa leaf powder obtained in step S2 into a 10L container; S4. Add enough 95% ethanol to the container in S3 to ensure that the powder is completely covered with ethanol; S5. Seal the container and soak it in a constant temperature environment for seven days; S6. After soaking for seven days, filter the solution using a 2L suction filtration device to remove undissolved solid residues; S7. To improve the extraction efficiency, the filtration process in step S6 was repeated three times, and the filtrate was replaced with new 95% ethanol each time to ensure that as many effective ingredients as possible were extracted; S8, combining the filtrates obtained by three filtrations; S9, using a vacuum decompression concentration device, the filtrate obtained in step S8 is concentrated at 45° C. This temperature can reduce the loss of heat-sensitive components; S10. After step S9 is concentrated until there is no alcohol taste, the equipment is turned off and the extract is allowed to cool naturally to room temperature; S11, the cooled extract in step S10 is dried using a vacuum dryer; S12, weighing the Moringa leaf ethanol extract obtained in step S11 to obtain 380 g of Moringa leaf ethanol extract. And storing it in a dry, dark, cool condition to maintain its stability and effectiveness.

[0027] In this example, the ethanol extract of Moringa leaves was used as a test object, and in vivo experiments on mice were performed to verify the improvement effect of the ethanol extract of Moringa leaves on mice with metabolism-related fatty liver disease.

[0028] Experimental methods:

[0029] (1) Mouse grouping:

[0030] Forty 8-week-old male C57BL / 6J mice weighing 18-20 g were purchased and kept at a constant temperature and humidity for one week. After that, they were divided into five groups (8 mice in each group) according to their body weight: normal control (Con) group, high-fat and high-cholesterol (HFC) model group, low-dose Moringa leaf ethanol extract intervention group (200 mg / kg), high-dose Moringa leaf ethanol extract intervention group (400 mg / kg), and positive drug rosiglitazone intervention group (10 mg / kg).

[0031] (2) Prepare low-dose intervention drugs with Moringa leaves:

[0032] Use a 1 / 10,000 day precision balance to accurately weigh 60 mg of Moringa leaf ethanol extract into a 5 mL ethylene oxide sterilized (EP) tube, use a high-precision pipette to add 3 mL of ultrapure water to the EP tube, and gently vortex and ultrasonically mix to completely dissolve the Moringa leaf ethanol extract to prepare a 20 mg / mL low-dose solution.

[0033] (3) Prepare high-dose intervention drugs with Moringa leaves:

[0034] Use a 1 / 10,000 day precision balance to accurately weigh 120 mg of Moringa leaf ethanol extract into a 5 mL ethylene oxide sterilized (EP) tube, use a high-precision pipette to add 3 mL of ultrapure water to the EP tube, and mix by gently vortexing and ultrasonic vortexing to completely dissolve the Moringa leaf ethanol extract to prepare a 40 mg / mL low-dose solution.

[0035] (4) Configure positive drugs:

[0036] 4 mg of rosiglitazone was weighed into a 5 mL ethylene oxide sterilized (EP) tube using a 100,000th precision balance, and 2‰ of dimethyl sulfoxide (DMSO) was added to the EP tube using a high-precision pipette, followed by 5% Tween 80 and saline, and then the solution in the EP tube was gently vortexed to ensure that rosiglitazone was completely dissolved. Thus, a precise 1 mg / mL concentration of rosiglitazone solution was prepared.

[0037] (5) Drug administration to mice:

[0038] The mice in the normal control (NC) group were fed with normal feed (purchased from Guangdong Experimental Animal Center). The other groups were fed with high-fat feed (HFC, containing 60% fat and 1.2% cholesterol, purchased from Research Diet, USA). The three intervention groups set in (1): low-dose Moringa leaf ethanol extract intervention group, high-dose Moringa leaf ethanol extract intervention group and positive drug rosiglitazone intervention group were given solutions prepared in (2) to (4) by gavage. The drugs were given at 10 a.m. every day. The drugs were given by gavage for 60 consecutive days.

[0039] (6) After step (5), the subjects were anesthetized and blood was collected 24 hours after drug withdrawal. The subjects were killed by cervical dislocation and autopsied. The abdominal anatomical diagram and fat weight of each group were recorded.

[0040] Experimental results:

[0041] The results are shown in Figure. Figure 1 It can be seen that after 60 days of feeding with high-fat and high-cholesterol feed, the liver weight of the model group mice increased by 50% compared with the blank control group, and the difference was significant (p < 0.01); the liver weight of the mice in the low-dose Moringa leaf group was reduced by 21.9% compared with the model group, and there was a significant difference (p < 0.05), and the liver weight of the mice in the high-dose Moringa leaf group was reduced by 31.3% compared with the model group, and there was a significant difference (p < 0.01), indicating that the high-dose intervention group of Moringa leaf ethanol extract had better effect than the low-dose intervention group, proving that Moringa leaf ethanol extract can regulate lipid deposition in the liver, prevent abnormal enlargement of the liver of mice fed with high-fat and high-cholesterol, improve liver fat deposition, and effectively improve MASLD and related metabolic disorders.

[0042] Depend on Figure 2It can be seen that (1) from the liver photographs: the normal group rats' livers were normal in size, with smooth capsules, sharp edges, soft texture and red color; the model group mice were significantly enlarged in size, their livers were milky white to varying degrees, the capsules were tight, the cut surface was greasy, and obvious fatty lesions were visible; the Moringa leaf ethanol extract group rats' livers were less enlarged, dark red in color, with slightly thick capsules and soft texture, and the improvement effect of the high-dose group was better than that of the low-dose group. (2) HE staining results showed that the liver cell structure of the normal group mice was normal, while the fatty liver cells of the model group mice were enlarged in size, and many round vacuoles of different sizes appeared in the cytoplasm of the liver cells, with varying degrees of fatty degeneration and local inflammatory cell infiltration; compared with the model group, the number of fatty vacuoles in the liver cells of the Moringa leaf ethanol extract group mice was significantly reduced, and the inflammatory cell infiltration phenomenon was improved. (3) Oil red O staining results showed that the fat content in the liver of the model mice increased significantly. Compared with the model group, the hepatocyte structure of the mice in the Moringa leaf ethanol extract group was significantly improved, and the fatty degeneration, vacuolar degeneration and lipid accumulation of the mouse liver were improved. At the same time, the morphology of the mouse liver was consistent with that of the normal group. In summary, the Moringa leaf ethanol extract has a positive effect on improving MASLD by reducing fat accumulation in the liver.

[0043] Depend on Figure 3 It can be seen that after 60 days of medication, the triglyceride content in the liver of the MASLD mouse model in this experiment was significantly increased compared with that in the control group (P<0.001), indicating lipid deposition in hepatocytes; the triglyceride content in the liver tissue of mice in the low-dose group of Moringa leaf ethanol extract showed a downward trend compared with the model (P<0.05), and the triglyceride content in the liver tissue of mice in the high-dose group of Moringa leaf ethanol extract was significantly decreased (P<0.001). The changes in triglyceride content clearly indicate that Moringa leaf ethanol extract can reduce the triglyceride content in the liver, and it is enhanced in a dose-dependent manner, and has the effect of reversing the pathological basis of MASLD.

[0044] Depend on Figure 4 It can be seen that compared with the control group, the free fatty acid content in the liver tissue of the MASLD mouse model in the experiment was significantly increased (P<0.01), which also indicated lipid deposition in the hepatocytes; the free fatty acid content in the liver tissue of the mice in the low-dose group of Moringa leaf ethanol extract showed a downward trend compared with the model group (P<0.05), and the free fatty acid content in the liver tissue of the mice in the high-dose group of Moringa leaf ethanol extract was significantly decreased (P<0.01). The change in the free fatty acid content clearly shows that Moringa leaf ethanol extract can reduce the free fatty acid content in the liver, showing the therapeutic effect of Moringa leaf ethanol extract on MASLD.

[0045] In summary, the mouse experiments can effectively demonstrate that Moringa leaf ethanol extract can effectively alleviate fatty liver disease, effectively improve metabolism-related fatty liver disease, and that Moringa leaf ethanol extract, as a source of both medicine and food, is itself safer.

[0046] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing an ethanol extract of Moringa leaves, characterized in that The following steps are involved: S1, drying the Moringa leaf material and crushing it into powder; S2, soak the powder in 95% ethanol for 7 days, select the supernatant for filtration, and repeat this process three times; S3. The filtrate is concentrated under vacuum at 45° C. until there is no ethanol smell, and then dried to obtain the Moringa leaf ethanol extract.

2. The use of the Moringa leaf ethanol extract prepared according to claim 1 for improving metabolism-related fatty liver disease in mice, characterized in that The following steps are involved: S1. Purchase 40 8-week-old male C57BL / 6J mice weighing 18-20 g of specific pathogen-free (SPF) grade; S2. After one week of feeding at constant temperature and humidity, the rats were divided into five groups according to their body weight (8 rats in each group): normal control (Con) group, high-fat and high-cholesterol (HFC) model group, low-dose Moringa leaf ethanol extract (L-MOL) intervention group (200 mg / kg), high-dose Moringa leaf ethanol extract (H-MOL) intervention group (400 mg / kg), and positive drug rosiglitazone (RSG) intervention group (10 mg / kg); S3. Except for the normal control group, the other groups were given a high-fat diet and intragastrically administered drugs at 10 a.m. every day for 60 consecutive days. S4, after step S3, the drug was stopped for 24 hours, blood was collected under anesthesia, the mice were killed by cervical dislocation and autopsied, the weight of the liver of the mice was recorded, and the fat accumulation was observed; S5, weighing a certain weight of the mouse liver tissue collected in step S5, making paraffin sections, staining with hematoxylin-eosin (HE) and oil red O, respectively, and observing the pathological sections; S6. After step S5, serum of mice is collected to measure the contents of triglycerides and free fatty acids in the mice.

3. A Moringa leaf ethanol extract, characterized in that The method according to claim 1 is used to improve the symptoms of mice with metabolic-related fatty liver disease induced by a high-fat and high-cholesterol diet.

4. The experimental results according to claim 2, characterized in that Moringa leaf ethanol extract can effectively improve the symptoms of metabolic-related fatty liver disease mice induced by a high-fat, high-cholesterol diet, specifically in terms of liver weight, liver free fatty acids, triglycerides, and liver staining.

5. A pharmaceutical composition using Moringa leaf ethanol extract, characterized in that The composition comprises the Moringa leaf ethanol extract according to claim 3 as an active ingredient.

6. An application of Moringa leaf ethanol extract, characterized in that The moringa leaf extract is used for treating and preventing metabolism-related fatty liver disease and related symptoms caused by a high-fat and high-cholesterol diet.

7. A preparation for liver protection, characterized in that , the preparation includes medicine, health care product or food, and the preparation contains Moringa leaf ethanol extract.

8. A Moringa leaf ethanol extract, characterized in that The extract is prepared by the method according to claim 1 and is used for the application described in any one of claims 2 to 7.

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