CN116617335A - A compound traditional Chinese medicine composition for preventing and treating vascular aging, its preparation method and application - Google Patents

Abstract

The invention discloses a traditional Chinese medicine compound composition for preventing and treating vascular aging, a preparation method and application thereof. The composition can effectively improve vascular aging, regulate lipid metabolism, reduce oxidative stress level, and improve pathological morphology and vascular homeostasis of aorta on the basis of preventing and treating atherosclerosis.

Description

A compound traditional Chinese medicine composition for preventing and treating vascular aging, its preparation method and application

technical field

The invention relates to the field of traditional Chinese medicine, in particular to a compound traditional Chinese medicine composition for preventing and treating vascular aging, a preparation method and application.

Background technique

The aging of blood vessels is an important link between aging and senile diseases. As an important link in the aging process of the human body, it accelerates the aging process of the body. Vascular aging (VA) refers to the degenerative changes in the structure and function of blood vessels due to aging, which is manifested in microvascular degeneration, decreased blood vessel density, disordered arrangement of smooth muscle cells, thickened intima and media, and luminal changes. Enlargement, increased collagen and decreased elastin deposition; functionally manifested as decreased angiogenesis, increased vascular stiffness, decreased diastolic sensitivity, and increased systolic sensitivity. VA not only increases the independent risk of cardiovascular diseases such as coronary heart disease and hypertension, but also plays an important role in the development and prognosis of neurodegenerative diseases and other cerebrovascular diseases, and is the driving factor of many age-related vascular diseases.

Aging is the main risk factor for the occurrence of atherosclerotic cardiovascular and cerebrovascular diseases. According to statistics, age-related atherosclerotic cardiovascular and cerebrovascular diseases account for nearly half of the global deaths. Vascular aging, which is closely related to age, is inextricably related to atherosclerosis. The two often coexist and share a common pathogenesis and pathological mechanism. Lipid deposition in atherosclerosis can damage the vascular endothelium, leading to an increase in pro-inflammatory factors and adhesion molecules in blood vessels, promoting vascular endothelial dysfunction, causing intima-media thickening, elastin rupture, collagen deposition, vasodilation and The increase of the inner diameter further reduces the elasticity of blood vessels and increases the hardness of blood vessels, thereby promoting the aging of blood vessels.

Atherosclerosis (AS) is a diffuse, relatively slowly progressive disease characterized by deposition of fat and/or fibrous tissue in the arterial intima and asymmetric thickening of the arterial vessel wall. It is the initiating link in the progression of cardiovascular and cerebrovascular diseases such as ischemic cardiomyopathy, myocardial infarction, and stroke. AS is caused by the joint interference of genetic factors and environmental factors. While its formation is affected by vascular cell aging, its occurrence and development will also affect and accelerate the aging process.

Traditional Chinese medicine believes that aging is closely related to meridian qi and blood, "the owner of a person is blood and qi and ears", and the maintenance of human life depends on the sufficiency and smooth flow of meridian qi and blood. If the "blood vessels are in good harmony", severe diseases will not be covered, and "spirit will live"; if the Qi and blood are deficient and the meridians are blocked, the normal physiological functions of the human body cannot be maintained, thereby accelerating aging. Vascular aging is mainly reflected in the abnormal structure and function of the vessel wall. With age, viscera are weakened, qi and blood are deficient, and collaterals are empty, so that the channels cannot be nourished normally; blood circulation is unfavorable, and the effect of collaterals "moisturizing bones and joints" decreases accordingly. "Incipient disease lies in the meridian, long-term disease lies in the collaterals", according to the meridian theory, vascular aging angelica belongs to the category of "collateral disease".

The traditional Chinese medicine compound is composed of ginseng extract, hawthorn extract, polygonum cuspidatum extract and red yeast rice powder. Among them, ginseng nourishes the deficiency and is a medicine for "replenishing the five internal organs and eliminating evil spirits". Blood stasis is a minister drug; Polygonum cuspidatum detoxifies and activates blood circulation is an adjuvant medicine; achievement.

Ginseng (Panax ginseng C.A.Mey), a perennial herb of the genus Araliaceae, Araliaceae, has the effects of invigorating qi and solidifying, invigorating the spleen and lungs, calming the mind and intelligence, nourishing blood and promoting body fluid; Polygonum cuspidatum Sieb.etZucc .), the dry rhizomes and roots of Polygonaceae plants have the functions of promoting dampness and reducing jaundice, clearing heat and detoxification, dissipating blood stasis and relieving pain, relieving cough and reducing phlegm; Hawthorn (Crataegus pinnatifida Bunge), also known as Shanliguo and Shanlihong, belong to the Rosaceae Hawthorn genus, It has the effects of digesting food and invigorating the stomach, promoting blood circulation and removing blood stasis, astringent and stopping dysentery; red yeast rice is a kind of red yeast rice made by parasitizing the mycelium of the fungus Monascus purpureus on the japonica rice. Branch, has the effects of promoting blood circulation and removing blood stasis, invigorating the spleen and eliminating food. Modern pharmacological studies have proved that ginsenoside saponin, the main component of ginseng, exerts anti-AS and anti-aging effects by enhancing GPX content, enhancing SOD activity, and reducing malondialdehyde content. The main component of hawthorn is flavonoid organic acid, which can increase the content of HDL. It has significant preventive and therapeutic effects on AS; it can also reduce TC, TG and CRP, and play an anti-AS role through anti-platelet aggregation. The alcohol extract of hawthorn and the total flavonoids of hawthorn can delay aging by resisting oxidative stress and increasing SOD activity. Polydatin in Polygonum cuspidatum can reduce the expression levels of TG, LDL and malondialdehyde in rats to play an anti-AS effect; another component in Polygonum cuspidatum, resveratrol, can activate SIRT, activate AMPK for anti-oxidation, and inhibit inflammation to inhibit aging . Red yeast rice plays an anti-AS role by reducing blood lipid and anti-VSMCs proliferation.

In view of this, the present invention is proposed.

Contents of the invention

The object of the present invention is to provide a traditional Chinese medicine compound composition for preventing and treating vascular aging and its preparation method and application, which can reduce blood lipids and prevent and treat atherosclerosis. In addition, it can also effectively improve vascular aging, and has the functions of regulating lipid metabolism, Reduce the level of oxidative stress and improve the function of pathological morphology of the aorta.

In order to solve the above problems, in the first aspect, the embodiment of the present invention provides a compound traditional Chinese medicine composition for preventing and treating vascular aging, including:

Ginseng extract, hawthorn extract, knotweed extract and red yeast rice powder;

Among them, the mass ratios of ginseng extract, hawthorn extract, knotweed extract and red yeast rice powder are as follows:

(6-12):(6-12):(6-12):(3-9).

Optionally, the mass ratio of ginseng extract, hawthorn extract, knotweed extract and red yeast rice powder is 9:9:9:6 in sequence.

Optionally, the mass content of total ginsenosides in the ginseng extract is 85-95%.

Optionally, the mass content of the total flavonoids of hawthorn in the hawthorn extract is 10-30%,

Optionally, the mass content of polydatin in the Polygonum cuspidatum extract is 95-99%.

Optionally, the mass content of lovastatin in the red yeast rice powder is 3-7%.

Optionally, the mass content of total ginsenosides in ginseng extract is 90%, the mass content of total hawthorn flavonoids in hawthorn extract is 20%, the mass content of polydatin in Polygonum cuspidatum extract is 98%, and the mass content of lovastatin in red yeast rice powder The mass content is 5.09%.

In the second aspect, the embodiment of the present invention provides a preparation method of the above-mentioned traditional Chinese medicine compound composition for preventing and treating vascular aging, including:

Ginseng, Hawthorn, Polygonum cuspidatum slices washed and dried;

Ginseng decoction pieces are cut into granules, ultrasonically crushed, put into a container, add water, and distill and extract under the conditions of temperature control 65°C-75°C and vacuum degree control 0.06mpa-0.07mpa. The time is 80-100min; the distilled liquid reaches the predetermined amount Collect; continue to add water, distill and extract again, distill out a predetermined amount of liquid, combine the two, concentrate the liquid, cool to normal temperature, and dry into powder to obtain the extract of ginseng;

Wash the hawthorn decoction pieces and put them in the container, then decoct for 20-40min, filter the precipitate; then add water to the filter residue, decoct for 10-30min again, and filter the precipitate again; combine the two decoctions, concentrate the liquid, and cool to room temperature; The polyamide resin column is eluted with deionized water and ethanol solution, the ethanol is recovered under reduced pressure, the pH value is adjusted to 6.0-6.5, and dried into powder to obtain the hawthorn extract;

The rhizome of Polygonum cuspidatum is dried and crushed, soaked in 60%-70% ethanol at room temperature, and the ethanol extract is concentrated to 1/3-1/7 of the original volume; the concentrated solution is cooled to room temperature and then filtered to remove impurities; The filtrate passes through a short and thick neutral alumina column, collects the effluent from the upper column, concentrates to no alcohol, cools at room temperature, and dries into powder to obtain the extract of Polygonum cuspidatum;

In the above steps, the extraction of ginseng, hawthorn, and knotweed three-flavored medicinal decoction pieces can also use supercritical fluid extraction; supercritical fluid extraction technology is a new type of separation technology in modern chemical industry separation, supercritical CO ₂ extraction is the use of CO ₂ As a solvent, the fluid density and dielectric constant of _CO2 in the supercritical state are large, and the solubility of the substance is very large, and it changes sharply with the change of pressure and temperature. Therefore, it is not only selective for the solubility of certain substances, but also Solvents and extracts are very easy to separate. Supercritical _CO2 extraction is especially suitable for the extraction of fat-soluble, high boiling point, and heat-sensitive substances, and it is also suitable for the fine separation of different components, that is, supercritical rectification.

The ginseng extract, the hawthorn extract, the knotweed extract and the pre-prepared red yeast rice powder are compounded in proportion to obtain the traditional Chinese medicine compound composition for preventing and treating vascular aging.

Optionally, the distillation extraction time of the ginseng decoction pieces is 90 minutes; the hawthorn decoction pieces are decocted for 30 minutes for the first time, and then decocted for 20 minutes again; the ethanol extract of the knotweed decoction pieces is concentrated to 1/5 of the original volume.

In the third aspect, the embodiments of the present invention also provide the application of the above-mentioned traditional Chinese medicine compound composition in the preparation of drugs for preventing and treating vascular aging.

Compared with the prior art, the present invention has the following beneficial effects: on the basis of preventing and treating atherosclerosis, the composition can also effectively improve vascular aging, regulate lipid metabolism, reduce oxidative stress levels, and improve aortic Function of Pathological Morphology.

Description of drawings

Fig. 1 is the arch tissue HE staining figure (I is 100 times, II is 200 times, A is a normal control group, D is a model group, E is Western medicine group, F is the low-dose Chinese medicine group, G is the high-dose Chinese medicine group);

Fig. 2 is the Masson staining diagram (I is 40 times, II is 400 times, A is the normal control group, D is the model group, and E is the arch tissue Masson staining figure of the mouse aorta pathological test of the Chinese medicine compound composition provided by the embodiment of the present invention. Western medicine group, F is the low-dose Chinese medicine group, G is the high-dose Chinese medicine group);

Fig. 3 is the experimental result diagram of the influence of the traditional Chinese medicine compound composition provided by the embodiment of the present invention on the blood lipid of ApoE ^-/- mice;

Fig. 4 is the experimental result diagram of the influence of the traditional Chinese medicine compound composition provided by the embodiment of the present invention on the oxidative stress index of ApoE ^-/- mice;

Fig. 5 is the experimental result diagram of the influence of the traditional Chinese medicine compound composition provided by the embodiment of the present invention on ApoE ^-/- mouse vascular aging-related indicators;

Fig. 6 is two kinds of traditional Chinese medicine compound compositions that the embodiment of the present invention provides to the arch tissue Masson staining comparison figure of mouse aortic pathology test (I is 40 times, II is 400 times, a is the normal control group, b is the model group , c is the rosuvastatin group, d is the two-drug compound group, e is the four-drug group);

Fig. 7 is the experimental result diagram of the influence of two kinds of traditional Chinese medicine compound compositions provided by the embodiment of the present invention on blood lipids of mice;

Fig. 8 is a graph showing the experimental results of the effects of two traditional Chinese medicine compound compositions on oxidative stress indicators in ApoE ^−/− mice provided in the embodiment of the present invention.

Detailed ways

The principle and spirit of the present invention will be described below with reference to several exemplary embodiments shown in the accompanying drawings. It should be understood that these implementations are described only to enable those skilled in the art to better understand and implement the present invention, but not to limit the scope of the present invention in any way.

The embodiment of the present invention provides a traditional Chinese medicine compound composition for preventing and treating vascular aging and its preparation method and application. The compound composition is composed of ginseng extract, hawthorn extract, knotweed extract and red yeast powder. During preparation, the decoction pieces of ginseng, hawthorn and knotweed can be weighed in proportion (9:12:9), washed and dried. Then, distilled extraction, water extraction, alcohol extraction and other methods can be used to extract the main active ingredients of ginsenosides, hawthorn flavonoids, and polydatins, and then compounded with commercially available red yeast powder in proportion (6) Formed, that is to get the Chinese medicine compound composition, the Chinese medicine compound composition (ginseng, hawthorn, Polygonum cuspidatum, red yeast decoction pieces, the ratio is 9:12:9:6) when preparing the medicine for preventing and treating vascular aging, you can add pharmaceutically Accepted excipients to prepare medicines in corresponding dosage forms.

Example 1

In this example, the Chinese medicine compound composition is composed of ginseng extract (total ginsenosides, content 90%), hawthorn extract (hawthorn total flavonoids, content 20%), Polygonum cuspidatum extract (polygonatin, content 98%), red yeast rice Micropowder (lovastatin, content 5.09%) is composed of mass ratio 9:9:9:6, provided by Xi'an Kailai Bioengineering Co., Ltd., production batch number: 20201220.

Example 2

In this example, the Chinese medicine compound composition is composed of ginseng extract (total ginsenosides, content 85%), hawthorn extract (hawthorn total flavonoids, content 10%), Polygonum cuspidatum extract (polygonadin, content 95%), red yeast rice Micropowder (lovastatin, content 3%) is composed of mass ratio 12:12:12:9, provided by Xi'an Kailai Bioengineering Co., Ltd., production batch number: 20201220.

Example 3

In this example, the Chinese medicine compound composition is composed of ginseng extract (total ginsenosides, content 95%), hawthorn extract (hawthorn total flavonoids, content 30%), Polygonum cuspidatum extract (polygonadin, content 99%), red yeast rice Micropowder (lovastatin, content 7%) is composed of mass ratio 6:6:6:3, provided by Xi'an Kailai Bioengineering Co., Ltd., production batch number: 20201220.

Example 4

This example is the preparation method of the composition in Example 1, which specifically includes the following steps: cut ginseng into granules, ultrasonically pulverize it, put it into a container and add water, control the temperature at 65°C-75°C, and control the degree of vacuum at 0.06mpa-0.07mpa Distillation extraction under the condition of 90min. When the distilled liquid is about 1/2, collect; continue to add 1/2 of the water, distill and extract again, distill 1/2 of the liquid, combine the two, concentrate the liquid, cool to room temperature, and dry into powder , to obtain the extract of ginseng (the main active ingredient is ginsenosides).

Hawthorn decoction pieces, washed and put into a container, then decocted for 30 minutes, filtered and precipitated. Then add water to the filter residue, decoct for 20 minutes again, and filter the precipitate again. Combine the two decoctions, concentrate the liquid, and cool to room temperature. Pass through a polyamide resin column (the main active ingredient is hawthorn flavonoids), elute with deionized water, elute with ethanol solution, recover ethanol under reduced pressure, adjust the pH value to 6.0-6.5, and dry it into powder to obtain the hawthorn extract. thing.

The rhizomes of Polygonum cuspidatum are dried and crushed, soaked in 60%-70% ethanol at room temperature, and the ethanol extract is concentrated to 1/5 of the original volume; the concentrated solution is cooled to room temperature and then filtered to remove impurities; the filtrate is passed through neutral A short and thick column of alumina (the main active ingredient is polydatin), collect the effluent from the upper column, concentrate to no alcohol, let it cool at room temperature, and dry it into powder to obtain the extract of Polygonum cuspidatum.

The red yeast rice is purchased commercially, which consists of ginseng extract, hawthorn extract, knotweed extract, and red yeast powder (9:9:9:6).

Example 5

The difference between this example and Example 4 is that the distillation and extraction time of ginseng decoction pieces is 80 minutes, the decoction time of hawthorn decoction pieces is 40 minutes for the first time, and 30 minutes for the second time, and the ethanol extract of Polygonum cuspidatum decoction pieces is concentrated to 1/3 of the original volume , and all the other process parameters are the same as in Example 4.

Example 6

The difference between this example and Example 4 is that the distillation and extraction time of ginseng decoction pieces is 100 minutes, the decoction time of hawthorn decoction pieces is 20 minutes for the first time, and 10 minutes for the second time, and the ethanol extract of Polygonum cuspidatum decoction pieces is concentrated to 1/7 of the original volume , and all the other process parameters are the same as in Example 4.

Test example 1

1. Materials and methods

1.1 Experimental animals and feeding

40 SPF grade ApoE ^-/- mice, 10 weeks old, half male and half male; 10 C57BL/6J mice with the same genetic background, 10 weeks old, half male and half male, body weight (18.0±5.0) g , are provided by Beijing Weitong Lihua Experimental Technology Co., Ltd., license number: SCXK (Beijing) 2016-0006. Animals were fed in a barrier-level animal room. Animals were free to eat and drink. The room temperature was controlled at (23-25)°C, the humidity was controlled at 50%-70%, 12 hours of light and 12 hours of darkness. The high-fat diet feed formula is 41% fat + 0.15% cholesterol + 58.85% basic feed, purchased from Beijing Keaoxieli Feed Co., Ltd., license number: SCXK (Beijing) 2019-0003, and the feed is sterilized by ultraviolet irradiation.

1.2 Drugs

The compound Chinese medicine is composed of ginseng extract (ginseng total saponins, content 90%), hawthorn extract (hawthorn total flavonoids, content 20%), Polygonum cuspidatum extract (polygonatin, content 98%), red yeast powder (lovastatin, content 5.09%) according to the mass ratio of 9:9:9:6 (corresponding to Example 1), provided by Xi'an Kailai Bioengineering Co., Ltd., production batch number: 20201220. Rosuvastatin calcium tablets (trade name: Keding, 10mg/tablet) are produced by AstraZeneca Pharmaceuticals (China) Co., Ltd., batch number: H200110563.

1.3 Experimental reagents and experimental equipment

High-density lipoprotein (HDL-C) kit (lot number: 834012), low-density lipoprotein (LDL-C) kit (lot number: 821019), triglyceride (TG) kit (lot number: 824402), total cholesterol (TCHO) kit (batch number: 843321), all of which were purchased from Roche Diagnostics (Germany) Co., Ltd. Oxidized low-density lipoprotein (ox-LDL) ELSIA kit (batch number: 20210316) was purchased from Beijing Xinbosheng Biotechnology Co., Ltd. Glutathione peroxidase (GSH-PX) kit (lot number: 20210512, 20210721) was purchased from Beijing Huaying Institute of Biotechnology. Reactive oxygen species (ROS) kit (batch number: 20210624-1) and nicotinamide adenine dinucleotide (NAD ⁺ ) kit (batch number: 20210719-2) were purchased from Nanjing Jiancheng Institute of Bioengineering. SIRT1 (NAD-dependent sirtuin-1) antibody (lot number: 43810), p21 (cell cycle-dependent protein kinase inhibitor 1A) antibody (lot number: 41939), p53 (human tumor suppressor gene p53) antibody ( Batch number: 822100526), provided by Abcam (UK) Biological Limited.

DR-200BS automatic enzyme label analyzer, produced by Wuxi Huawei Delang Instrument Co., Ltd. Roche/E601 fully automatic electrochemiluminescence immunoassay analyzer was produced by Roche (Switzerland).

1.4 Animal grouping

The experiment was divided into 5 groups, half male and half male. Ten 10-week-old C57BL/6J mice belonged to the normal group (C57 mice + normal diet). 40 10-week-old ApoE ^-/- mice were randomly divided into model group (ApoE ^-/- mice + high-fat diet), western medicine group (rosuvastatin 2.6mg/kg body weight/day + high-fat diet) , the low-dose compound Chinese medicine group in section 1.2 (4.29g/kg body weight/day + high-fat diet), the high-dose compound Chinese medicine group (8.58g/kg body weight/day + high-fat diet) in section 1.2, 10 in each group Rats were fed with corresponding diet and gavaged with corresponding medicine or equal volume of distilled water for 12 weeks. During the animal experiments, the animal ethics requirements of the "Guidelines for the Ethical Review of Experimental Animal Welfare in Beijing" were strictly followed, and were approved by the Animal Ethics Committee of Xiyuan Hospital, China Academy of Chinese Medical Sciences (ethical batch number: 2021XLC007-3).

1.5 Detection of serological indicators

The mice in each group were fed with the corresponding feed or gavaged for 12 weeks, and the samples were collected. The mice were fasted and deprived of water for 12 hours before the samples were collected. After weighing, the mice were anesthetized by intraperitoneal injection of 2% pentobarbital sodium according to the body weight of each mouse. Blood was collected through the orbit, centrifuged at 3000r·min-1, 4°C for 15min, the serum was separated, and the contents of TC, TG, HDL and LDL were detected by an automatic biochemical analyzer.

1.6 Observation of pathological morphology of aorta

After blood collection, the mice were dissected under sterile conditions, the chest cavity and abdominal cavity were quickly opened, the aorta and aortic arch tissue were separated, fixed in 4% paraformaldehyde, serially sliced with a manual rotary microtome with a thickness of 3 μm, and then HE stained and Masson staining. Observe HE-stained aortic arch tissue under 100X and 200X light microscope, and observe Masson-stained aortic arch tissue under 40X and 400X light microscope.

1.7 Determination of ox-LDL content by ELISA method

Strictly operate according to the instructions of the ELISA kit, measure the absorbance (OD) value at a wavelength of 450nm on a microplate reader, and calculate the concentration value of each value.

1.8 Determination of ROS, GSH, GPX, NAD ⁺ content by colorimetry

In strict accordance with the instructions of the colorimetric kit, the contents of ROS, GSH, GPX, and NAD ⁺ in the serum of mice in each group were determined.

1.9 Western Blot detection of vascular aging pathway related proteins SIRT1, p53, p21, NOX4 expression

Select 5 mouse aortic tissue specimens from each group for tissue protein extraction, and biquinolinic acid disodium salt (BCA) for protein quantitative detection; according to the loading requirements of gel electrophoresis (SDS-PAGE), use RIPA and Adjust the protein concentration with 5× reducing sample buffer, boil and denature for 5 minutes; according to the molecular weight of the target protein, prepare 12% separating gel respectively, the concentration of the stacking gel is 5%, electrophoresis conditions: the stacking gel constant pressure 90V, about 20min, the separation gel Constant voltage 160V, use pre-stained protein marker to determine the stop time of electrophoresis; transfer membrane, block, primary antibody incubation: dilute the primary antibody with 3% BSA-TBST, 1:20000 dilution, incubate at room temperature for 10min, put it at 4℃ overnight, wash the membrane , Secondary antibody incubation: add goat anti-rabbit IgG(H+L)HRP or goat anti-mouse IgG(H+L)HRP, 1:10000, shake gently at room temperature for 40min, wash membrane, ECL reaction, film exposure, developing, fixing .

1.10 Statistical Analysis

SPSS 23.0 software was used for statistical analysis, and the obtained data were all subjected to normality test, and the mean ±

ˉ

The standard deviation (x±s) indicates that when the data are normally distributed, one-way analysis of variance is used for comparison between groups, and Tukey’s method is used for pairwise comparison when the variance is homogeneous; Welch analysis of variance is used for uneven variance, and Games is used for pairwise comparison -Howell's method, P<0.05 was considered statistically significant.

2. Results

2.1 Ginseng Yimai Fang (i.e. the Chinese medicine compound composition of the embodiment of the present invention, the same below) on the impact of ApoE ^-/- mice aortic pathomorphology

Combined with Figure 1, HE staining: normal group: the structure of the aortic intima was complete, no plaque and lipid deposition were seen, the smooth muscle strips of the media were arranged in an orderly longitudinal direction, and there was no hyperplasia. Group D: Lipid deposition was seen under the aortic intima, and the intima and media thickened significantly. Compared with group D, in groups E, F, and G, there was a little lipid deposition in the intima, the media was slightly thickened, the structure of each layer was clearer, and the arrangement was more orderly.

Combined with Figure 2, Masson staining: Group A: the structure of the aortic intima is smooth, the media cells are arranged regularly, and the elastic fibers are arranged in an orderly manner. Group D: The thickness of the intima is uneven, fat deposits can be seen, the elastic fibers are significantly reduced, and the collagen fibers are significantly increased. Compared with group D, the media of groups E, F, and G were slightly thickened and the content of collagen fibers was less.

2.2 Effects of traditional Chinese medicine compound on blood lipids in ApoE ^-/- mice

The experimental results showed that compared with the normal group, the TC, TG, HDL, LDL, and ox-LDL indexes of the mice in the model group were all significantly increased (P<0.01); compared with the model group, the TC, TG, ox- The LDL indexes decreased (P<0.05, P<0.01), the ox-LDL in the low-dose Chinese medicine group decreased significantly (P<0.01), and the TC and ox-LDL indexes in the high-dose Chinese medicine group decreased (P<0.01); There was no statistical difference between them (P>0.05). The results are shown in Figure 3.

2.3 Effects of traditional Chinese medicine compound on oxidative stress indicators in ApoE ^-/- mice

Compared with the normal group, the ROS index of the mice in the model group was significantly increased (P<0.01), while the NAD ⁺ , GSH, and GPX indexes were significantly reduced (P<0.01); In the group, ROS decreased significantly (P<0.01), and NAD ⁺ , GSH increased significantly (P<0.05, P<0.01). See Figure 4.

2.4 Effects of traditional Chinese medicine compound on ApoE ^-/- mice vascular aging pathway related indicators

Compared with the normal group, SIRT1 in the model group was significantly decreased (P<0.05, P<0.01), and p21 and p53 indexes were significantly increased (P<0.05, P<0.01); compared with the model group, p21 and NOX4 in the western medicine group were significantly increased (P<0.05, P<0.01), and p53 decreased significantly (P<0.05); p21 index significantly decreased (P<0.01) and NOX4 index significantly increased (P<0.01) in the high-dose Chinese medicine group. See Figure 5.

3. Conclusion

VA is an important pathophysiological basis for the aging of various organ systems in the human body, and its pathological manifestations are decreased compliance and increased stiffness of elastic arteries. AS is an arteriosclerotic disease caused by a variety of risk factors. Its pathology shows lipid deposition on the vascular endothelium, intimal thickening, and fibrous tissue hyperplasia. Both have the same pathological manifestations such as intimal thickening and arterial stiffness increase during the development process. Lipid deposition is the most prominent pathological feature in AS, while the main pathological feature of VA is the imbalance of elastic fibers and collagen fibers in the vessel wall, and the content of collagen fibers increases. By observing HE staining and Masson staining, the Chinese medicine compound low and high dose groups of the embodiment of the present invention can reduce the deposition of lipids in blood vessels and reduce the deposition of collagen fibers in the blood vessel wall, suggesting that the Chinese medicine compound group can inhibit ApoE ^-/- mouse AS and VA development.

Lipid metabolism disorder and hyperlipidemia are the main risk factors of AS. The results of this study show that the blood TC, TG, HDL, LDL, ox-LDL and other indicators of ApoE ^-/- mice are significantly higher than those of the normal control group after being fed with a high-fat diet for 12 weeks. After 12 weeks of intervention in the high-dose group, the blood TC and ox-LDL of ApoE ^-/- mice were significantly reduced, suggesting that the Chinese medicine compound group of the embodiment of the present invention can lower blood lipids and resist vascular aging by reducing the levels of TC and ox-LDL .

Oxidative damage is the main cause of endothelial cell structure and dysfunction, and the damage of endothelial cell structure and function is the key joint in the early occurrence and development of AS and VA. The study found that the ROS level of ApoE ^-/- mice was significantly increased after 12 weeks of high-fat diet feeding, and the GSH and GPX indicators were significantly decreased. The ROS index of the Chinese medicine compound low and high dose groups of the example was significantly reduced after the intervention, and the GSH index was significantly increased, and the Chinese medicine high dose group was better than the rosuvastatin group in reducing ROS, suggesting that the Chinese medicine compound group of the embodiment of the present invention can pass Reduce the ROS level and increase the GSH content in ApoE ^-/- mice, thereby exerting the anti-oxidative stress effect.

Under oxidative stress conditions, NOX4 generates a large amount of ROS to attack mitochondrial DNA, causing DNA oxidative damage, leading to mitochondrial dysfunction. SIRT1 is a protein with NAD ⁺ -dependent deacetylase activity. It is highly expressed on the endothelium and has the functions of regulating vascular endothelium and homeostasis, regulating cell cycle, and regulating aging. It is called a "longevity gene". The p53/p21 signaling pathway is involved in cell cycle regulation, apoptosis and senescence. The results of this experiment show that after 12 weeks of high-fat diet feeding in ApoE ^-/- mice, the expression of NAD ⁺ and SIRT1 decreased significantly, the expression of p53 and p21 increased significantly, and the VA index did not change significantly after the intervention of rosuvastatin. The expressions of NAD ⁺ and NOX4 were significantly increased, and the expression of p21 was significantly decreased after the intervention of the low and high dose groups of traditional Chinese medicine compound in the example, suggesting that the mechanism of the Chinese medicine compound group of the embodiment of the present invention intervening in ApoE ^-/- mice vascular aging may be related to the up-regulation of NAD ⁺ expression, correlated with down-regulation of p21 expression.

In summary, the traditional Chinese medicine compound composition of the embodiment of the present invention can effectively improve the vascular aging of ApoE ^-/- mice, and its mechanism may be related to regulating lipid metabolism, reducing oxidative stress levels, improving aortic pathomorphology, and up-regulating Aortic NAD ⁺ expression was associated with downregulation of p21 expression.

Test example 2

1. Materials and methods

1.1 Experimental animals and feeding

40 SPF grade ApoE ^-/- mice, 10 weeks old, half male and half male; 10 C57BL/6J mice with the same genetic background, 10 weeks old, half male and half male, body weight (18.0±5.0) g , are provided by Beijing Weitong Lihua Experimental Technology Co., Ltd., license number: SCXK (Beijing) 2016-0006. Animals were fed in a barrier-level animal room. Animals were free to eat and drink. The room temperature was controlled at (23-25)°C, the humidity was controlled at 50%-70%, 12 hours of light and 12 hours of darkness. The high-fat diet feed formula is 41% fat + 0.15% cholesterol + 58.85% basic feed, purchased from Beijing Keaoxieli Feed Co., Ltd., license number: SCXK (Beijing) 2019-0003, and the feed is sterilized by ultraviolet irradiation.

1.2 Drugs

The compound traditional Chinese medicine of two herbs is composed of Polygonum cuspidatum extract (polydatin, content 98%), hawthorn extract (hawthorn total flavonoids, content 20%), with a mass ratio of 10:5, provided by Xi’an Kailai Bioengineering Co., Ltd. Production batch number: 20201225.

The four-flavor medicine compound compound Chinese medicine consists of ginseng extract (total ginsenosides, content 90%), hawthorn extract (total flavonoids from hawthorn, content 20%), Polygonum cuspidatum extract (polydatin, content 98%), red yeast rice powder ( Lovastatin, content 5.09%) is composed of mass ratio 9:9:9:6, provided by Xi'an Kailai Bioengineering Co., Ltd., production batch number: 20201220.

Rosuvastatin calcium tablets (trade name: Keding, 10mg/tablet) are produced by AstraZeneca Pharmaceuticals (China) Co., Ltd., batch number: H200110563. .

1.3 Experimental reagents and experimental equipment

High-density lipoprotein (HDL-C) kit (lot number: 834012), low-density lipoprotein (LDL-C) kit (lot number: 821019), triglyceride (TG) kit (lot number: 824402), total cholesterol (TCHO) kit (batch number: 843321), all of which were purchased from Roche Diagnostics (Germany) Co., Ltd. Oxidized low-density lipoprotein (ox-LDL) ELSIA kit (batch number: 20210316) was purchased from Beijing Xinbosheng Biotechnology Co., Ltd. Glutathione peroxidase (GSH-PX) kit (lot number: 20210512, 20210721) was purchased from Beijing Huaying Institute of Biotechnology. Reactive oxygen species (ROS) kit (batch number: 20210624-1) and nicotinamide adenine dinucleotide (NAD ⁺ ) kit (batch number: 20210719-2) were purchased from Nanjing Jiancheng Institute of Bioengineering. SIRT1 (NAD-dependent sirtuin-1) antibody (lot number: 43810), p21 (cell cycle-dependent protein kinase inhibitor 1A) antibody (lot number: 41939), p53 (human tumor suppressor gene p53) antibody ( Batch number: 822100526), provided by Abcam (UK) Biological Limited.

DR-200BS automatic enzyme label analyzer, produced by Wuxi Huawei Delang Instrument Co., Ltd. Roche/E601 fully automatic electrochemiluminescence immunoassay analyzer was produced by Roche (Switzerland).

1.4 Animal grouping

The experiment was divided into 5 groups, half male and half male. Ten 10-week-old C57BL/6J mice belonged to the normal group (C57 mice + normal diet). 40 10-week-old ApoE ^-/- mice were randomly divided into model group (ApoE ^-/- mice + high-fat diet), western medicine group (rosuvastatin 2.6mg/kg body weight/day + high-fat diet) , the two-flavor compound Chinese medicine group in Section 1.2 (8.58g/kg body weight/day + high-fat diet), the four-flavor compound Chinese medicine group in Section 1.2 (8.58g/kg body weight/day + high-fat diet), each group 10 Rats were fed with corresponding diet and gavaged with corresponding medicine or equal volume of distilled water for 12 weeks. During the animal experiments, the animal ethics requirements of the "Guidelines for the Ethical Review of Experimental Animal Welfare in Beijing" were strictly followed, and were approved by the Animal Ethics Committee of Xiyuan Hospital, China Academy of Chinese Medical Sciences (ethical batch number: 2021XLC007-3).

1.5 Detection of serological indicators

The mice in each group were fed with the corresponding feed or gavaged for 12 weeks, and the samples were collected. The mice were fasted and deprived of water for 12 hours before the samples were collected. After weighing, the mice were anesthetized by intraperitoneal injection of 2% pentobarbital sodium according to the body weight of each mouse. Blood was collected from the orbit, centrifuged at 3000r min-1, 4°C for 15 min, the serum was separated, and the contents of TC, TG, HDL and LDL were detected by an automatic biochemical analyzer.

1.6 Observation of pathological morphology of aorta

After blood collection, the mice were dissected under sterile conditions, the chest cavity and abdominal cavity were quickly opened, the aorta and aortic arch tissue were separated, fixed in 4% paraformaldehyde, serially sliced with a manual rotary microtome with a thickness of 3 μm, and then HE stained and Masson staining. Observe HE-stained aortic arch tissue under 100X and 200X light microscope, and observe Masson-stained aortic arch tissue under 40X and 400X light microscope.

1.7 Determination of ox-LDL content by ELISA method

Strictly operate according to the instructions of the ELISA kit, measure the absorbance (OD) value at a wavelength of 450nm on a microplate reader, and calculate the concentration value of each value.

1.8 Determination of ROS, GSH, GPX, NAD ⁺ content by colorimetry

In strict accordance with the instructions of the colorimetric kit, the contents of ROS, GSH, GPX, and NAD ⁺ in the serum of mice in each group were determined.

1.9 Statistical analysis

SPSS 23.0 software was used for statistical analysis, and the obtained data were all subjected to normality test, with mean ± standard deviation It means that when the data are normally distributed, one-way analysis of variance is used for comparison between groups, and Tukey's method is used for pairwise comparison when the variance is homogeneous; Welch analysis of variance is used for uneven variance, and Games-Howell l method is used for pairwise comparison, with P <0.05 means the difference is statistically significant.

2. Results

2.1 The effect of two kinds of traditional Chinese medicine compatibility on the pathological morphology of ApoE ^-/- mice aorta

HE staining showed that the aortic intima of the mice in the normal group (a) had a complete structure, no plaques and lipid deposition were seen, and the smooth muscle strips of the media were arranged in an orderly manner without hyperplasia. In the model group (b), lipid deposition was observed under the aortic intima, and the intima and media were significantly thickened. Compared with the model group, in the two-drug compound, four-drug compound and rosuvastatin group (c) there was a little lipid deposition in the intima, the media was slightly thickened, and the structure of each layer was clearer and more orderly. The degree of blood vessel improvement in the four-drug compound group (e) was significantly better than that in the two-drug compound group (d). The results are shown in Figure 6.

Masson staining showed that the aortic intima in the normal group (a) was smooth, the media cells were arranged regularly, and the elastic fibers were arranged in an orderly manner. In the model group, the thickness of the aortic intima was uneven, fat deposition was observed, elastic fibers were significantly reduced, and collagen fibers were significantly increased. Compared with the model group (b), the media of the two-drug compound (d), the four-drug compound (e) and the rosuvastatin calcium tablet group (c) were slightly thickened, and the content of collagen fibers was less. The degree of improvement of vascular collagen in the compound group was significantly better than that in the two-drug compound group, the results are shown in Figure 7.

2.2 Effects of two kinds of traditional Chinese medicine compound on blood lipids of ApoE ^-/- mice

The experimental results showed that, compared with the normal group, the TC, TG, HDL, LDL, and ox-LDL indexes of the ApoE ^-/- mice in the model group were all significantly increased (P<0.01); The TC, TG, and ox-LDL indexes of the Siweiyao Compound and Rosuvastatin Tablets groups decreased (P<0.05, P<0.01), and the LDL of the Siweiyao Compound and Rosuvastatin Calcium Tablets group decreased significantly (P<0.05, P<0.01). 0.01, <0.01). The results are shown in Figure 8.

2.3 The effect of two kinds of traditional Chinese medicine compound on the oxidative stress index of ApoE ^-/- mice

Compared with the normal group, the ROS indexes of the ApoE ^-/- mice in the model group were significantly increased (P<0.01), while the NAD ⁺ , GSH, and GPX indexes were significantly reduced (P<0.01); The ROS in the Siweiyao Compound, Siweiyao Compound and Rosuvastatin Tablets groups decreased significantly (P<0.01, P<0.01), and the NAD ⁺ and GSH in the Siweiyao Compound and Rosuvastatin Calcium Tablets groups increased significantly (P<0.01, P<0.01). <0.05, P<0.01), the NAD ⁺ and GSH of the two herbs compound tended to increase, and there was no statistical difference (P>0.05, P>0.05).

3. Conclusion

Studies have shown that age-related vascular aging is inextricably related to atherosclerosis. The two often coexist and have a common pathogenesis and pathological mechanism, including oxidative stress, inflammation, telomeres, mitochondria Dysfunction, etc. Lipid deposition in atherosclerosis can damage the vascular endothelium, leading to an increase in pro-inflammatory factors and adhesion molecules in the blood vessel, promoting vascular endothelial dysfunction, causing thickening of the intima-media, elastin rupture, and collagen deposition , vascular dilation and increased internal diameter, further reducing vascular elasticity, increasing vascular stiffness, thereby promoting vascular aging; and vascular aging caused by increased arterial stiffness, central elastic artery dilation, and increased intima and media thickness will further aggravate endothelial function. damage, and promote the development of atherosclerosis; the two interact with each other in a vicious circle, and jointly induce the occurrence and development of cardiovascular and cerebrovascular diseases.

The results show that the compound recipe of two herbs (polygonum cuspidatum, hawthorn) and the compound recipe of four herbs (ginseng, knotweed, hawthorn, red yeast rice) of the embodiment ^{of the} present invention have effects on the hyperlipidemia TC, TC, Indexes such as TG and ox-LDL all have significant inhibitory action, for index effectiveness, the effect of the compound prescription of four herbs of the embodiment of the present invention is more obvious, especially the compound prescription of four herbs of the embodiment of the present invention has significant effect on LDL LDL (low-density lipoprotein cholesterol), also often referred to as "bad" cholesterol, is closely related to an increased risk of heart disease, and controlling LDL levels is usually also a part of a low-cholesterol diet. focus. The hawthorn in the four herbs of the embodiment of the present invention contains substances such as citric acid, malic acid, ascorbic acid, etc., and has the effects of lowering fat, reducing turbidity, and consuming fat. Red yeast rice contains natural statin components, which have the effects of lowering blood fat and blood pressure. The combination of four herbs can exert better pharmacological effects of lowering blood fat and anti-vascular aging.

Oxidative stress damage is an important factor leading to vascular aging. Both the two-drug compound and the four-drug compound in the embodiment of the present invention can significantly reduce ROS. For NAD ⁺ and GSH indicators that affect vascular aging, the embodiment of the present invention The four-herb compound has a significant effect on increasing the effect, while the two-herb compound has no statistical difference in the effect on NAD ⁺ , GSH. For the main pathological factors of vascular aging are deficiency, phlegm, stasis, and toxin, the treatment principle of the four-flavored medicine compound in the embodiment of the present invention is "supplementing qi and activating blood, resolving phlegm and detoxifying", in which ginseng nourishes deficiency and damage, hawthorn reduces turbidity and lipids , Polygonum cuspidatum detoxifies and activates blood circulation, red yeast rice promotes blood circulation and removes blood stasis, together play the effects of replenishing deficiency, promoting blood circulation and removing blood stasis, detoxifying and removing turbidity, and can exert the pharmacological effect of anti-vascular aging.

In summary, both the two-drug compound and the four-drug compound in the embodiment of the present invention have a certain effect of lowering blood fat and preventing and treating atherosclerosis, but for effectively improving the vascular aging of ApoE ^-/- mice, the present invention The four-flavored medicine compound of the embodiment shows more effective pharmacological effects, and has the functions of regulating lipid metabolism, reducing oxidative stress level, and improving pathological morphology of aorta.

In this paper, specific examples are used to illustrate the inventive concept in detail, and the descriptions of the above embodiments are only used to help understand the core idea of the present invention. It should be pointed out that for those skilled in the art, any obvious modification, equivalent replacement or other improvement should be included in the protection scope of the present invention without departing from the inventive concept.

Claims (10)

1. The Chinese herbal compound composition for preventing and treating vascular aging is characterized by comprising the following components:

ginseng extract, haw extract, polygonum cuspidatum extract and red Qu Weifen;

wherein the mass ratio of the ginseng extract, the hawthorn extract, the polygonum cuspidatum extract and the monascus micropowder is as follows:

(6-12)：(6-12)：(6-12)：(3-9)。

2. the traditional Chinese medicine compound composition for preventing and treating vascular aging according to claim 1, wherein the mass ratio of ginseng extract, hawthorn extract, polygonum cuspidatum extract and monascus micropowder is as follows: 9:9:9:6.

3. the Chinese herbal compound composition for preventing and treating vascular aging according to claim 1, wherein the total ginsenoside content of the ginseng extract is 85-95%.

4. The Chinese herbal compound composition for preventing and treating vascular aging according to claim 1, wherein the total flavone content of hawthorn in the hawthorn extract is 10-30%.

5. The compound Chinese medicinal composition for preventing and treating vascular aging according to claim 1, wherein the content of polydatin in the polygonum cuspidatum extract is 95-99%.

6. The traditional Chinese medicine compound composition for preventing and treating vascular aging according to claim 1, wherein the lovastatin content in the monascus micropowder is 3-7%.

7. The compound Chinese medicinal composition for preventing and treating vascular aging according to any one of claims 3 to 6, wherein the total ginsenoside content in the ginseng extract is 90%, the total flavone content in the hawthorn extract is 20%, the polydatin content in the polygonum cuspidatum extract is 98%, and the lovastatin content in the monascus micropowder is 5.09%.

8. The method for preparing the traditional Chinese medicine compound composition for preventing and treating vascular aging of claim 1, which is characterized by comprising the following steps:

cleaning and drying three medicinal decoction pieces of ginseng, hawthorn and polygonum cuspidatum;

cutting ginseng decoction pieces into particles, carrying out ultrasonic grinding, putting the particles into a container, adding water, and carrying out distillation extraction under the conditions of controlling the temperature to 65-75 ℃ and the vacuum degree to 0.06-0.07 mpa for 80-100min; collecting distilled liquid when the distilled liquid reaches a preset amount; continuously adding water, distilling again to extract, distilling to obtain predetermined amount of liquid, mixing the two liquids, concentrating the liquid, cooling to room temperature, and drying to obtain Ginseng radix extract;

cleaning fructus crataegi decoction pieces, placing into a container, decocting for 20-40min, and filtering to obtain precipitate; adding water into the filter residue, decocting for 10-30min again, and filtering to obtain precipitate; mixing the two decoctions, concentrating, and cooling to room temperature; passing through polyamide resin column, eluting with deionized water, eluting with ethanol solution, recovering ethanol under reduced pressure, adjusting pH to 6.0-6.5, and drying to obtain fructus crataegi extract;

drying and crushing giant knotweed rhizome decoction pieces, soaking the giant knotweed rhizome decoction pieces in 60 to 70 percent of ethanol at normal temperature, and concentrating ethanol extract to the liquid amount of 1/3 to 1/7 of the original volume; cooling the concentrated solution to room temperature, filtering, and removing impurities; passing the filtrate through neutral alumina short and thick column, collecting effluent liquid, concentrating until no alcohol exists, cooling at normal temperature, and drying to obtain rhizoma Polygoni Cuspidati extract;

in the above steps, supercritical fluid extraction can be used for extracting three medicinal decoction pieces of Ginseng radix, fructus crataegi, and rhizoma Polygoni Cuspidati;

the ginseng extract, the hawthorn extract, the polygonum cuspidatum extract and the red yeast rice micropowder prepared in advance are compounded according to a proportion to obtain the traditional Chinese medicine compound composition for preventing and treating vascular aging.

9. The method for preparing a Chinese herbal compound composition for preventing and treating vascular aging according to claim 8, wherein the distillation extraction time of ginseng decoction pieces is 90min; the haw decoction pieces are decocted for 30min for the first time and 20min again; the ethanol extract of the giant knotweed decoction pieces is concentrated to a liquid amount of 1/5 of the original volume.

10. The use of the compound Chinese medicine composition according to claim 1 in preparing medicines for preventing and treating vascular aging.