CN111544422A

CN111544422A - Application of policosanol in preparation of cholesterol absorption inhibitor and/or liver protection preparation

Info

Publication number: CN111544422A
Application number: CN202010486342.4A
Authority: CN
Inventors: 黎鹏; 项辉; 孟飞; 黄俊生; 黄冬婷; 汤静洁
Original assignee: Guangdong Institute of Bioengineering Guangzhou Cane Sugar Industry Research Institute
Current assignee: Guangdong Institute of Bioengineering Guangzhou Cane Sugar Industry Research Institute
Priority date: 2020-06-01
Filing date: 2020-06-01
Publication date: 2020-08-18

Abstract

The invention discloses application of policosanol in preparation of a cholesterol absorption inhibitor and/or a preparation for protecting liver. The policosanol provided by the invention can inhibit intestinal absorption of cholesterol and promote excretion of cholesterol; meanwhile, the policosanol can reduce the accumulation of Triglyceride (TG) in the liver, improve the pathological change condition of the liver, treat alcoholic liver/fatty liver and protect the liver.

Description

Application of policosanol in preparation of cholesterol absorption inhibitor and/or liver protection preparation

The technical field is as follows:

the invention belongs to the technical field of food and medicine, and particularly relates to application of policosanol in preparation of a cholesterol absorption inhibitor and/or a preparation for protecting liver.

Background art:

the policosanol is a mixture composed of various long-chain primary aliphatic alcohols, and is mainly octacosanol. Policosanol is mainly extracted from sugar cane wax, beeswax and rice bran, and has antiinflammatory, antioxidant, hyperglycemia and hypertension improving, and cardiovascular disease and cerebrovascular disease preventing effects.

Dyslipidemia is a chronic disease very common at present, and is specifically an abnormality in blood lipid levels, including cholesterol (TC) and Triglyceride (TG) levels. Research proves that dyslipidemia is related to the onset of hypertension, diabetes and cardiovascular diseases, which seriously threatens human health. The clinically applied lipid-lowering drugs are mainly statins, and after a large amount of statins are treated by many patients, the cholesterol level in the body cannot be effectively controlled, so that the cholesterol absorption inhibitor plays an increasingly important role in treating hypercholesterolemia. Liver diseases are one of the most common diseases clinically harming human health, and the incidence of fatty liver diseases or alcoholic liver injury in China is increasing in recent years. Therefore, prevention and treatment of liver diseases are becoming important.

The invention content is as follows:

the invention aims to overcome the defects of the prior art and provides the application of policosanol in preparing a cholesterol absorption inhibitor and/or a preparation for protecting the liver, and the policosanol can inhibit intestinal absorption of cholesterol and promote the excretion of the cholesterol; meanwhile, the policosanol can reduce the accumulation of Triglyceride (TG) in the liver, improve the pathological change condition of the liver and protect the liver.

The invention provides application of policosanol in preparing a cholesterol absorption inhibitor and/or a preparation for protecting liver. Policosanol is derived from plant extract or synthetic product such as sugar cane wax, beeswax, and rice bran.

Preferably, the cholesterol absorption inhibitor is a preparation for inhibiting the absorption of cholesterol by intestinal tracts and promoting the excretion of cholesterol by organisms; the liver-protecting preparation is a liver-protecting preparation by reducing accumulation of triglycerides in the liver. The policosanol provided by the invention can prevent and/or treat cholesterol level disorder or liver injury under the influence of high-fat diet and/or alcohol. Alcohol and high-fat diet can promote the absorption of cholesterol by animals, and the policosanol can obviously increase the excretion of the cholesterol of the animals or inhibit the absorption of the cholesterol by intestinal tracts.

The cholesterol absorption inhibitor provided by the invention can be used for regulating the cholesterol level of human beings and other mammals, other mammals comprise mice, rabbits, pigs, cattle, sheep, dogs and the like, and the functional preparation containing the policosanol can also be used for protecting the liver, and is particularly suitable for cholesterol level disorder, fatty liver disease, alcoholic liver injury and the like of mammals caused by intake of a large amount of high-fat food and/or alcohol through a digestive system.

Preferably, the dosage form of the cholesterol absorption inhibitor and/or the preparation for protecting liver is a liquid preparation, a solid preparation or a semisolid preparation.

Further preferably, the liquid preparation is an injection, a solution, a suspension, an emulsion or an aerosol, the semisolid preparation is an ointment, and the solid preparation is a tablet, a capsule, a pill, a powder injection, a sustained release preparation or a microparticle drug delivery system.

Preferably, the cholesterol absorption inhibitor and/or the liver-protecting agent is a food, a health product, a pharmaceutical product, an additive or a feed.

The second object of the present invention is to provide a cholesterol absorption inhibitor comprising an effective amount of polycosanol or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier.

The third purpose of the invention is to provide a preparation for protecting liver, which comprises effective amount of polycosanol or the medicinal salt thereof as an active ingredient and a pharmaceutically acceptable carrier.

The invention has the beneficial effects that:

1. the high-fat diet/alcohol can promote the absorption of the cholesterol by animal bodies, and the policosanol provided by the invention has certain effect on the influence of the high-fat diet/alcohol on the cholesterol, specifically, the policosanol can inhibit the absorption of the cholesterol by intestinal tracts and increase the excretion of the cholesterol of the animal bodies.

2. The policosanol provided by the invention reduces the accumulation of Triglyceride (TG) in the liver, improves the pathological change condition of the liver and has the effect of protecting the liver.

3. According to the invention, the policosanol is used for inhibiting intestinal absorption of cholesterol and promoting excretion of cholesterol, and meanwhile, the policosanol can reduce accumulation of Triglyceride (TG) in the liver, improve the pathological change condition of the liver and protect the liver.

Description of the drawings:

FIG. 1 is a HE staining of rat liver in example 3 (eyepiece. times.10, objective. times.20);

FIG. 2 is an oil red O staining pattern of rat liver in example 3 (eyepiece. times.10, objective. times.20).

The specific implementation mode is as follows:

the following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Unless otherwise specified, the experimental materials and reagents in the invention are all conventional commercial products in the technical field.

The invention relates to a method for measuring cholesterol in feces by adopting high performance liquid chromatography, wherein the conditions are that a chromatographic column is ODS-C18(250 x 4.6mm, 5 mu m), and a mobile phase is methanol: isopropanol (80:20, V/V), detection wavelength of 210nm, flow rate of 1.0mL/min, column temperature of 35 deg.C, and sample injection amount of 10 μ L.

The invention relates to measurement of cholesterol (TC), Triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) in serum by using a full-automatic blood fat tester.

Example 1:

1.1 materials

The high-fat feed comprises, by mass, 20% of sucrose, 15% of lard, 1.2% of cholesterol, 0.2% of sodium cholate, 10% of casein, 0.6% of calcium hydrophosphate, 0.4% of stone powder, 0.4% of premix and 52.2% of basal feed.

The policosanol solution is prepared by the following steps: adding anhydrous ethanol into a container filled with policosanol according to the volume of 10% of the prepared volume, heating in a boiling water bath for 1min to fully dissolve the policosanol, adding 80 drops of Tween, fully shaking, adding water to the total volume, carrying out vortex oscillation for 15min, and carrying out ultrasound for 30min to obtain the policosanol solution. The adding amount of the Tween 80 is 1 drop of Tween 80 in every 1.0mg of policosanol, and the mass concentration of the policosanol solution is 0.5-5.0 mg/mL.

Meanwhile, the solution without adding policosanol is prepared to be used as a solvent (adding 10 percent of absolute ethyl alcohol of the prepared volume).

1.2 modeling, grouping and index detection

36 male KM mice are adopted, 5-6 weeks old and marked, all mice freely drink and eat water, the environmental sanitation and good ventilation of a laboratory are kept, the temperature is 18-22 ℃, the relative humidity is 50% -60%, the illumination is carried out for 12 hours, and the circulation of the light and the night is carried out. One week after adaptive feeding, all mice were randomly divided into 6 groups of 6 mice each, a normal control group, a model control group, a vehicle group, a policosanol low dose group, a policosanol medium dose group and a policosanol high dose group, wherein the normal control group was fed with a conventional feed for 7 days, and the other groups were continuously fed with a high-fat feed for 7 days. On the 8 th day, the normal control group and the model control group were gavaged with physiological saline, the vehicle group was gavaged with vehicle, the administration doses of the policosanol low dose group, the policosanol medium dose group and the policosanol high dose group were 10mg/kg, 20mg/kg and 100mg/kg, respectively, and the gavage volumes of the 6 groups of mice were 20 mL/kg. Feces of each group of mice were collected for 6h, 12h, 18h and 24h, respectively, to determine the cholesterol content, and all mice were given regular free water and food intake during the feces collection process. After the experiment is finished, 2% sodium pentobarbital is injected into the abdominal cavity of an anesthetized mouse according to the dose of 50mg/kg, the inferior vena cava is used for taking blood, all the blood is placed for 20min at the normal temperature, then the blood is centrifuged for 15min at 3000r/min, so that fresh serum without hemolysis is obtained, and the serum is stored in a refrigerator at the temperature of 4 ℃ and is used for measuring TC and TG.

2. Results of the experiment

The effect of policosanol on fecal cholesterol and blood lipids in mice fed with high-fat diet for 7 days is shown in table 1. Compared with the normal control group, the concentration of fecal cholesterol is increased by more than 10 times, and the blood fat TC and TG are both obviously increased (P is less than 0.05). In the mice of the vehicle-dissolving agent group, the concentration of cholesterol in excrement is obviously reduced within 12 hours of gastric lavage, and the alcohol promotes the absorption of cholesterol, so that the concentration of cholesterol in excrement is obviously reduced. The cholesterol output of mice in the policosanol low-dose group, the policosanol medium-dose group and the policosanol high-dose group is increased within 12 hours, and the fecal cholesterol concentration is increased; compared with the menstruum group, the TC content in the blood has no obvious influence, but the TG content in the blood of mice in the middle-dose group and the high-dose group of the polycosanol is reduced (both P is less than 0.05), which shows that the polycosanol has certain lipid-lowering effect.

TABLE 1 Effect of policosanol on fecal cholesterol and blood lipid in mice fed with high-fat diet for 7 days

Note: p-10, P-20 and P-100 respectively represent a policosanol low dose group, a policosanol medium dose group and a policosanol high dose group in the experiment. Feces were all feces collected from a group of animals, with only one measurement. The lipid index is expressed as mean ± standard deviation, n ═ 6. P <0.05 compared to normal controls; in comparison to vehicle group, # # indicates P < 0.05.

Example 2:

1. experimental methods

1.1 materials

The high-fat feed comprises 20% of sucrose, 15% of lard, 1.2% of cholesterol, 0.2% of sodium cholate, 10% of casein, 0.6% of calcium hydrophosphate, 0.4% of stone powder, 0.4% of premix and 52.2% of basic feed.

The policosanol solution is prepared by the following steps: adding anhydrous ethanol into a container filled with policosanol according to the volume of 10% of the prepared volume, heating in a boiling water bath for 1min to fully dissolve the policosanol, adding 80 drops of Tween, fully shaking, adding water to the total volume, carrying out vortex oscillation for 15min, and carrying out ultrasound for 30min to obtain the policosanol solution. The adding amount of the Tween 80 is 1 drop of Tween 80 in every 1.0mg of policosanol, and the mass concentration of the policosanol solution is 1.0-5.0 mg/mL.

Meanwhile, the solution without adding policosanol is prepared to be used as a solvent (adding absolute ethanol with the preparation volume of 10 percent).

1.2 modeling, grouping and index detection

Taking 30 male SD rats 180-220 g, marking, and freely drinking and eating all rats, keeping the environment of a laboratory sanitary and well ventilated, wherein the temperature is 18-22 ℃, the relative humidity is 50% -60%, and the light irradiation is carried out for 12 hours and the circulation is carried out at night. After one week of adaptive feeding, the animals are randomly divided into 6 groups of 5 animals, including a normal control group, a model control group, a solvent group, a policosanol low-dose group, a policosanol medium-dose group and a policosanol high-dose group. The five groups were fed with high-fat diet except the normal control group with basal diet. Gavage was timed daily for 30 days while eating. The normal control group and the model control group are intragastrically filled with normal saline, the solvent group is intragastrically filled with solvent, the administration doses of the three dose groups of the polycosanol low dose group, the polycosanol medium dose group and the polycosanol high dose group are respectively 10mg/kg, 20mg/kg and 50mg/kg, and the intragastrically filled volume is 10 mL/kg. Weigh daily and record weight changes. During the dosing period, daily (24h) fecal cholesterol levels were measured in rats per group once every 7d and collected for a total of 4 weeks.

2. Results of the experiment

When the animals are fed with the high-fat feed, the cholesterol content in the animal feces is greatly increased, and the cholesterol content in the rat feces also tends to increase along with the prolonging of the feeding time of the high-fat feed, and the cholesterol content in the feces of the model control group at the 4 th week is obviously increased compared with that at the 1 st week as shown in the table 2. The concentration of fecal cholesterol in the rats in the solvent group (10% ethanol) is higher than that in the model control group 24 hours per week on the whole, which shows that the alcohol (10mL/kg of intragastric administration amount) has great influence on the metabolism of the cholesterol in the animal body and promotes the absorption of the cholesterol in the body. The content of cholesterol in the feces of rats in the three doses of policosanol and the contents of the cholesterol in the feces of the rats in the menstruum group and the model control group (such as the measurement results of the week 2 and the week 4) show that the policosanol has a certain regulating effect on the fat metabolism of animals fed with the high-fat feed, and can regulate the cholesterol level under the influence of alcohol.

TABLE 2 Effect of policosanol continuous administration on fecal cholesterol content of rats fed with high-fat diet for 30 days

Note: p-10, P-20 and P-50 respectively represent the low, medium and high doses of policosanol in the rat experiment. Feces were all feces collected from a group of animals and three replicates were prepared for this experiment and expressed as mean ± standard error, n-3.

Example 3:

1. experimental methods

1.1 materials

1.2 modeling, grouping and index detection

Taking 70 male SD rats 120-150 g, marking, and allowing all rats to freely drink and eat water, keeping the environmental sanitation and good ventilation of a laboratory, wherein the temperature is 18-22 ℃, the relative humidity is 50% -60%, and the light irradiation is carried out for 12 hoursAnd cycling through the night. One week after adaptive feeding, the groups were randomly divided into 7 groups of 10, including a normal control group (normal group), a model control group (model group), a vehicle group, a policosanol low dose group, a policosanol medium dose group and a policosanol high dose group, and the rest group was a simvastatin group. The normal control group was fed with basal diet and the remaining groups were fed with high-fat diet, and the study was performed daily with gavage for 16 weeks at regular intervals while the diet was started. The normal control group and the model control group are intragastrically filled with normal saline, the solvent group is intragastrically filled with solvent, the administration doses of the polycosanol low dose group, the polycosanol medium dose group and the polycosanol high dose group are respectively 10mg/kg, 20mg/kg and 50mg/kg, the administration dose of the simvastatin group is 20mg/kg, and the intragastrically filled volume is 10 mL/kg. Weigh daily and record weight changes. Rats were collected every 7d a day-night (24h) feces 5 times starting at week 12. The experiment was carried out to 120d, the rats were removed, anesthetized with 2% sodium pentobarbital at a dose of 50mg/kg by intraperitoneal injection, the abdominal cavity was opened along the midline of the abdomen, and 2cm of the abdominal cavity was taken²Left and right sized liver pieces were fixed with 4% paraformaldehyde followed by HE and oil red O staining. Another 50mg liver is washed with normal saline and placed in a refrigerator at-80 ℃ for standby. After the liver was treated, TC and TG were measured.

1.3 results of the experiment

As shown in table 3, the TC levels were reduced in both the policosanol low dose group and the policosanol medium dose group, and the TG level was reduced by about 38% in the simvastatin group, compared to the vehicle group. The TG levels of the policosanol low-dose group, the policosanol medium-dose group and the policosanol high-dose group are respectively reduced by about 43%, 36% and 31% (P <0.01), and the reduction is inversely proportional to the administration dose, so that the accumulation of TG in the liver can be reduced by administering the policosanol to rats for a long time.

TABLE 3 rat liver TC, TG levels

Note: the concentration is in mmol/L and the values are expressed as mean ± standard error (n ═ 10). #, P <0.01, compared to the normal group; p <0.05, P <0.01, compared to vehicle group.

At the same time, the results of liver HE staining (FIG. 1) and oil red O staining (FIG. 2) also clearly show the accumulation of lipid droplets in liver cells of rats in each group. The arrow in FIG. 1 indicates vacuole, and the normal control group had almost no vacuole and no steatosis; more vacuoles in the model control group are completely fused, and liver steatosis with mixed large-vesicular and medium-vesicular steatosis is formed; the vacuoles of the menstruum group are fusing, and part of the vacuolated fat which is fused can be seen according to the rarity; in the policosanol low-dose group, the policosanol medium-dose group and the policosanol high-dose group, although wide microvesicle-like steatosis exists, no obvious macrovesicular steatosis is seen. In fig. 2, the arrow position indicates lipid droplets, the normal control group has only a very small distribution of lipid droplets, and the other groups have relatively the largest distribution of lipid droplets in the vehicle group, and the model control group and the simvastatin group have the smallest distribution of lipid droplets in the low policosanol dose group, the medium policosanol dose group and the high policosanol dose group.

In addition, the present invention calculates the ratio of vacuole area of HE sections of liver to positive area of oil red O sections (see Table 4). Because certain gaps exist among cells of liver tissues, the vacuole area ratio is larger than the positive area ratio, and in contrast, the positive area ratio can more accurately reflect the severity of the fatty liver. Compared with the solvent group, the positive area ratios of the simvastatin group, the policosanol low-dose group, the policosanol medium-dose group and the policosanol high-dose group are respectively reduced by about 12%, 40%, 25% and 27% (P is less than 0.05), and the policosanol has a protective effect on the liver.

Table 4 rat liver section specific area ratio (%)

Note: the area ratio is obtained by selecting five regions for each section by a five-point sampling method, calculating the void area and positive area (fat drop) ratio of each region by imagej, and taking the average value as the area ratio of the section. Values are expressed as mean ± standard error (n ═ 3). #, P <0.01, compared to the normal group; p <0.05, compared to vehicle group.

While the use of the policosanols provided by the present invention in the preparation of cholesterol absorption inhibitors and/or hepatoprotective agents has been described in detail, the above examples are only provided to aid the understanding of the technical aspects of the present invention and the core concept thereof, and it should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principle of the present invention, and such improvements and modifications also fall within the protection scope of the appended claims.

Claims

1. Application of policosanol in preparing cholesterol absorption inhibitor and/or liver protecting preparation is provided.

2. Use according to claim 1, characterized in that: the cholesterol absorption inhibitor is a preparation for inhibiting the absorption of cholesterol by intestinal tracts and promoting the excretion of cholesterol by organisms.

3. Use according to claim 1, characterized in that: the liver-protecting preparation is a liver-protecting preparation by reducing accumulation of triglycerides in the liver.

4. Use according to claim 1, characterized in that: the dosage form of the cholesterol absorption inhibitor or the preparation for protecting the liver is a liquid preparation, a solid preparation or a semisolid preparation.

5. Use according to claim 4, characterized in that: the liquid preparation is injection, solution, suspension, emulsion or aerosol, the semisolid preparation is paste, and the solid preparation is tablet, capsule, pill, powder injection, sustained release preparation or microparticle drug delivery system.

6. Use according to claim 1, characterized in that: the cholesterol absorption inhibitor or the preparation for protecting liver is food, health product, medicine or feed.

7. A cholesterol absorption inhibitor characterized by comprising an effective amount of policosanol or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable carrier.

8. A preparation for protecting liver, which is characterized by comprising effective amount of policosanol or pharmaceutically acceptable salts thereof as an active ingredient and a pharmaceutically acceptable carrier.