CN101756984B - Application of pyrroloquinoline quinone in treatment and prevention of atherosclerosis - Google Patents

Abstract

The invention provides application of pyrroloquinoline quinone in treatment and prevention of atherosclerosis, and further provides a health-care product composition or a pharmaceutical composition containing pyrroloquinoline quinone. The pyrroloquinoline quinone disclosed by the invention can inhibit the oxidation of low-density lipoprotein, protect endothelial cells and reduce the content of lipid in endothelium and the area of atherosclerotic plaques. Therefore, the pyrroloquinoline quinone has good application prospect in preventing and/or treating the formation and development of atherosclerosis.

Description

Application of pyrroloquinoline quinone in treatment and prevention of atherosclerosis

technical field

The present invention relates to the field of vascular diseases. More specifically, the present invention relates to the application of pyrroloquinoline quinone in the treatment and/or prevention of atherosclerosis.

Background technique

Atherosclerosis is a lesion that invades arteries and blood vessels. It causes coronary heart disease, cerebrovascular disease and peripheral vascular disease, which seriously endangers human health. Its morbidity and mortality rank among the forefront of various diseases. Epidemiological surveys show that the incidence of cardiovascular disease in some developed countries is close to 10%, and the annual death toll in the world accounts for 10% of the total population death toll. There are more than 7 million patients with cardiovascular and cerebrovascular diseases in my country, and there are even more patients with atherosclerotic lesions, and their morbidity and mortality are increasing year by year, while the age of onset is decreasing. Therefore, the development of drugs for the prevention and/or treatment of atherosclerosis will not only effectively meet the clinical needs, but will also have a broad market.

Atherosclerosis is a chronic inflammatory reaction of the arterial wall, which can last for decades without symptoms, and can eventually develop due to two reasons: first, the compensatory enlargement of the arterial lumen, and repeated rupture of atherosclerotic plaques Heal to form emboli, block the arterial lumen, and cause insufficient blood supply to vital organs; second, if the arterial lumen increases excessively compensatoryly, an aneurysm may occur. Therefore, preventing atherosclerotic plaque formation and delaying disease progression is very important for improving morbidity and mortality.

Atherosclerosis can be caused by a variety of factors, major ones include the accumulation of macrophages and leukocytes, the stimulation of low-density lipoprotein (LDL), and the clearance of lipids and cholesterol due to the lack of high-density lipoprotein insufficient.

Due to the complexity of the pathogenesis of atherosclerosis and the long course of the disease, there is still a lack of very effective and safe methods for the treatment of atherosclerosis. Therefore, there is an urgent need in this field to develop safe and effective drugs and health products for atherosclerosis.

Pyrroloquinoline quinone (Pyrroloquinoline Quinone, PQQ) is the first small molecular compound discovered from microorganisms, and it also exists in higher eukaryotes. PQQ is a reddish-brown powder and is a water-soluble compound. It exists in our daily diet, is widely distributed in various tissues and organs of the human body, and exists in breast milk. The molecular formula of PQQ is as follows:

PQQ is the prosthetic group of many important enzymes in bacteria, which can affect the function of the respiratory chain and the level of free radicals in the body. Studies have shown that mice lacking PQQ grow slowly, have poor reproductive ability, and are prone to joint inflammation, which may be one of the essential vitamins in the body.

Studies have confirmed that PQQ is a substance that is very beneficial to human health. Multiple functions of PQQ have been observed through animal experiments: Purified PQQ acts as an antioxidant to protect the liver from CCl4 or alcohol damage. Purified PQQ reduced glucocorticoid-induced cataract formation during chick embryo development, possibly by restoring glutathione levels and reducing responsiveness to corticosteroids. PQQ directly oxidizes the NMDA redox site of receptors, protects cells from NMDA toxicity, thereby preventing cerebral ischemia and hypoxia, and avoiding severe shock in animal models. At the same time, PQQ can also protect the heart against myocardial ischemia and myocardial infarction. For the nervous system, PQQ has antioxidant capacity and neuroprotective effects.

However, prior to the present invention, no research has been done in the art on the use of PQQ active substances for the prevention and/or treatment of atherosclerosis.

Contents of the invention

One of the objects of the present invention is to provide a safe and effective composition for preventing and/or treating atherosclerosis.

In the first aspect of the present invention, the application of pyrroloquinoline quinone active substance in the preparation of a composition for treating and/or preventing atherosclerosis or its symptoms in a subject is provided.

In one embodiment of the present invention, the symptoms of atherosclerosis are selected from the group consisting of atherosclerotic plaque thrombosis, ischemic syndrome caused by atherosclerosis, coronary heart disease caused by atherosclerosis , stroke from atherosclerosis, abdominal aortic aneurysm from atherosclerosis, or peripheral arterial disease from atherosclerosis.

In another embodiment of the present invention, the composition is a health product composition or a pharmaceutical composition.

In another embodiment of the invention, said subject is a mammal. In a preferred example, the mammal is a human.

In another embodiment of the present invention, the pyrroloquinoline quinone active substance accounts for 0.001-99.9 wt% of the total weight of the composition.

In a preferred example, the pyrroloquinoline quinone active substance accounts for 1-95wt%, preferably 5-90wt%, more preferably 10-80wt% of the total weight of the composition.

In another embodiment of the present invention, the dosage form of the composition includes: tablet, powder, injection, granule, syrup, capsule, solution, suppository or ointment.

In a preferred example, the composition is in unit dosage form or multi-dosage form, wherein the content of pyrroloquinoline quinone active substance is 1-4000 mg/dose, preferably 100-2000 mg/dose.

In another preferred embodiment, the composition is administered at 0.001-10 mg/kg body weight, preferably 0.01-5 mg/kg body weight, preferably 0.01-1 mg/kg body weight.

In another embodiment of the invention, the composition also comprises other active substances for the treatment and/or prevention of atherosclerosis or its symptoms.

In a preferred example, the other active substances for treating and/or preventing atherosclerosis or its symptoms are selected from: drugs that dilate blood vessels; substances that regulate blood lipids, such as statins, cholestyramine, niacin, Saturated fatty acids, etc.; antiplatelet substances, such as aspirin, dipyridamole, ticlide, etc.; thrombolytic substances, such as urokinase, recombinant tissue plasminogen activator, heparin, etc.; other active substances, such as choline, egg Phospholipids, Calamet, vitamin E, carotenoids, vitamin B, vitamin C or coenzyme Q10.

In a second aspect the present invention provides a composition comprising:

(1) effective amount of pyrroloquinoline quinone active substance;

(2) other active substances for the treatment and/or prevention of atherosclerosis or its symptoms; and

(3) A pharmaceutically acceptable carrier.

In a preferred example, the pyrroloquinoline quinone active substance accounts for 0.001-99.9wt%, preferably 1-95wt%, more preferably 5-90wt%, more preferably 10-80wt% of the total weight of the composition.

In another preferred example, the other active substances for treating and/or preventing atherosclerosis or its symptoms are one or more substances selected from the group consisting of drugs that dilate blood vessels; substances that regulate blood lipids, such as statins anti-platelet substances, such as aspirin, dipyridamole, ticlide, etc.; thrombolytic substances, such as urokinase, recombinant tissue plasminogen activator, heparin etc.; other active substances, such as choline, lecithin mixture, calcium beauty mixture, vitamin E, carotenoids, vitamin B, vitamin C, coenzyme Q10, etc.

In another embodiment of the present invention, the composition is a health product composition or a pharmaceutical composition.

In another embodiment of the present invention, the weight ratio of component (1) and component (2) is 0.01:100～100:0.01.

In a preferred example, the weight ratio of component (1) to component (2) is 1:50-50:1, more preferably 1:10-10:1.

In a third aspect of the present invention, a method for preventing and/or treating atherosclerosis or its symptoms in a subject is provided, the method comprising administering to the subject a pyrroloquinoline quinone active substance or a drug containing the active substance combination.

In one embodiment of the present invention, the symptoms of atherosclerosis are selected from the group consisting of atherosclerotic plaque thrombosis, ischemic syndrome caused by atherosclerosis, coronary heart disease caused by atherosclerosis , stroke from atherosclerosis, abdominal aortic aneurysm from atherosclerosis, or peripheral arterial disease from atherosclerosis.

In another embodiment of the present invention, the composition is a health product composition or a pharmaceutical composition. In another embodiment of the invention, said subject is a mammal. In a preferred example, the mammal is a human.

In another embodiment of the present invention, the pyrroloquinoline quinone active substance accounts for 0.001-99.9 wt% of the total weight of the composition. Preferably it is 1-95 wt%, more preferably 5-90 wt%, even more preferably 10-80 wt%.

In another embodiment of the present invention, the method further comprises the administration of other active substances for the treatment and/or prevention of atherosclerosis or its symptoms.

In a preferred example, the other active substances for treating and/or preventing atherosclerosis or its symptoms are selected from: drugs that dilate blood vessels; substances that regulate blood lipids, such as statins, cholestyramine, niacin, Saturated fatty acids, etc.; antiplatelet substances, such as aspirin, dipyridamole, ticlide, etc.; thrombolytic substances, such as urokinase, recombinant tissue plasminogen activator, heparin, etc.; other active substances, such as choline, egg Phospholipids, Calamet, vitamin E, carotenoids, vitamin B, vitamin C or coenzyme Q10.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

Figure 1: Inhibition curve of PQQ on copper-induced low-density lipoprotein (LDL) oxidation.

Abscissa: time (unit: minute) for inducing LDL oxidation with copper sulfate;

Ordinate: absorbance of conjugated dienoic acid at 234 nm.

Figure 2: Inhibitory effect of PQQ on peroxide-induced endothelial cell injury (mean ± S.D., n = 4).

Abscissa: different groups;

Ordinate: measured value of MTT experiment;

( ^* : P<0.01, Group 5, Group 6 versus Group 4).

Figure 3: The effect of PQQ on reducing cholesterol content in arteries of atherosclerotic rat models (mean ± S.D., n = 6).

Abscissa: different groups;

Ordinate: cholesterol content in the aorta;

( ^** : P<0.01, group 3, group 4 compared with group 2; ##: P<0.01, group 2 compared with group 1).

Figure 4: Effect of PQQ on the area of sclerotic plaque in rat atherosclerosis model (mean ± S.D., n = 6).

Abscissa: different groups;

Vertical axis: ratio of atherosclerotic plaque area to aortic sinus area;

( ^** : P<0.01, group 3, group 4 compared with group 2; ##: P<0.01, group 2 compared with group 1).

Detailed ways

The present invention is based on the following research results: pyrroloquinoline quinone (PQQ) can inhibit the oxidation of low-density lipoprotein, protect endothelial cells, and in a rat model of atherosclerosis, can reduce the lipid content and atheroma in the endothelium. The area of hardened plaque. Therefore, PQQ has good application value in preventing and/or treating the formation and development of atherosclerosis.

related definition

Herein, the term "comprising" means that various ingredients can be used together in the composition of the present invention. Accordingly, the terms "consisting essentially of" and "consisting of" are included in the term "comprising".

Herein, the term "effective amount" refers to the amount of ingredients sufficient to obtain the desired effect without undue adverse side effects (such as toxicity, irritation and allergic reaction), ie, a reasonable benefit/risk ratio when used in the manner of the present invention. Obviously, the specific "effective amount" varies with various factors, such as the age and condition of the subject to be administered.

The Structure and Known Functions of Pyrroloquinoline Quinone (PQQ)

Herein, the term "pyrroloquinoline quinone" or "PQQ" refers to pyrroloquinoline quinone or a salt or ester thereof, representative salts include (but not limited to): sodium salt, potassium salt, zinc salt, and the like. Herein, the terms "PQQ active substance" or "pyrroloquinoline quinone active substance" are used interchangeably, and both include pyrroloquinoline quinone, nutraceutical, physiologically or pharmaceutically acceptable salts, esters, hydrated compounds, solvates, isomers or other derivative forms, or mixtures thereof. The term "PQQ derivative" refers to a product containing pyrroloquinoline quinone as a main structure or obtained by metabolism of pyrroloquinoline quinone as long as it also has the effect of pyrroloquinoline quinone in the present invention.

As stated in the background art section: PQQ exists in the daily diet, is widely distributed in various tissues and organs of the human body, and exists in breast milk. Moreover, it has also been identified as a coenzyme for redox reactions, has vitamin activity, and may be a new type of B vitamin.

Therefore, PQQ is a natural compound for the human body, has no toxic and side effects, and ensures its drug safety. Moreover, the prevention and/or treatment of atherosclerosis by supplementing PQQ can also produce positive effects such as liver protection and nervous system protection.

Not limited to the mechanism, PQQ may prevent and/or treat atherosclerosis through a variety of main mechanisms: (1) inhibit the oxidation of low-density lipoprotein; (2) protect endothelial cells; (3) reduce the lipid content in the endothelium and (4) reducing the size of atherosclerotic plaque.

combination

The present invention provides a composition, which comprises: (a) pyrroloquinoline quinone, a pharmaceutically or physiologically acceptable salt and/or ester thereof; and (b) a pharmaceutically or physiologically acceptable carrier.

The composition in the present invention can be health product or medicine, which can be used for effective prevention and/or treatment of atherosclerosis. Usually, when the composition is a health care product, the dose of PQQ contained therein is lower than that of the pharmaceutical composition.

As used herein, the term "pharmaceutically or physiologically acceptable carrier" refers to a carrier used in health care products or pharmaceutical preparations, including various excipients and diluents, which are not essential active ingredients themselves, and have no or without undue toxicity. Suitable vectors are well known to those of ordinary skill in the art. For example, pharmaceutically acceptable carriers are described in detail in "Remington's Pharmaceutical Sciences" (Mack Pub. Co., N.J. 1991).

Pharmaceutically acceptable carriers in compositions can contain liquids such as water, saline, glycerol and ethanol. In addition, there may also be auxiliary substances in these carriers, such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting agents or emulsifiers, flavoring agents, pH buffering substances, and the like. Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably at a pH of about 6-8.

The active substance (a) in the composition of the present invention accounts for 0.001-99.9wt% of the total weight of the composition; preferably 1-95wt%, more preferably 5-90wt%, more preferably 10-80wt%. The balance is substances such as carriers and other additives.

In another preferred embodiment of the present invention, the composition is in unit dosage form or in multiple dosage form. As used herein, the term "unit dosage form" refers to the preparation of the composition of the present invention into a dosage form required for single administration or use for the convenience of taking or use, including but not limited to various solid dosage forms (such as tablets), liquid dosage forms, etc. Doses, Capsules, Sustained Release Formulations.

In another preferred embodiment of the present invention, 1-6 doses of the composition of the present invention are administered per day, preferably 1-3 doses are administered; when the dose is high, it is preferred to take 1 dose per day. In a preferred example of the present invention, 0.001-10 mg/kg body weight of the composition of the present invention is administered to the subject every day, preferably 0.01-5 mg/kg body weight, more preferably 0.01-1 mg/kg body weight.

It is to be understood that the effective dose of the active substance used can be determined according to the individual condition of the subject in need of prophylaxis or treatment (e.g. subject's weight, age, physical condition, desired effect), which is within the judgment of a skilled physician or nutritionist Inside.

The composition of the present invention can be solid (such as granules, tablets, freeze-dried powder, suppository, capsule, sublingual tablet) or liquid (such as oral liquid, solution or syrup) or other suitable forms.

medicine box or health care box

The present invention also provides a medicine box or health care box, which contains: (A) a container containing an effective amount of pyrroloquinoline quinone, its pharmaceutically or physiologically acceptable salt and/or ester; and (B) A container containing an effective amount of an acetylcholinesterase inhibitor. The medicine box or health care box is used to improve memory and cognitive ability, prolong the life expectancy of the subject, and reduce the production of β-amyloid protein in the brain, thereby preventing or treating senile degenerative neurological diseases, preferably for preventing or treating senile degenerative neurological diseases. dementia.

As used herein, the term "health kit" refers to a combination of containers containing the active substance PQQ that can provide a subject with a health care effect, which can be provided to a subject in need as a health care product.

Application method

The composition, medicine kit or healthcare kit of the present invention can be administered by conventional routes, including (but not limited to): oral administration, spraying, intranasal, or transdermal routes. Oral or injectable administration is preferred. The form of the composition should match the mode of administration. The dosage of the composition of the present invention is usually about 0.001-10 mgPQQ/kg body weight per day, preferably about 0.01-5 mgPQQ/kg body weight, preferably 0.01-1 mgPQQ/kg body weight, based on the weight of the active substance PQQ.

The composition, medicine kit or health care box of the present invention can be used directly, and can also be combined with other substances for preventing or treating atherosclerosis (such as 0.0005-0.1 gram/kg body weight/day; preferably 0.001-0.05 gram/kg body weight/day ) used in combination or in combination. These substances include, but are not limited to: dilating blood vessels, regulating blood lipids, antiplatelets, dissolving thrombus and anticoagulant substances, preferably isosorbide mononitrate, statins, cholestyramine, niacin, unsaturated fatty acids, aspirin, dipyridamole , Ticlide, etc., urokinase, recombinant tissue-type plasminogen activator, heparin, etc.

The composition of the present invention can also be combined with other methods or means commonly used in the treatment or prevention of atherosclerosis besides drug therapy. These methods or means include but are not limited to: surgical treatment, acupuncture, reasonable diet, etc.

When these substances or methods are administered in combination or in conjunction with PQQ, it is preferable to have an effect superior to that of administering these substances or methods alone.

Advantages of the invention

Advantages of the present invention include but are not limited to:

(a) It was discovered for the first time that PQQ can inhibit the oxidation of low-density lipoprotein, protect endothelial cells, reduce the lipid content in endothelium and reduce the area of atherosclerotic plaque, so it can be used to prevent or treat atherosclerosis;

(b) PQQ exists in the living body, is a natural compound, and has no toxic and side effects on the human body, so the use of PQQ to prevent or treat atherosclerosis has high safety;

(c) The addition of PQQ can also supplement the endogenous pyrroloquinoline quinone in the body, and alleviate the symptoms caused by the lack of pyrroloquinoline quinone in the body.

Example

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, usually follow the conventional conditions or the conditions suggested by the manufacturer. Percentages and parts are by weight unless otherwise indicated. Cell incubation conditions were 37°C, 5% CO ₂ unless otherwise stated.

Unless otherwise defined, all professional and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. In addition, any methods and materials similar or equivalent to those described can also be applied in the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

Example 1: Inhibitory Effect of PQQ on Low Density Lipoprotein (LDL) Oxidation

experimental method:

1. Preparation of LDL: Take fresh plasma from healthy adults (obtained from volunteer donations), obtain an EDTA-containing LDL solution by density gradient sequence ultracentrifugation (reference 5: Lindgren, Jensen et al. 1969), and dissolve the solution in PBS The solution (pH7.5) was dialyzed and desalted (4°C, 4 hours), the resulting LDL solution was diluted to 200 mg/L, and the protein content was determined according to the Lowry method;

2. LDL oxidation experiment: Dilute the LDL solution obtained in step 1 with 5 μM CuSO ₄ to a final concentration of 15 μg/ml to induce LDL oxidation (Reference 1: Esterbauer, Striegl et al., 1989). 25 μg/ml and 50 μg/ml of PQQ were added to the reaction system, and the LDL oxidation curve was drawn by measuring the absorbance of conjugated dienoic acid at 234 nm at different time points (UV spectrophotometer, BECKMAN, DU 640).

Experimental results and analysis:

The oxidation curve of LDL is shown in Figure 1.

The results showed that compared with the control group, PQQ could significantly inhibit the oxidation of LDL, and the inhibitory effect was more obvious at high doses.

The above results show that: PQQ can inhibit the oxidation of LDL in vitro, and the effect is more significant at high doses.

Example 2: The protective effect of PQQ on endothelial cells

experimental method:

1. Cells and culture conditions: Under sterile conditions, the umbilical cords of newborn fetuses (obtained from Shanghai Obstetrics and Gynecology Hospital) were taken immediately after delivery from healthy puerperas, digested with 0.1% collagenase, and the obtained human umbilical vein endothelial cells were prepared according to Conventional adherent cell culture method for subculture (reference 4: Jaffe, Nachman et al. 1973);

The composition of the cell culture medium is: 1 g of DMEM medium, 0.36 g of NaHCO ₃ , 1000 u each of penicillin and streptomycin, 10 mL of fetal bovine serum, and add water to 100 mL. Cultivate in 37°C, 5% CO ₂ incubator, change the medium after 24 hours, and subculture and digest when the cell density is about 80%, and take the 2-3 passage cells for experiments;

2. PQQ pretreatment: culture umbilical vein endothelial cells in a 96-well plate, add PQQ to the culture medium to a final concentration of 50 μg/ml and 100 μg/ml, and incubate at 37°C for 30 minutes;

3. H ₂ O ₂ treatment of endothelial cells: add H 2 O 2 to the cell culture medium of PQQ pretreatment and control group to a final concentration of 1 mM, and conduct subsequent experiments after 90 minutes of treatment;

4. MTT method to detect the protective effect of PQQ on human umbilical vein endothelial cells:

MTT detection was performed according to the method described in the literature (reference 3: Hansen, Nielsen et al. 1989), and the damage degree and growth state of the cells were judged by measuring the activity of mitochondrial dehydrogenase. After the cells in the 96-well plate were treated with H ₂ O ₂ for 90 minutes, 50 μl of MTT (1 mg/ml) was added to each well, and cultured at 37°C for 4 hours. The absorbance at 490 nm was read on an instrument (Molecular Devices, SpectraMax Plus384).

Experimental results and analysis:

The detection results of the MTT method are shown in Figure 2, wherein group 1 is the control group, group 2 is the simple PQQ low-dose group (final concentration 50 μg/ml), and group 3 is the simple PQQ high-dose group (final concentration 100 μg/ml) , group 4 was the injury group (adding final concentration of 1mM H ₂ O ₂ ), group 5 was the PQQ low-dose group (same as the injury group, adding PQQ with a final concentration of 50 μg/ml in advance), and group 6 was the PQQ high-dose group (same as the injury group) group, pre-added PQQ with a final concentration of 100 μg/ml).

It can be seen from the figure that there is no significant difference among group 1, group 2 and group 3 (P>0.05), but there is a significant difference between group 5, group 6 and group 4 (*, P<0.01).

The above results showed that: PQQ can significantly inhibit the damage of endothelial cells caused by peroxide.

Example 3: The effect of PQQ on reducing the content of cholesterol in the arteries of atherosclerosis rat model

experimental method:

1. Take several SD male rats aged 6 weeks and weighing about 220g, and raise them under standard conditions;

2. The rats were randomly divided into 4 groups, 6 in each group. The groups are as follows:

Group 1: Control group: ordinary SD rats fed with feed;

Group 2: High cholesterol diet group (HCD);

Group 3: High cholesterol plus low-dose PQQ group, PQQ was given with meals.

Group 4: High cholesterol plus high-dose PQQ group, PQQ was given with meals.

Wherein, the composition of high cholesterol diet in group 2-group 4 is: 2% cholesterol, 3% lard, 0.5% bile acid and 0.2% propylthiouracil, add in common feed;

The oral dose of PQQ in the low-dose PQQ group was: 0.5 mg/kg/d;

The oral dose of PQQ in the high-dose PQQ group was: 1.0 mg/kg/d.

3. Except for the control group, each group of rats was orally administered vitamin D2 at a dose of 300,000 IU/kg/day to induce the formation of atherosclerotic plaques;

4. After feeding for 12 weeks, measure the cholesterol content in the rat aorta tissue (reference 2: Gamble, Vaughan et al. 1978):

The aorta of each group of rats was taken, and the adipose tissue and other connective tissues outside the aortic wall were removed with ophthalmic forceps, washed with PBS and weighed. Put the aorta into a centrifuge tube, add 2ml homogenate, homogenate at 4°C, centrifuge at 1700g for 5min, transfer the supernatant to another clean test tube, add 0.4ml 8.8g/L KCl solution, make the supernatant Divided into water layer and fat layer. Transfer the lipid layer to another centrifuge tube, dry it with nitrogen at 37°C, add 0.2ml of absolute ethanol to each tube, and store it at -20°C until testing.

Cholesterol content was determined by enzymatic method, 0.1ml sample was added to 0.9ml cholesterol detection analysis solution (0.1U/ml cholesterol oxidase, 1U/ml peroxidase, 0.01U/ml cholesterol esterase, 0.5ml/L TritonX- 100, 1mM sodium cholate, 0.6mg/ml mandelic acid, dissolved in 0.1M sodium phosphate buffer at pH 7.4), incubated at 37°C for 1h, and detected by a fluorescence spectrophotometer (Shimadzu, AA-670, Kyoto) Fluorescence intensity, the excitation wavelength for measuring the fluorescence intensity is 325nm, and the emission wavelength is 410nm. The slit width is 10nm for excitation and 5nm for emission.

Experimental results and analysis:

The cholesterol content in the aorta of the control group and each experimental group of atherosclerosis model rats is shown in FIG. 3 .

The results showed that: compared with the control group 1, the cholesterol content in the aorta of the model rats (group 2) given a high-cholesterol diet and induced by vitamin D2 was significantly increased (##, P<0.01); compared with the group 2, The administration of PQQ (group 3, group 4) significantly reduced the cholesterol content in the arteries of rat models of atherosclerosis (**, P<0.01), and the effect was more obvious at high doses.

The above results showed that: PQQ can effectively inhibit the uptake of cholesterol in arteries.

Example 4: The effect of PQQ on reducing the area of sclerotic plaque in a rat model of atherosclerosis

experimental method:

1. As the method shown in embodiment 3, SD rat is divided into 4 groups: control group (normal feeding group, group 1), high-cholesterol diet group (group 2), high cholesterol plus low-dose PQQ group (group 3), high cholesterol plus high-dose PQQ group (group 4);

2. After 12 weeks of feeding, the rats were sacrificed, the aortic atherosclerotic plaque was observed under a microscope, and its area was calculated (Reference 6: Paigen, Morrow et al. 1987). The oil red staining method was used to make the atherosclerotic plaque appear red. Fully automatic image analysis system (digital camera: model Penguin 600CL, Pixera Inc., USA; Leica automatic microscope: model DMLA, Leica Inc., Germany; software Simple PCI: v5.2, Compix Inc., USA) analyzed each The aortic sinus atherosclerotic plaque area and the aortic sinus lumen area of the rats in the experimental group were expressed by the ratio of the atherosclerotic plaque surface/lumen area, and the size of the atherosclerotic plaque in each mouse was expressed by The average value was used to represent the size of the atherosclerotic plaque area in each group.

Experimental results and analysis:

The ratio of atherosclerotic plaque area/lumen area in the arteries of rats in the control group and each test group is shown in FIG. 4 .

Compared with control group 1, the area of atherosclerotic plaque in the aorta of model rats given high cholesterol diet (group 2) and induced by vitamin D2 was significantly increased (##, P<0.01); Compared with (group 2), the administration of PQQ significantly reduced the area of atherosclerotic plaque in the arteries of the atherosclerotic rat model, and the effect was more obvious at high doses (**, P<0.01).

The above results show that: PQQ can significantly inhibit the area of atherosclerotic plaque induced by high-fat diet.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

references

1. Esterbauer, H., G. Striegl et al. (1989). Continuous monitoring of in vitro oxidation of human low density lipoprotein, "Continuous monitoring of in vitro oxidation of human low density lipoprotein." FreeRadic Res Commun 6(1):67-75;

2. Gamble, W., M. Vaughan et al. (1978). Method for determination of free and total cholesterol in micro-or nanogram amounts suitable for studies with cultured cells. "Procedure for determination of free and total cholesterol in micro-or nanogram amounts suitable for studies with cultured cells."JLipid Res 19(8) :1068-70;

3. Hansen, M.B., S.E. Nielsen et al. (1989). "Re-examination and further development of aprecise and rapid dye method for measuring cell growth/cell kill." J Immunol Methods, 119(2): 203 -10;

4. Jaffe, E.A., R.L. Nachman et al. (1973). Culture of human endothelial cells derived from umbilical veins, "Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunological criteria." JClin Invest 52(11): 2745-56;

5. Lindgren, F.T., L.C. Jensen et al. (1969). Flotation rates, molecular weights and hydrated densities of the low-density lipoproteins. "Lipids 4(5): 337-44;

6. Paigen, B., A. Morrow et al. (1987). Quantitative assessment of atherosclerotic lesions in mice, "Quantitative assessment of atherosclerotic lesions in mice." Atherosclerosis 68(3):231-40.

Claims (5)

1. Application of pyrroloquinoline quinone active substance in the preparation of a composition for treating and/or preventing atherosclerosis or its symptoms in a subject, wherein the symptoms of atherosclerosis are selected from the group consisting of: atherosclerotic plaque thrombosis Peripheral arterial disease caused by, or atherosclerosis. 2. The application according to claim 1, wherein the composition is a health product composition or a pharmaceutical composition. 3. The use of claim 1, wherein the subject is a mammal. 4. The application according to claim 1, characterized in that the pyrroloquinoline quinone active substance accounts for 0.001-99.9wt% of the total weight of the composition. 5. The application according to claim 1, wherein the dosage form of the composition comprises: tablet, powder, injection, granule, syrup, capsule, solution, suppository or ointment.

Images