Application of spicate clerodendranthus herb extract in preparation of medicine for preventing or treating non-alcoholic fatty liver disease

Abstract

The invention discloses an application of a spicate clerodendranthus herb extract in preparation of a medicine for preventing or treating a non-alcoholic fatty liver disease. The spicate clerodendranthus herb extract prepared by combining an ultrasonic extraction method with a system extraction method can relieve free fatty acid induced lipid droplet accumulation in liver cells, abnormal lipid metabolism and cell damage; the pharmaceutical composition also can relieve obesity, dyslipidemia, liver fatty degeneration and inflammation of rats induced by high-fat feed feeding, and shows good anti-NAFLD pharmacological activity. Therefore, the spicate clerodendranthus herb extract can be widely applied to preparation of the medicine for preventing or treating the non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to new uses of natural products, and specifically to the use of a cat's whisker grass extract in the preparation of a drug for preventing or treating non-alcoholic fatty liver disease. Background Art

[0002] The disease spectrum of non-alcoholic fatty liver disease (NAFLD) includes non-alcoholic hepatic steatosis, non-alcoholic steatohepatitis (NASH), liver fibrosis and cirrhosis, which eventually lead to hepatocellular carcinoma. In my country, NAFLD has become the number one chronic liver disease and the primary cause of abnormal liver biochemical indicators in health examinations, and has now become a serious public health problem in my country. Resmetirom, the first NASH specific drug approved by the US FDA this year, can only improve 25-30% of NASH patients in Phase III clinical trials. Therefore, there is a huge and unmet clinical need in this field. Chinese herbal medicine has been tested by the long-term medical practice of the Chinese nation and is an important means for my country to prevent and treat diseases and protect people's health. There is sufficient basis and broad prospects for seeking active ingredients for the prevention and treatment of NASH.

[0003] Orthosiphon stamineus Benth. is a medicinal plant of the genus Orthosiphon in the family Lamiaceae. It is also known as kidney tea and cat's whisker. It is known as "the best in the world" and is rich in a variety of active ingredients. Orthosiphon stamineus Benth. has a cooling and anti-inflammatory effect and can be used as medicine. Traditionally, it has been widely used to treat urinary system diseases such as nephritis, cystitis and urinary stones. Recent studies have found that Orthosiphon stamineus Benth. can also relieve diseases such as diabetes, arthritis, and Alzheimer's disease, but the pharmacological effect of Orthosiphon stamineus Benth. on anti-NAFLD has not yet been reported. Summary of the invention

[0004] The purpose of the present invention is to provide an application of a cat's whisker grass extract in the preparation of a medicament for preventing or treating non-alcoholic fatty liver disease, which has strong pharmacological activity and meets the use requirements.

[0005] To achieve the above-mentioned purpose, the present invention provides the use of a Miscanthus pubescens extract in the preparation of a drug for preventing or treating non-alcoholic fatty liver disease.

[0006] Furthermore, the non-alcoholic fatty liver disease is hepatocyte NAFLD induced by free fatty acids.

[0007] Furthermore, the non-alcoholic fatty liver disease is hepatocyte NAFLD induced by a high-fat diet.

[0008] Furthermore, the cat's whiskers extract is prepared by the following method:

[0009] (1) Selecting dried medicinal materials of cat's whiskers and grinding them into cat's whiskers powder by a grinder;

[0010] (2) taking a certain amount of cat's whiskers powder and immersing it in an ethanol solution, ultrasonically extracting it at room temperature for a period of time, and then filtering and collecting the filtrate;

[0011] (3) immersing the filter residue obtained in step (2) in an ethanol solution for ultrasonic extraction for a period of time at room temperature, and filtering and collecting the filtrate;

[0012] (4) The filtrates obtained in step (2) and step (3) are combined and concentrated under reduced pressure to form a solid, which is then dried in a ventilated drying cabinet to obtain a crude extract of cat's whiskers grass.

[0013] Furthermore, the crude extract of cat's whiskers is dissolved in an aqueous solution, and then extracted with an equal volume of dichloromethane. The obtained extract is concentrated under reduced pressure to evaporate the solvent to obtain a solid, which is the cat's whiskers extract.

[0014] Preferably, in step (2), the concentration of the ethanol solution is 55%, the mass volume ratio between the cat's whiskers powder and the ethanol solution is 1 g:40 mL, and the ultrasonic extraction is performed for 40 min.

[0015] Preferably, in step (3), the concentration of the ethanol solution is 55%, the mass volume ratio between the filter residue and the ethanol solution is 1 g:40 mL, and the ultrasonic extraction is performed for 40 min.

[0016] Preferably, the mass volume ratio between the crude extract of Misquiterpene strychnifolia and the aqueous solution is 1g:100mL.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The cat's whiskers extract prepared by the ultrasonic extraction method combined with the system extraction method adopted in the present invention can alleviate the accumulation of fat droplets in liver cells, abnormal lipid metabolism and cell damage induced by free fatty acids; it can also alleviate obesity, abnormal lipid metabolism, fatty degeneration and inflammation of the liver induced by high-fat diet in rats, showing good anti-NAFLD pharmacological activity. It can be seen that the cat's whiskers extract will have a wide range of applications in the preparation of drugs for preventing or treating non-alcoholic fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the effect of cat's whiskers extract on intracellular lipid droplet accumulation induced by free fatty acids; control is the control group, FFA is the model group, DCF-OSB is the cat's whiskers extract administration group, and OSB is the cat's whiskers crude extract administration group;

[0020] Figure 2Schematic diagram of the effect of cat's whiskers extract on intracellular lipid metabolism indicators induced by free fatty acids; (a) TG, (b) TC, (c) LDL-C;

[0021] Figure 3 Schematic diagram of the effect of cat's whisker grass extract on intracellular ALT (a) and AST (b) levels induced by free fatty acids;

[0022] Figure 4 Schematic diagram of the effect of cat's whiskers extract on the expression of intracellular pro-inflammatory factors induced by free fatty acids; (a) TNF-α, (b) IL-1β, (c) IL-6;

[0023] Figure 5 This is a trend chart of the weight changes of rats per week; control group vs model group, #p<0.05, ##p<0.001, ###p<0.001; drug administration group vs model group, *p<0.05, **p<0.001, ***p<0.001;

[0024] Figure 6 Schematic diagram of the effect of lipid metabolism index levels in rat serum; (a) TG, (b) TC, (c) LDL-C;

[0025] Figure 7 Schematic diagram of the effects of ALT (a) and AST (b) levels in rat serum;

[0026] Figure 8 This is a schematic diagram of the HE staining results of rat liver tissue pathological sections; Normal is the normal group, HFD is the high-fat diet group, DCF-OSB is the group administered with cat's whiskers extract, and OSB is the group administered with cat's whiskers crude extract;

[0027] Fig. 9 Schematic diagram of the levels of SOD activity (a), GPx activity (b) and MDA content (c) in rat liver tissue;

[0028] Fig.10 It is a schematic diagram of the levels of pro-inflammatory factors TNF-α (a), IL-1β (b), and IL-6 (c) in rat liver tissue;

[0029] Figure 2-7 , Figure 9-10 In the table, *p<0.05, **p<0.001, ***p<0.001. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0031] The Miscanthus sinensis extract used in the following examples was prepared by the following preparation method, and other raw materials and reagents used, unless otherwise specified, were from conventional commercial stores.

[0032] 1. Cat's Whiskers Extract

[0033] The dried medicinal material of cat's whiskers was ground into powder by a grinder, 50 g of the powder was weighed and immersed in 2.0 L of 55% ethanol solution, ultrasonically extracted at room temperature for 40 min, and then the filtrate was collected by filtration; the filter residue was immersed in 2.0 L of 55% ethanol solution again at room temperature for ultrasonic extraction for 40 min, and the filtrate was collected by filtration. The filtrates were combined and concentrated to solids under reduced pressure, and then dried in a ventilated drying cabinet to obtain 3.3 g of crude extract.

[0034] 2. Extraction of crude extract of cat's whiskers

[0035] 5 g of crude extract of cat's whiskers was dissolved in 500 ml of aqueous solution, then extracted with an equal volume of dichloromethane, and then the extract was concentrated under reduced pressure to evaporate the solvent to obtain a solid, which was the cat's whiskers extract.

[0036] Example 1

[0037] Alleviating effect of Misquito herb extract on free fatty acid-induced NAFLD in hepatocytes

[0038] 1. Materials and Reagents

[0039] Sodium oleate, sodium palmitate, Oil Red O, bovine serum albumin (BSA) and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich, human hepatocyte cell line LO2 cells were purchased from ATCC, 1640 medium for cell culture, penicillin-streptomycin double antibody solution and fetal bovine serum (FBS) were purchased from GIBCO Invitrogen, triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) detection kits were purchased from Nanjing Jiancheng Bioengineering Institute, and inflammatory factor TNF-α, IL-1β and IL-6 gene primers were purchased from BGI.

[0040] 2. Experimental Methods

[0041] LO2 cells were cultured in 1640 culture medium (containing 10% FBS, 100U / mL penicillin and 100μg / mL streptomycin) and placed in a 37°C incubator with 5% CO2. The cells were divided into a control group (NC), a model group (FFA), an OSB group, and a DCF-OSB group. After cell attachment, the cells in the control group were replaced with 1640 culture medium, and the cells in the model group and the treatment group were replaced with 1640 culture medium containing 1mM fatty acid (sodium oleate and sodium palmitate molar ratio of 2:1) to induce the cell NAFLD model. The cells in the treatment group were added with cat's whiskers crude extract (final concentration of 50μg / mL) and cat's whiskers extract (final concentration of 25μg / mL), and the cells were cultured for 24h. After the cell treatment, Oil Red O solution staining was used to evaluate the accumulation of intracellular lipid droplets. The intracellular TG, TC, ALT and AST contents were detected according to the instructions of the kit. RT-QPCR was used to detect the mRNA expression levels of TNF-α, IL-1β and IL-6 in the cells.

[0042] The results showed that OSB and DCF-OSB could significantly reduce the accumulation of intracellular lipid droplets induced by free fatty acids. Figure 1 ), significantly reducing the levels of intracellular TG, TC and LDL-C ( Figure 2 ), significantly reducing intracellular ALT and AST levels ( Figure 3 ), significantly inhibiting the expression of intracellular pro-inflammatory factors TNF-α, IL-1β and IL-6 ( Figure 4 The above results indicate that cat's whiskers extract can effectively alleviate free fatty acid-induced lipid droplet accumulation, abnormal lipid metabolism and inflammation in NAFLD model cells.

[0043] Example 2

[0044] Alleviation effect of Misquiterium wilfordii extract on high-fat diet-induced NAFLD in rats

[0045] Male Sprague-Dawley rats were selected and housed under standard conditions (temperature 22-25°C, humidity 55-70%) with a 12-hour light / dark cycle. Rats (8 weeks old, 250-300 g) were randomly divided into 4 groups (6 rats in each group): (i) normal group (0.5% sodium carboxymethylcellulose); (ii) high-fat diet (HFD) group (0.5% sodium carboxymethylcellulose); (iii) DCF-OSB group (25 mg / kg cat's whiskers extract); (iv) OSB group (50 mg / kg cat's whiskers crude extract). Rats in the normal group were given a normal diet, and rats in the other groups were given HFD as described above. All drugs (i.e., cat's whiskers crude extract, cat's whiskers extract) were ground and suspended in 0.5% sodium carboxymethylcellulose. The weight information of rats was recorded weekly. From the 5th week, each rat was gavaged with the corresponding drug once a day until the end of the 12th week.

[0046] All rats were fasted for 12 hours after the last administration and then anesthetized by intraperitoneal injection of 3% sodium pentobarbital (2 ml / kg, ip). Blood was collected through the abdominal aorta of the rats to prepare serum samples, and serum TG, TC, ALT, AST and LDL-C were then measured. Rat liver tissue was collected and pathological histological examination was performed using hematoxylin-eosin (H&E) staining. SOD activity, MDA content and GSH-Px activity in the liver were determined using the corresponding kits. The levels of TNF-α, IL-1β and IL-6 in the liver were determined using an enzyme-linked immunosorbent assay (ELISA) kit.

[0047] The results showed that compared with the normal diet, the high-fat diet caused the rats to gain weight rapidly; and compared with the high-fat diet group, the administration of cat's whiskers from the 10th week significantly inhibited the weight gain of rats caused by the high-fat diet ( Figure 5 ). A high-fat diet led to a significant increase in the levels of TG, TC, and LDL-C in the serum of rats; compared with the high-fat diet group, administration of cat's whiskers significantly reduced the levels of TG, TC, and LDL-C ( Figure 6 ), indicating that cat's whiskers can effectively alleviate the abnormal lipid metabolism induced by a high-fat diet. Compared with the high-fat diet group, the ALT and AST levels in the serum of rats after cat's whiskers administration were significantly reduced ( Figure 7 ), indicating that cat's whiskers can alleviate liver cell damage induced by a high-fat diet. Liver tissue pathological analysis showed that compared with the high-fat diet group, the fatty degeneration and inflammatory cell infiltration of the rat liver after cat's whiskers administration were significantly alleviated ( Figure 8 ). Compared with the high-fat diet group, the activities of SOD and GPx antioxidant enzymes in the liver tissue of rats after administration of cat's whiskers were significantly increased, and the level of lipid peroxide (MDA) was significantly reduced ( Fig. 9), indicating that cat's whiskers can effectively alleviate the oxidative stress of liver tissue induced by high-fat diet. Compared with the high-fat diet group, the levels of TNF-α, IL-1β and IL-6 in the liver tissue of rats after cat's whiskers administration were significantly reduced ( Fig.10 ), indicating that cat's whiskers can effectively alleviate liver tissue inflammation induced by a high-fat diet.

[0048] from Figure 1-10 It can be seen that the effect of the crude extract is equivalent to that of the extract, but the dosage of the crude extract is twice that of the extract, indicating that the active ingredients are mainly present in the dichloromethane component. In the future, the use of the dichloromethane component can reduce the dosage and reduce the intake of inactive ingredients to reduce side effects.

Claims

1. Application of cat's whiskers extract in the preparation of drugs for preventing or treating non-alcoholic fatty liver disease.

2. The use of the Miscanthus sinensis extract according to claim 1 in the preparation of a drug for preventing or treating non-alcoholic fatty liver disease, characterized in that: The non-alcoholic fatty liver disease is hepatocyte NAFLD induced by free fatty acids.

3. The use of the Miscanthus sinensis extract according to claim 1 in the preparation of a drug for preventing or treating non-alcoholic fatty liver disease, characterized in that: The non-alcoholic fatty liver disease is hepatocyte NAFLD induced by a high-fat diet.

4. The use of the Miscanthus sinensis extract according to claim 1 in the preparation of a drug for preventing or treating non-alcoholic fatty liver disease, characterized in that: The cat's whiskers extract is prepared by the following method: (1) Selecting dried medicinal materials of cat's whiskers and grinding them into cat's whiskers powder by a grinder; (2) taking a certain amount of cat's whiskers powder and immersing it in an ethanol solution, ultrasonically extracting it at room temperature for a period of time, and then filtering and collecting the filtrate; (3) immersing the filter residue obtained in step (2) in an ethanol solution for ultrasonic extraction for a period of time at room temperature, and filtering and collecting the filtrate; (4) The filtrates obtained in step (2) and step (3) are combined and concentrated under reduced pressure to form a solid, which is then dried in a ventilated drying cabinet to obtain a crude extract of cat's whiskers grass.

5. Use of the Miscanthus sinensis extract according to claim 4 in the preparation of a drug for preventing or treating non-alcoholic fatty liver disease, characterized in that: The crude extract of cat's whiskers is dissolved in an aqueous solution, and then extracted with an equal volume of dichloromethane. The obtained extract is concentrated under reduced pressure to evaporate the solvent to obtain a solid, which is the cat's whiskers extract.

6. Use of the Miscanthus sinensis extract according to claim 4 in the preparation of a drug for preventing or treating non-alcoholic fatty liver disease, characterized in that: In step (2), the concentration of the ethanol solution is 55%, the mass volume ratio between the cat's whiskers grass powder and the ethanol solution is 1 g:40 mL, and the ultrasonic extraction is performed for 40 minutes.

7. Use of the Miscanthus sinensis extract according to claim 4 in the preparation of a drug for preventing or treating non-alcoholic fatty liver disease, characterized in that: In step (3), the concentration of the ethanol solution is 55%, the mass volume ratio between the filter residue and the ethanol solution is 1 g:40 mL, and the ultrasonic extraction is performed for 40 min.

8. Use of the Miscanthus sinensis extract according to claim 5 in the preparation of a drug for preventing or treating non-alcoholic fatty liver disease, characterized in that: The mass volume ratio between the crude extract of cat's whiskers and the aqueous solution is 1g:100mL.

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