CN111671784A

CN111671784A - A product for preventing and treating non-alcoholic fatty liver disease

Info

Publication number: CN111671784A
Application number: CN202010740885.4A
Authority: CN
Inventors: 王刚; 赵文宇; 顾震南; 翟齐啸; 陆文伟; 赵建新; 张灏; 陈卫
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-07-29
Filing date: 2020-07-29
Publication date: 2020-09-18

Abstract

The invention discloses a product for preventing and treating non-alcoholic fatty liver disease, and belongs to the technical field of biological medicine. The myristica fragrans extract prepared by the method can inhibit synthesis of fatty acid LO2 of liver cells, inhibit expression of fatty acid synthase gene FASN of LO2 of liver cells and sterol regulatory element binding protein gene SREBP-1c, improve liver functions, regulate lipid metabolism, improve liver inflammatory response, enhance immune functions of mice, regulate liver lipid metabolism, reduce liver lipid accumulation and help prevent and/or treat nonalcoholic fatty liver.

Description

A product for preventing and treating non-alcoholic fatty liver disease

Technical Field

The invention relates to a product for preventing and treating non-alcoholic fatty liver, belonging to the technical field of biological medicine.

Background

Non-alcoholic fatty liver disease is a disease characterized by excessive accumulation of fat in hepatocytes. It is usually associated with severe obesity and chronic low grade inflammation. The clinicopathological syndrome of nonalcoholic fatty liver disease is characterized by diffuse hepatocytes with hepatocellular globulocystic, intrahepatic inflammation and progressive fibrosis. According to the pathological changes of the non-alcoholic fatty liver, the liver can be divided into simple steatosis, non-alcoholic steatohepatitis and fatty liver cirrhosis, and fibrosis even develops into liver cancer. Persistent liver fatty infiltration and inflammatory responses can increase the incidence of cirrhosis and liver cancer. In addition, non-alcoholic fatty liver promotes the development of dyslipidemia and significantly increases the incidence of cardiovascular disease. The mechanism by which the non-alcoholic fatty liver process occurs is not clear, although it is generally believed that it consists of at least two components, the "secondary strike" theory. Liver fat accumulation caused by disorders of fat metabolism is a common mechanism among the various causes causing liver steatosis, followed by immune cell activation and inflammatory cytokine production.

Studies have shown that obese patients with non-alcoholic fatty liver have decreased lipid metabolism but increased fatty acid synthesis. The fatty acid synthase gene (FASN) and the upstream sterol regulatory element binding protease-1 c (SREBP-1c) play an important role in fatty acid metabolism, activation of upstream phosphoadenosine protease (AMPK) accelerates energy metabolism, and inhibition of FASN leads to a decrease in the fatty acid synthesis pathway. Therefore, the method has become a powerful target for intervening the non-alcoholic fatty liver caused by excessive lipid accumulation. Sustained excess of free fatty acids is involved in the pathogenesis of steatosis and leads to non-alcoholic fatty liver-related metabolic complications, such as inflammatory reactions. Prolonged exposure of the liver to excess free fatty acids can lead to accumulation of intracellular triglycerides and impair insulin function, further affecting blood glucose levels. Excessive fatty acids promote TG secretion in the form of VLDL and increase hepatic neolipogenesis, leading to hepatotoxicity and inducing the liver to continuously produce inflammatory factors, affecting liver function. By inhibiting fatty acid synthesis, the proliferation and growth of liver cancer cells can be inhibited, and the survival rate is reduced. Therefore, the lipid metabolism is regulated to become a powerful means for losing weight and lowering lipid, inhibiting inflammatory reaction, recovering liver function and improving non-alcoholic fatty liver.

At present, non-alcoholic fatty liver disease can be prevented to some extent by limiting dietary energy intake (e.g. low sugar, low fat, etc.) or by exercising in moderate amounts, but the extent of intervention is limited. With the acceleration of modern life rhythm and the increase of social working pressure, irregular diet and sleep increase the incidence of the non-alcoholic fatty liver disease. However, the drug therapy is accompanied by a series of side effects, and the drug therapy causes a certain degree of damage to the body. Therefore, there is a need to develop a product for preventing and treating non-alcoholic fatty liver disease to solve the problem.

Disclosure of Invention

The invention aims to solve the technical problems of providing a novel health care food which can reduce weight, blood fat and liver lipid accumulation; can also regulate inflammatory reaction and reduce the generation of complications; and has little toxic and side effects, and can be used for preventing/treating non-alcoholic fatty liver.

The invention provides application of a myristica fragrans extract in preparation of a product for preventing/treating non-alcoholic fatty liver disease.

In one embodiment, the myristica fragrans extract is prepared by the following steps:

(1) pulverizing the roasted semen Myristicae, grinding, and sieving;

(2) leaching the sieved nutmeg powder in the step (1) for at least 10 hours by using an ethanol solution, and repeating for 2-3 times;

(3) mixing the extracts, vacuum filtering, and concentrating to obtain semen Myristicae extract.

In one embodiment, the myristica fragrans extract is dissolved in a solvent.

In one embodiment, the solvent includes, but is not limited to, dimethyl sulfoxide (DMSO) or sodium carboxymethyl cellulose (CMC) with a mass fraction of 0.5%.

In one embodiment, the myristica fragrans extract is prepared as follows:

(1) pulverizing the roasted nutmeg, grinding with a mechanical grinder, and sieving with a 200-mesh sieve;

(2) leaching the sieved nutmeg powder in the step (1) with 75% ethanol at 37 ℃ for 12h, filtering and recovering dregs, and repeating the leaching step for 3 times;

(3) mixing the 3 times of extracts, and vacuum filtering;

(4) concentrating the filtered liquid in a rotary evaporator, evaporating to dryness, and dissolving in a solvent; the solvent includes but is not limited to dimethyl sulfoxide (DMSO) or sodium carboxymethyl cellulose (CMC) with a mass fraction of 0.5%.

In one embodiment, the prevention/treatment of non-alcoholic fatty dryness is embodied in at least one of (a) to (f):

(a) reducing inflammatory response caused by free fatty acids;

(b) inhibiting hepatocyte lipid synthesis and macrophage inflammatory response;

(c) reducing blood fat;

(d) reducing blood glucose and improving glucose tolerance;

(e) reducing liver lipid synthesis;

(f) regulating liver lipid metabolism and/or reducing liver lipid accumulation;

(g) improving liver function;

(h) inhibit the proliferation of liver cancer cell and reduce the survival rate of cancer cell.

In one embodiment, the product is a functional food.

The second object of the present invention is to provide a pharmaceutical composition for preventing and treating non-alcoholic fatty liver disease, which comprises the aforementioned myristica fragrans extract.

In one embodiment, the myristica fragrans extract is dissolved in an ethanol solution, dimethyl sulfoxide (DMSO) or a 0.5% by weight solution of sodium carboxymethylcellulose (CMC).

In one embodiment, the myristica fragrans extract is present in the pharmaceutical composition in an amount of not less than 200 mg/kg.

In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

In one embodiment, the carrier is one or more of a pharmaceutically acceptable filler, wetting agent, disintegrant, binder, or lubricant.

In one embodiment, the filler is one or more of microcrystalline cellulose, lactose, mannitol, starch, or dextrin; the wetting agent is one or more of ethanol or glycerol; the disintegrant is one or more of sodium carboxymethyl starch, cross-linked povidone or low-substituted hydroxypropyl cellulose; the adhesive is one or more of starch paste, syrup, maltose, refined honey or liquid glucose; the lubricant is one or more of magnesium stearate, sodium fumarate stearate, talcum powder or silicon dioxide.

The invention also claims the use of the nutmeg extract in the preparation of a health product for non-medical purposes.

Has the advantages that:

(1) the invention takes nutmeg as RAW material to prepare nutmeg extract which can inhibit the synthesis of fatty acid of liver cells LO2, reduce inflammatory reaction of mouse macrophage RAW264.7 caused by free fatty acid, and inhibit the expression of mouse liver cells LO2 fatty acid synthetase gene FASN and sterol regulatory element binding protein gene SREBP-1 c. Compared with a control group, the expression quantity of the FASN gene and the SREBP-1c gene is obviously reduced, so that the product has better inhibition effect on in-vitro mouse hepatocyte lipid synthesis and macrophage inflammatory reaction.

(2) Can effectively inhibit the in vitro proliferation of the mouse liver cancer cell Hepa1-6, and is obviously superior to the reported inhibition level.

(3) The myristica fragrans extract disclosed by the invention can be used for reducing the weight of a mouse, reducing blood fat, obviously reducing liver lipid, improving the blood sugar level and the liver function and has a strong effect of preventing and/or treating nonalcoholic fatty liver.

(4) After the mouse is subjected to intragastric administration, the content of inflammatory factors IL-6 and TNF alpha in the liver of the mouse after the myristica fragrans extract prepared by the method is obviously reduced at 16 weeks of a non-alcoholic fatty liver disease model. Therefore, the myristica fragrans extract can improve the liver inflammatory reaction and enhance the immune function of mice.

(5) Protein expression levels of FASN and SREBP-1c in the mouse liver after the maceration of the myristica fragrans extract prepared by the method are obviously reduced compared with a model control group mouse, and the expression levels of pAMPK-alpha/AMPK-alpha are obviously increased compared with the control group mouse.

Drawings

Figure 1 effect of different myristol extract concentrations on LO2 hepatocyte survival.

FIG. 2 the effect of different concentrations of myristyl alcohol extract on the FASN and SREBP-1c genes of LO2 hepatocytes.

FIG. 3 shows the effect of different concentrations of myristyl alcohol extract on the expression level of FASN gene in LO2 hepatocytes.

FIG. 4 shows the effect of different concentrations of myristyl alcohol extract on the expression level of SREBP-1c gene of LO2 hepatocytes.

FIG. 5 shows the effect of myristyl alcohol extract on the expression level of TNF alpha gene, a mouse macrophage inflammatory factor.

FIG. 6 shows the effect of myristyl alcohol extract on the expression level of mouse macrophage inflammatory factor IL-6 gene.

FIG. 7 effect of myristyl alcohol extract on food intake of non-alcoholic fatty liver disease model mice.

FIG. 8 the effect of myristyl alcohol extract on body weight of non-alcoholic fatty liver disease model mice.

FIG. 9 the effect of myristyl alcohol extract on the amount of weight change in non-alcoholic fatty liver disease model mice.

FIG. 10 effect of myristyl alcohol extract on blood glucose levels in non-alcoholic fatty liver disease model mice.

FIG. 11 Effect of myristyl alcohol extract on oral glucose tolerance curves in non-alcoholic fatty liver disease model mice.

Figure 12 effect of myristyl alcohol extract on area under oral glucose tolerance curve of non-alcoholic fatty liver disease model mice.

FIG. 13 effect of myristyl alcohol extract on liver lipid accumulation in non-alcoholic fatty liver model mice.

FIG. 14 Effect of myristyl alcohol extract on serum low density lipoprotein levels in non-alcoholic fatty liver model mice.

FIG. 15 effect of myristyl alcohol extract on serum high density lipoprotein levels in non-alcoholic fatty liver disease model mice.

FIG. 16 Effect of myristyl alcohol extract on serum cholesterol levels in non-alcoholic fatty liver model mice.

FIG. 17 Effect of myristyl alcohol extract on serum alanine aminotransferase levels in non-alcoholic fatty liver model mice.

FIG. 18 effect of myristyl alcohol extract on serum alkaline phosphatase levels in non-alcoholic fatty liver model mice.

Figure 19 effect of myristyl alcohol extract on the level of inflammatory factor TNF α in the liver of non-alcoholic fatty liver model mice.

FIG. 20 Effect of myristyl alcohol extract on the level of inflammatory factor IL-6 in the liver of non-alcoholic fatty liver model mice.

FIG. 21 shows the effect of myristyl alcohol extract on the relative expression level of FASN gene in liver of non-alcoholic fatty liver disease model mouse.

FIG. 22 shows the effect of myristyl alcohol extract on the relative expression level of SREBP-1c gene in liver of non-alcoholic fatty liver disease model mouse.

FIG. 23 shows the effect of myristyl alcohol extract on the expression level of FASN, SREBP-1c and β -actin in liver of non-alcoholic fatty liver disease model mouse.

FIG. 24 Effect of myristyl alcohol extracts on pAMPK- α/AMPK- α in the liver of non-alcoholic fatty liver model mice.

(P < 0.05; P < 0.01; significance is represented by abc: no significant difference between identical letters, significant difference between different letters.)

Detailed Description

Nutmeg referred to in the following examples was purchased from Tongrentang, Beijing;

ELISA kits for detection of IL-1 β (cat # DY401-05), IL-6 (cat # DY406-05), TNF α (cat # DY410-05) and IL-10 (cat # DY417-05) referred to in the examples were purchased from R & D;

LO2 cells and Raw264.7 cells mentioned in the examples were purchased from Nanjing Ke Bai Biotech Co., Ltd; mouse hepatoma cell line Hepa1-6 was purchased from Kyobo, Shanghai.

DMEM medium and RPMI-1640 medium (cat # C11965092 and C11875500B) referred to in the examples were purchased from Gibco, USA;

the 60% high fat diet referred to in the examples was purchased from south-lead Techlophene (cat # TP 23300).

The method for establishing the non-alcoholic fatty liver model comprises the following steps:

taking 5-week-old female C57BL/6 mice, feeding the mice with a 60% high-fat model feed every day after feeding the mice adaptively for 1 week under the conditions of room temperature of 20-26 ℃, humidity of 40-70% and 12h/12h day and night alternation and free feeding and drinking, observing the change of the weight and the food intake of the mice, observing the change of the weight, the liver weight, plasma triglyceride, blood sugar and the like when the model is established for 10-12 weeks, and determining whether to establish a non-alcoholic fatty liver model.

Example 1: preparation of nutmeg alcohol extract

Pulverizing roasted semen Myristicae (purchased from Beijing Tongrentang) with a mechanical grinder, sieving with 200 mesh sieve, repeatedly extracting with 75% ethanol at 37 deg.C for three times, filtering, recovering residue, and leaching for 12 hr each time. Mixing the three extracts, and vacuum filtering. The filtered liquid is concentrated in a rotary evaporator, evaporated to dryness and weighed, respectively dissolved in dimethyl sulfoxide (DMSO) and 0.5% sodium carboxymethylcellulose (CMC) for cell experiments and animal experiments, and refrigerated at 4 ℃ for later use. The major components of the extract were also identified by two-dimensional gas chromatography-time of flight mass spectrometry (GC X GC-TOF-MS) (Table 1).

TABLE 1 composition of the major components of the Myristica fragrans extract

Example 2: influence of myristica fragrans alcohol extract on liver cell lipid synthesis related genes FASN and SREBP-1c

The human liver cell line LO2 was cultured in the presence of 5% (v/v) CO₂Was cultured in a 37 ℃ incubator and grown in DMEM medium containing 10% FBS, 100U/mL penicillin and 100. mu.g/mL streptomycin. Until the cell density of LO2 cells reached 80%, transfer to 96-well plates at a density of 5000 cells per well. After 24 hours of incubation, treatment with different concentrations of ethanol extract of nutmeg was divided into four dose groups (25, 50, 100 and 200 mg/L). Control cells (CON group) were added with solvent (1% DMSO) only. Each set is provided with three parallel holes repeatedly. Cell proliferation was measured by MTT colorimetry for 72 hours (i.e., 48 hours after stimulation). The medium was aspirated, the cells were carefully rinsed three times with PBS, and then medium containing MTT was added and left for 4 hours. Finally, DMSO was added to dissolve the blue-violet crystalline Formazan (Formazan) in the cells. Cell viability was identified in a microplate reader by measuring the absorption at a wavelength of 570 nm. Another set of LO2 cells was seeded in 6-well plates and treated with different doses (25, 50 and 100mg/L) of ethanol extract of nutmeg. The positive control group was treated with Curcumin (Curcumin), a Fatty Acid Synthase (FAS) inhibitor. After 48 hours of culture, the medium was discarded, and the cells were washed three times with PBS and mixed with TRIzol. Total RNA of LO2 cells was extracted by Trizol method, and after reverse transcription, the expression levels of fatty acid synthetase FASN and sterol regulatory element binding protein gene SREBP-1c were determined by PCR (the results are shown in FIGS. 1-4).

The detection result is as follows: as shown in fig. 1, survival rate of LO2 decreased to different degrees in the four dose groups (25, 50, 100 and 200mg/L), and at 50mg/L, survival rate was significantly reduced, and at 100mg/L, survival rate of LO2 cells was 49.4%, that is, 100mg/L was below IC50, and effective concentration of myristol extract was present, and concentration gradient was set for the next experiment.

As seen in fig. 2-4, another set of LO2 cells was treated with different doses (25, 50 and 100mg/L) of ethanol extract of nutmeg in 6-well plates. The positive control group was treated with Curcumin (Curcumin), a fatty acid synthesis inhibitor. After 48h, the FASN gene of LO2 cells was significantly down-regulated in all four treatment groups, wherein at 100mg/L, the gene expression was 19.5% of that of CON group, and the down-regulation was 5.12 times; the expression level of the Curcumin group is 23.9 percent of that of the CON group, and is reduced by 4.18 times. The SREBP-1c gene is remarkably reduced in the 100mg/L group and the Curcumin group, and is respectively 19.1 percent and 31.5 percent. As can be seen from fig. 2-4, the myristyl alcohol extract can significantly inhibit lipid synthesis of LO2 cells, and is superior to the fatty acid synthase inhibitor Curcumin.

Example 3: effect of Myristica fragrans alcohol extract on in vitro proliferation of liver cancer cells

The mouse liver cancer cell line Hepa1-6 was cultured in the presence of 5% (v/v) CO₂Was cultured in a 37 ℃ incubator and grown in DMEM medium containing 10% FBS, 100U/mL penicillin and 100. mu.g/mL streptomycin. When the cell density of the Hepa1-6 cells reached 80%, transfer to a 96-well plate at a density of 5000 cells per well. After 24 hours of incubation, treatments with different concentrations of ethanol extract of nutmeg were divided into three dose groups (25, 50, 100 mg/L). Control cells (CON group) were added with solvent (1% DMSO) only. Six parallel holes are repeatedly arranged in each group. Cell proliferation was measured by MTT colorimetry after 18h and 36h of stimulation, respectively. The medium was aspirated, the cells were carefully rinsed three times with PBS, and then medium containing MTT was added and left for 4 hours. Finally, DMSO was added to dissolve the blue-violet crystalline Formazan (Formazan) in the cells. Cell viability was identified in a microplate reader by measuring the absorption at a wavelength of 570 nm. (the detection results are shown in Table 2).

The detection result is as follows: as can be seen from Table 2, the inhibition rates of Hepa1-6 cells were increased to different degrees in the three dose groups (25, 50, 100mg/L) under stimulation of 18h and 36h, with half-cell inhibition rates of 50mg/L or less and 47.05mg/L at 18h and 30mg/L or less and 28.96mg/L at 36 h. As shown in Table 2, AEN effectively inhibited the proliferation of hepatoma cells in vitro.

TABLE 2 inhibition of proliferation of murine hepatoma cells Hepa1-6 by Myristica fragrans extracts

Example 4: influence of myristica fragrans alcohol extract on mouse macrophage inflammatory response related genes IL-6 and TNF alpha

Mouse mononuclear macrophage RAW264.7 in 5% (v/v) CO₂The method comprises the following steps of culturing in a 37 ℃ incubator, growing in a DMEM culture medium containing 10% FBS, 100U/mL penicillin and 100 mu g/mL streptomycin, transferring to a 6-well plate when the cell density of RAW264.7 cells reaches 80%, obtaining a well plate full of RAW264.7 cells, dividing the well plate full of RAW264.7 cells into four groups, namely a CON group, an AEN group, an FFA group and an AEN + FFA group, adding a solvent (1 thousandth DMSO) into the well plate of the CON group, adding 100mg/L of myristyl alcohol extract into the well plate of the AEN group, adding a mixture of oleic acid and palmitic acid in a molar ratio of 2: 1 into the well plate of the FFA group, adding 100mg/L of myristyl alcohol extract and a mixture of oleic acid and palmitic acid (2: 1) into the well plate of the AEN + FFA group, discarding the culture medium after 48 hours, washing the cells for three times, extracting the cells with TRIzol, extracting RAW264.7 cells by a Trizol method, detecting the total RNA and the total RNA expression of TNF- α genes through PCR.

As can be seen from FIG. 5, there was no significant change in the addition of AEN to macrophage RAW264.7 (AEN group), the increase of inflammatory factor TNF- α was 2.34-fold higher than that of control group (FFA group) under the stimulation of oleic acid and palmitic acid mixture, and the TNF- α gene expression was down-regulated after the addition of AEN (AEN + FFA group), which was 27.0% lower than that of FFA group and was 1.71-fold higher than that of control group.

As can be seen from FIG. 6, there was no significant change in the addition of AEN to macrophage RAW264.7 (AEN group), the increase in IL-6, an inflammatory factor, was increased by 2.09-fold in the FFA group in response to the stimulation with a mixture of oleic acid and palmitic acid (FFA group), and the IL-6 gene expression was decreased by 22.8% in comparison with the FFA group (AEN + FFA group) and by 1.61-fold in comparison with the control group after the addition of AEN.

From fig. 5-6, AEN can significantly down-regulate the inflammatory response of mouse macrophages caused by free fatty acids, and does not stimulate inflammatory response to normal macrophages.

Example 5: effect of Myristica fragrans alcohol extract on lipid metabolism and blood glucose levels in non-alcoholic fatty liver disease mice

Taking 5-week-old female C57BL/6 mice, feeding the mice under the conditions of room temperature of 20-26 ℃, humidity of 40-70%, day and night alternation of 12h/12h, free feeding and drinking for 1 week, then dividing the mice into 3 groups according to the average weight, 10 mice in each group, feeding one group with common low-fat feed, feeding the other two groups with 60% high-fat feed, and determining the establishment of a model after feeding for 10-12 weeks. The 3 groups were set as blank control group (CON), model group (HF), and nutmeg alcohol extract group (HF + AEN), respectively. Diluting the myristica fragrans alcohol extract with sodium carboxymethylcellulose (0.5%) solution to a concentration of 200mg/kg to obtain myristica fragrans alcohol extract diluent, and preparing the myristica fragrans alcohol extract group for intragastric administration. The model group and the blank control group were filled with only carboxymethyl cellulose sodium (0.5%) solution as gastric solvent.

The experimental time was 5 weeks: the CON group mice were gavaged with sodium carboxymethylcellulose (0.5%) solution 200ul per day to observe changes in body weight and food intake, the HF group mice were gavaged with sodium carboxymethylcellulose (0.5%) solution 200ul per day to observe changes in body weight and food intake, and the HF + AEN group mice were observed with dilutions of myristyl alcohol extract 200ul per day to observe changes in body weight and food intake. Before the experiment is finished, the serum is taken to measure biochemical indexes by adopting a spectrophotometry method, and after the mouse is sacrificed, the liver, fat and other organ tissues are dissected and taken to be pathological sections. (the results of the detection are shown in FIGS. 7 to 16)

As can be seen from fig. 7, the food intake of the mice did not change significantly before and after gavage, indicating that the myristica fragrans extract did not significantly inhibit or increase the food intake of the mice.

From fig. 8-9, before gavage, the body weight of the mice in the high-fat diet group was significantly increased compared to the blank control group, and 43.4% compared to the control group, after gavage, the body weight of the mice in the HF + AEN group began to be significantly reduced, and at the end of the 16 th week experiment, the body weight was reduced by 34.1% compared to the HF group in the model group. From FIGS. 8-9, it can be seen that AEN has an effective alleviating effect on the weight gain of mice induced by high-fat diet.

As can be seen from fig. 10 to 12, at week 16, the blood glucose levels of the mice in the high-fat model group were significantly increased by 44.9% compared to the blank control group, and the blood glucose levels of the mice in the AEN-treated group were decreased by 28.1% compared to the high-fat model group. The OGTT curve shows that the blood sugar of the HF group mice is above 7 at 0min, the blood sugar rises to 20.72 +/-3.45 at 15min, and is increased by 35.1% compared with the blank control group, then the blood sugar of the three groups of mice begins to drop, at 120min, the blood sugar of the CON group mice drops to below 7, the blood sugar of the HF + AEN group recovers to the level of 6.4 +/-0.82, and the blood sugar of the HF group recovers to the level of 8.81 +/-1.32. As can be seen by the area AUC under the mouse OGTT curve, the HF group is obviously increased and is increased by 36.2 percent compared with the control group, and the HF + AEN group is obviously reduced and is reduced by 20.2 percent compared with the HF group. As can be seen from fig. 10 to 12, the non-alcoholic fatty liver disease mice had high blood glucose levels and impaired glucose tolerance, which resulted in imbalance of blood glucose regulation, and the myristica fragrans extract was effective in lowering blood glucose levels and improving glucose tolerance.

As can be seen from FIG. 13, by pathological liver section (HE staining: left; oil red staining: right, it can be seen that, compared with CON group (top), the liver of mice in HF group (middle) shows steatosis, i.e. vacuoles (lipid droplets) or orange lipid droplets with different sizes appear, the boundary is clear, the cell nucleus is squeezed to one side, the volume of liver cells is increased, the cell nucleus is swollen, the cell nucleus is obvious, and the local inflammatory reaction appears, the area of vacuoles (lipid droplets) of liver tissues of mice in AEN group (bottom) is reduced, the inflammatory reaction is reduced, and the lipid accumulation of liver has obvious relief, and from FIGS. 11-13, AEN can regulate the blood lipid level of non-alcoholic fatty liver mice.

From fig. 14 to 16, it can be seen that, when the serum levels of low-density lipoprotein (LDL), high-density lipoprotein (HDL), and serum cholesterol (TG) were analyzed by biochemical indicators of the mouse serum, the levels of LDL and TC were increased in the serum of the HF group as compared with the control group, and the levels of LDL and TC were significantly decreased in the HF + AEN group, which were lower in the HF group, and the levels of HDL were significantly increased in the HF + AEN group.

From FIGS. 17 to 18, alanine Aminotransferase (ALT) and alkaline phosphatase (ALP) in the liver function index of the mouse were increased in the HF group to different degrees, indicating that a certain degree of impaired liver function and inflammatory reaction occurred, and AEN was effective in restoring liver function, down-regulated by 50.7% and 12.3%, respectively.

From fig. 7-18, it can be seen that the myristyl alcohol extract can reduce weight and fat, regulate lipid metabolism, improve liver function, lower blood glucose level, and improve glucose tolerance.

Example 6: influence of Myristica fragrans Houtt extract on inflammatory factors IL-6 and TNF alpha in liver of non-alcoholic fatty liver disease mouse

The experimental procedure was as follows: the CON group mice were gavaged with sodium carboxymethylcellulose (0.5%) solution 200ul per day to observe changes in body weight and food intake, the HF group mice were gavaged with sodium carboxymethylcellulose (0.5%) solution 200ul per day to observe changes in body weight and food intake, and the HF + AEN group mice were observed with dilutions of myristyl alcohol extract 200ul per day to observe changes in body weight and food intake. After the experiment is finished, the liver of the mouse is dissected and taken out after the mouse is killed, the liver is ground into liver homogenate, and the content of inflammatory factors IL-6 and TNF alpha in the liver of the mouse is detected by an ELISA kit.

From fig. 19-20, it can be seen that at week 16 of feeding, proinflammatory factors IL-6 and TNF α in the liver of non-alcoholic fatty liver model mice were significantly increased, AEN effectively reduced the level of inflammatory factors in the liver of mice, by 25.6% and 24.9% respectively, compared to HF group, and the inflammatory response in the liver of mice was alleviated.

Example 7: influence of myristica fragrans alcohol extract on expression level of protein related to liver fat metabolism of non-alcoholic fatty liver mice

The experimental procedure was as follows: the CON group mice were gavaged with sodium carboxymethylcellulose (0.5%) solution 200ul per day to observe changes in body weight and food intake, the HF group mice were gavaged with sodium carboxymethylcellulose (0.5%) solution 200ul per day to observe changes in body weight and food intake, and the HF + AEN group mice were observed with dilutions of myristyl alcohol extract 200ul per day to observe changes in body weight and food intake. After the experiment is finished, the liver of the mouse is dissected and taken out after the mouse is killed, the liver homogenate is ground, the liver tissue is dissolved by RIPA buffer solution containing protease inhibitor, and the protein expression quantity of FASN, SREBP-1c and pAMPK-alpha/AMPK-alpha of the mouse is measured by Western blot method after the protein is extracted.

From FIGS. 21 to 23, it is shown that the protein expression levels in the liver of the mouse are significantly higher than those of the blank control group, and that AEN can significantly reduce the protein expression levels of liver FASN and SREBP-1c, and lower the lipid synthesis rate, thereby reducing the lipid accumulation in the liver. This function is achieved by activation of upstream target AMP-dependent protein kinase-alpha (AMPK-alpha). pAMPK-alpha/AMPK-alpha is an index for evaluating the degree of activation of the AMPK system. As can be seen in FIG. 24, the ratio of pAMPK- α/AMPK- α was significantly increased in the HF + AEN group compared to the HF group. From fig. 21 to 24, the myristica fragrans alcohol extract can regulate the expression level of the protein related to the liver fat metabolism of the mouse to reduce the liver lipid accumulation condition, so that the process of the non-alcoholic fatty liver disease of the mouse can be effectively improved.

Example 8

The myristica fragnance extract was prepared according to the method of example 1.

The nutmeg extract is combined with a pharmaceutically acceptable carrier or combined with various solid or liquid pharmaceutical excipients and/or auxiliary agents to form a suitable administration form or dosage form for human use.

One or more of the pharmaceutically acceptable filler, wetting agent, disintegrant, binder or lubricant.

The filler is one or more of microcrystalline cellulose, lactose, mannitol, starch or dextrin; the wetting agent is one or more of ethanol or glycerol; the disintegrant is one or more of sodium carboxymethyl starch, cross-linked povidone or low-substituted hydroxypropyl cellulose; the adhesive is one or more of starch paste, syrup, maltose, refined honey or liquid glucose; the lubricant is one or more of magnesium stearate, sodium fumarate stearate, talcum powder or silicon dioxide.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. The application of the myristica fragrans extract in preparing products for preventing and/or treating non-alcoholic fatty liver diseases is characterized in that the myristica fragrans extract is prepared by the following steps:

(1) pulverizing the roasted semen Myristicae, grinding, and sieving;

2. The use according to claim 1, wherein the myristica fragrans extract is dissolved in a solvent.

3. Use according to claim 1 or 2, wherein the product comprises but is not limited to a food, a pharmaceutical or a nutraceutical product.

4. Use according to any one of claims 1 to 3, wherein the prevention and/or treatment of non-alcoholic fats is at least one of (a) to (f):

(a) reducing inflammatory response caused by free fatty acids;

(b) inhibiting hepatocyte lipid synthesis and macrophage inflammatory response;

(c) reducing blood fat;

(d) reducing blood glucose and improving glucose tolerance;

(e) reducing liver lipid synthesis;

(f) regulating liver lipid metabolism and/or reducing liver lipid accumulation;

(g) improving liver function;

5. A pharmaceutical composition comprising an extract of Myristica fragrans Houtt; the nutmeg extract is prepared by the following steps:

(1) pulverizing the roasted semen Myristicae, grinding, and sieving;

(3) mixing the extracts obtained in step (2), vacuum filtering, and concentrating to obtain semen Myristicae extract.

6. The pharmaceutical composition of claim 5, wherein the myristyl alcohol extract is present in an amount of no less than 200 mg/kg.

7. The pharmaceutical composition of claim 5 or 6, further comprising a pharmaceutically acceptable carrier.

8. The pharmaceutical composition of claim 7, wherein the carrier is one or more of a pharmaceutically acceptable filler, wetting agent, disintegrant, binder, or lubricant.

9. The pharmaceutical composition of claim 8, wherein the filler is one or more of microcrystalline cellulose, lactose, mannitol, starch, or dextrin; the wetting agent is one or more of ethanol or glycerol; the disintegrant is one or more of sodium carboxymethyl starch, cross-linked povidone or low-substituted hydroxypropyl cellulose; the adhesive is one or more of starch paste, syrup, maltose, refined honey or liquid glucose; the lubricant is one or more of magnesium stearate, sodium fumarate stearate, talcum powder or silicon dioxide.

10. Use of Myristica fragrans Houtt extract in the preparation of non-medical health products.