阅读说明：本技术 一种化合物、中药益生菌发酵产物及其在制备具有降血脂作用的药物或保健品中的应用 (Compound, traditional Chinese medicine probiotic fermentation product and application of compound and traditional Chinese medicine probiotic fermentation product in preparation of medicine or health-c) 是由 刘志刚 刘杰 刘晓宇 于 2019-11-25 设计创作，主要内容包括：本发明涉及生物医学技术领域,具体公开了一种化合物、中药益生菌发酵产物及其在制备具有降血脂作用的药物或保健品中的应用。所述的化合物具有式(Ⅰ)所示的结构。所述的中药益生菌发酵产物,包含式(Ⅰ)化合物；其制备方法包含如下步骤：(1)取中药原料用益生菌进行发酵,得发酵液；(2)将发酵液进行干燥后即得所述的中药益生菌发酵产物。本发明所述的式(Ⅰ)化合物以及中药益生菌发酵产物具有优异的降血脂作用,可以用于制备药物和保健品。(The invention relates to the technical field of biomedicine, and particularly discloses a compound, a traditional Chinese medicine probiotic fermentation product and application of the compound and the traditional Chinese medicine probiotic fermentation product in preparation of a medicine or a health-care product with a blood fat reducing effect. The compound has a structure shown in a formula (I). The traditional Chinese medicine probiotic fermentation product comprises a compound shown in a formula (I); the preparation method comprises the following steps: (1) taking the traditional Chinese medicine raw materials, and fermenting with probiotics to obtain fermentation liquor; (2) drying the fermentation liquor to obtain the traditional Chinese medicine probiotic fermentation product. The compound shown in the formula (I) and the traditional Chinese medicine probiotic fermentation product have excellent blood fat reducing effect, and can be used for preparing medicines and health-care products.)

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof;

2. a process for the preparation of a compound of formula (i) as claimed in claim 1, comprising the steps of:

(1) taking the traditional Chinese medicine raw materials, and fermenting with probiotics to obtain fermentation liquor;

(2) drying the fermentation liquor to obtain a crude product;

(3) separating the crude product by chromatography to obtain a compound of formula (I);

the traditional Chinese medicine raw materials in the step (1) are traditional Chinese medicine raw materials comprising honey, hawthorn, astragalus membranaceus, salvia miltiorrhiza, schisandra chinensis, ginkgo leaves and kudzu roots; or Chinese medicinal materials including Mel, fructus crataegi extract, radix astragali extract, Saviae Miltiorrhizae radix extract, fructus Schisandrae chinensis extract, folium Ginkgo extract and radix Puerariae extract;

the probiotics in the step (1) are selected from one or a mixture of more than two of lactobacillus acidophilus, bifidobacterium infantis and bifidobacterium breve.

3. The preparation method according to claim 2, wherein the specific method of step (1) is: mixing the traditional Chinese medicine raw materials, the probiotic dry powder and water, and fermenting for 16-30 hours at the temperature of 20-30 ℃ and the pH of 5-6 to obtain fermentation liquor;

the weight and dosage ratios of the traditional Chinese medicine raw materials, the probiotic dry powder and the water are as follows: 20-40: 8-15: 40-60.

4. The process according to claim 2, wherein the chromatographic technique in step (3) is a preparative HPLC technique; the specific conditions of the preparative HPLC technique are as follows: adopting a reversed phase chromatographic column; selecting an ultraviolet detector, wherein the detection wavelength is 220-230 nm; using a trifluoroacetic acid aqueous solution with the volume percent of 0.05-0.2 as a mobile phase A, using a trifluoroacetic acid acetonitrile solution with the volume percent of 0.05-0.2 as a mobile phase B, wherein the mobile phase A: mobile phase B75: 25; and (3) collecting the part corresponding to the chromatographic peak for 9.3-9.9 min, concentrating and drying to obtain the compound shown in the formula (I).

5. A traditional Chinese medicine probiotic fermentation product is characterized by comprising a compound shown in a formula (I).

6. The method for preparing the traditional Chinese medicine probiotic fermentation product according to claim 5, characterized by comprising the following steps:

(1) taking the traditional Chinese medicine raw materials, and fermenting with probiotics to obtain fermentation liquor;

(2) drying the fermentation liquor to obtain the traditional Chinese medicine probiotic fermentation product;

the traditional Chinese medicine raw materials in the step (1) are traditional Chinese medicine raw materials comprising honey, hawthorn, astragalus membranaceus, salvia miltiorrhiza, schisandra chinensis, ginkgo leaves and kudzu roots; or Chinese medicinal materials including Mel, fructus crataegi extract, radix astragali extract, Saviae Miltiorrhizae radix extract, fructus Schisandrae chinensis extract, folium Ginkgo extract and radix Puerariae extract;

the probiotics in the step (1) are selected from one or a mixture of more than two of lactobacillus acidophilus, bifidobacterium infantis and bifidobacterium breve.

7. The preparation method according to claim 5, wherein the specific method of step (1) is: mixing the traditional Chinese medicine raw materials, the probiotic dry powder and water, and fermenting for 16-30 hours at the temperature of 20-30 ℃ and the pH of 5-6 to obtain fermentation liquor;

the weight and dosage ratios of the traditional Chinese medicine raw materials, the probiotic dry powder and the water are as follows: 20-40: 8-15: 40-60.

8. Use of a compound of formula (i) as defined in claim 1 or a pharmaceutically acceptable salt thereof or a fermentation product of a probiotic of a traditional Chinese medicine as defined in claim 6 in the manufacture of a medicament or a health product.

9. The use according to claim 8, wherein the medicament is a medicament having hypolipidemic effect, or a medicament having anti-inflammatory effect, or a medicament for reducing accumulation of serum metabolites caused by hyperlipidemia, or a medicament for reducing morphological and functional impairment of vascular endothelial cells;

the health care product has the function of reducing blood fat, or the health care product for relieving the accumulation of serum metabolites caused by hyperlipidemia, or the health care product for relieving the morphological and functional damage of vascular endothelial cells.

10. The use according to claim 9, wherein the medicament or health product comprises a compound of formula (i) or a pharmaceutically acceptable salt thereof or a probiotic fermentation product of a traditional Chinese medicine and a carrier; the mass fraction of the compound of formula (I) or the medicinal salt thereof or the traditional Chinese medicine probiotic fermentation product is 5-95%.

Technical Field

The invention relates to the technical field of biomedicine, in particular to a compound, a traditional Chinese medicine probiotic fermentation product and application thereof in preparing a medicine or health-care product with a blood fat reducing effect.

Background

Cardiovascular disease (CVD) is a circulatory disease mainly characterized by abnormal function of the heart and blood vessels, including coronary heart disease, cardiomyopathy, atherosclerosis, hypertension, and cardiac insufficiency due to various causes, and has been the first cause of death in developed countries. The main treatment methods comprise drug treatment, interventional therapy, surgical treatment and the like. The epidemiological investigation and research data of China show that: dyslipidemia is a risk factor for the onset of coronary heart disease, and has the same action intensity as that of western people; the increase of the serum total cholesterol level of people in China not only increases the risk of coronary heart disease, but also increases the risk of ischemic stroke. The united states society of cardiology/american society of cardiology (AHC/ACC) published 4 guidelines for the prevention of cardiovascular-related diseases in 2013, and these 4 new guidelines are 2013AHC/ACC cardiovascular risk assessment guideline, 2013AHC/ACC cardiovascular risk reduction lifestyle management guideline, 2013AHC/ACC cardiovascular risk reduction hypercholesterolemia treatment guideline, and 2013AHC/ACC adult overweight and obesity management guideline, respectively. Therefore, attention must be paid to the prevention and treatment of dyslipidemia as early as possible.

In experimental research and clinical application, the compound traditional Chinese medicine has very wide application in reducing blood fat, a formula generally adopts a method of functional factor complementation, and the action way of reducing blood fat can follow a plurality of mechanisms simultaneously, thereby achieving the purpose of quickly reducing blood fat. The action mechanism of the traditional Chinese medicine for reducing blood fat can be considered from the following aspects: reducing or inhibiting the absorption of TC in the intestinal tract; inhibit the synthesis of TC and TG; affecting the distribution, transfer and elimination of blood fat; increase bile acid excretion, promote TC excretion, etc. The mechanism of action of many compound herbs may be the result of the combination of the above actions.

The major probiotics at present include lactobacilli, streptococci and bifidobacteria, which are the normal constituents of microorganisms in the human and animal intestinal tracts and have various health-promoting effects. Yogurt and fermented milk are attracting much attention as carriers for culturing probiotics, and probiotic fermented milk products such as milk, yogurt, and cheese have been reported to have a significant hypolipidemic effect in vivo models. Clinical experiments prove that various strains such as lactobacillus rhamnosus, lactobacillus plantarum, lactobacillus acidophilus, lactobacillus casei, lactobacillus paracasei, bifidobacterium longum, bifidobacterium, enterococcus faecalis, lactobacillus reuteri, lactobacillus gasseri, bifidobacterium breve and lactobacillus amylovorus can effectively regulate lipid in serum. So far, a great number of documents report the blood fat reducing effect of different probiotics at home and abroad. However, at present, reports of developing lipid-lowering drugs by fermenting probiotics for traditional Chinese medicines are rare and need further research.

Disclosure of Invention

In view of the above, the present invention firstly provides a new compound, and further research shows that the new compound has the effects of reducing blood fat and resisting inflammation.

In addition, the invention also provides a traditional Chinese medicine probiotic fermentation product which also has the effects of reducing blood fat and resisting inflammation.

In addition, the invention also provides a preparation method and application of the novel compound and a traditional Chinese medicine probiotic fermentation product.

The detailed technical scheme of the invention is as follows:

a compound of formula (I) or a pharmaceutically acceptable salt thereof;

the compound of formula (i) is named:

3,4,5-trimethoxy-N-(3-(2-(4-methoxybenzamido)-3-phenylpropanamido)propyl)benzamide

preferably, the pharmaceutically acceptable salt is a pharmaceutically acceptable acid addition salt, wherein the acid is selected from the group consisting of: citric acid, tartaric acid, lactic acid, acetic acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, pyruvic acid, fumaric acid, maleic acid, hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, and sulfuric acid.

The present invention also provides a process for the preparation of a compound of formula (i) comprising the steps of:

(1) taking the traditional Chinese medicine raw materials, and fermenting with probiotics to obtain fermentation liquor;

(2) drying the fermentation liquor to obtain a crude product;

(3) separating the crude product by chromatography to obtain a compound of formula (I);

the traditional Chinese medicine raw materials in the step (1) are traditional Chinese medicine raw materials comprising honey, hawthorn, astragalus membranaceus, salvia miltiorrhiza, schisandra chinensis, ginkgo leaves and kudzu roots; or Chinese medicinal materials including Mel, fructus crataegi extract, radix astragali extract, Saviae Miltiorrhizae radix extract, fructus Schisandrae chinensis extract, folium Ginkgo extract and radix Puerariae extract;

the probiotics in the step (1) are selected from one or a mixture of more than two of lactobacillus acidophilus, bifidobacterium infantis and bifidobacterium breve.

The extracts of the present invention are all conventional pharmaceutical raw materials and can be purchased. Or extracting with ethanol by conventional method (such as heating reflux extraction, ultrasonic extraction or leaching).

Preferably, the weight ratio of honey, hawthorn, astragalus membranaceus, salvia miltiorrhiza, schisandra chinensis, ginkgo leaves and kudzu roots in the traditional Chinese medicine raw materials in the step (1) is 1-5: 1-5: 1-5: 1-5: 1-5: 1-5: 1 to 5.

Most preferably, the weight ratio of honey, hawthorn, astragalus mongholicus, salvia miltiorrhiza, schisandra chinensis, ginkgo leaves and kudzu roots in the traditional Chinese medicine raw materials in the step (1) is 1: 1: 1: 1: 1: 1: 1.

preferably, the weight ratio of honey, hawthorn extract, astragalus extract, salvia extract, schisandra extract, ginkgo biloba extract and kudzu root extract in the traditional Chinese medicine raw materials in the step (1) is 1-5: 1-5: 1-5: 1-5: 1-5: 1-5: 1 to 5.

Most preferably, the weight ratio of honey, hawthorn extract, astragalus extract, salvia extract, schisandra extract, ginkgo biloba extract and kudzu root extract in the traditional Chinese medicine raw materials in the step (1) is 1: 1: 1: 1: 1: 1: 1.

preferably, the probiotics in the step (1) are probiotic dry powder, wherein the weight ratio of lactobacillus acidophilus dry powder to bifidobacterium infantis dry powder to bifidobacterium breve dry powder is 1-5: 1-5: 1 to 5.

Most preferably, the probiotic bacteria in step (1) are probiotic bacteria dry powder, wherein the weight ratio of lactobacillus acidophilus dry powder to bifidobacterium infantis dry powder to bifidobacterium breve dry powder is 1: 1: 1.

preferably, the specific method of step (1) is: mixing the traditional Chinese medicine raw materials, the probiotic dry powder and water, and fermenting for 16-30 hours at the temperature of 20-30 ℃ and the pH of 5-6 to obtain fermentation liquor;

the weight and dosage ratios of the traditional Chinese medicine raw materials, the probiotic dry powder and the water are as follows: 20-40: 8-15: 40-60.

Most preferably, the specific method of step (1) is: mixing the traditional Chinese medicine raw materials, probiotic dry powder and water, and fermenting for 24h at the temperature of 25 ℃ and the pH of 5.5 to obtain fermentation liquor;

the weight and dosage ratios of the traditional Chinese medicine raw materials, the probiotic dry powder and the water are as follows: 30: 10: 50.

preferably, the chromatographic technique described in step (3) is a preparative HPLC technique; the specific conditions of the preparative HPLC technique are as follows: adopting a reversed phase chromatographic column; selecting an ultraviolet detector, wherein the detection wavelength is 220-230 nm; using a trifluoroacetic acid aqueous solution with the volume percent of 0.05-0.2 as a mobile phase A, using a trifluoroacetic acid acetonitrile solution with the volume percent of 0.05-0.2 as a mobile phase B, wherein the mobile phase A: mobile phase B75: 25; and (3) collecting the part corresponding to the chromatographic peak for 9.3-9.9 min, concentrating and drying to obtain the compound shown in the formula (I).

Most preferably, the specific conditions of the preparative HPLC technique are: adopting an ODS (oxide dispersion strengthened) reversed-phase chromatographic column; selecting an ultraviolet detector, wherein the detection wavelength is 220 nm; using a 0.1 volume percent trifluoroacetic acid aqueous solution as a mobile phase A, using a 0.1 volume percent trifluoroacetic acid acetonitrile solution as a mobile phase B, wherein the ratio of the mobile phase A: mobile phase B75: 25; and (3) collecting the part corresponding to the chromatographic peak for 9.3-9.9 min, concentrating and drying to obtain the compound shown in the formula (I).

The invention also provides a traditional Chinese medicine probiotic fermentation product which comprises the compound shown in the formula (I).

The invention also provides a preparation method of the traditional Chinese medicine probiotic fermentation product, which comprises the following steps:

(1) taking the traditional Chinese medicine raw materials, and fermenting with probiotics to obtain fermentation liquor;

(2) drying the fermentation liquor to obtain the traditional Chinese medicine probiotic fermentation product;

the traditional Chinese medicine raw materials in the step (1) are traditional Chinese medicine raw materials comprising honey, hawthorn, astragalus membranaceus, salvia miltiorrhiza, schisandra chinensis, ginkgo leaves and kudzu roots; or Chinese medicinal materials including Mel, fructus crataegi extract, radix astragali extract, Saviae Miltiorrhizae radix extract, fructus Schisandrae chinensis extract, folium Ginkgo extract and radix Puerariae extract;

the probiotics in the step (1) are selected from one or a mixture of more than two of lactobacillus acidophilus, bifidobacterium infantis and bifidobacterium breve.

Preferably, the weight ratio of honey, hawthorn, astragalus membranaceus, salvia miltiorrhiza, schisandra chinensis, ginkgo leaves and kudzu roots in the traditional Chinese medicine raw materials in the step (1) is 1-5: 1-5: 1-5: 1-5: 1-5: 1-5: 1 to 5.

Most preferably, the weight ratio of honey, hawthorn, astragalus mongholicus, salvia miltiorrhiza, schisandra chinensis, ginkgo leaves and kudzu roots in the traditional Chinese medicine raw materials in the step (1) is 1: 1: 1: 1: 1: 1: 1.

preferably, the weight ratio of honey, hawthorn extract, astragalus extract, salvia extract, schisandra extract, ginkgo biloba extract and kudzu root extract in the traditional Chinese medicine raw materials in the step (1) is 1-5: 1-5: 1-5: 1-5: 1-5: 1-5: 1 to 5.

Most preferably, the weight ratio of honey, hawthorn extract, astragalus extract, salvia extract, schisandra extract, ginkgo biloba extract and kudzu root extract in the traditional Chinese medicine raw materials in the step (1) is 1: 1: 1: 1: 1: 1: 1.

preferably, the probiotics in the step (1) are probiotic dry powder, wherein the weight ratio of lactobacillus acidophilus dry powder to bifidobacterium infantis dry powder to bifidobacterium breve dry powder is 1-5: 1-5: 1 to 5.

Most preferably, the probiotic bacteria in step (1) are probiotic bacteria dry powder, wherein the weight ratio of lactobacillus acidophilus dry powder to bifidobacterium infantis dry powder to bifidobacterium breve dry powder is 1: 1: 1.

preferably, the specific method of step (1) is: mixing the traditional Chinese medicine raw materials, the probiotic dry powder and water, and fermenting for 16-30 hours at the temperature of 20-30 ℃ and the pH of 5-6 to obtain fermentation liquor;

the weight and dosage ratios of the traditional Chinese medicine raw materials, the probiotic dry powder and the water are as follows: 20-40: 8-15: 40-60.

Most preferably, the specific method of step (1) is: mixing the traditional Chinese medicine raw materials, probiotic dry powder and water, and fermenting for 24h at the temperature of 25 ℃ and the pH of 5.5 to obtain fermentation liquor;

the weight and dosage ratios of the traditional Chinese medicine raw materials, the probiotic dry powder and the water are as follows: 30: 10: 50.

the invention also provides an application of the compound of formula (I) or a pharmaceutically acceptable salt thereof or the traditional Chinese medicine probiotic fermentation product in preparation of medicines or health products.

Preferably, the medicament is a medicament with the function of reducing blood fat, or a medicament with the function of anti-inflammation, or a medicament for reducing the accumulation of serum metabolites caused by hyperlipemia, or a medicament for reducing the morphological and functional damage of vascular endothelial cells; the health care product has the function of reducing blood fat, or the health care product for relieving the accumulation of serum metabolites caused by hyperlipidemia, or the health care product for relieving the morphological and functional damage of vascular endothelial cells.

Preferably, the medicine or health care product contains a compound shown in the formula (I) or a medicinal salt thereof or a traditional Chinese medicine probiotic fermentation product and a carrier; the mass fraction of the compound of formula (I) or the medicinal salt thereof or the traditional Chinese medicine probiotic fermentation product is 5-95%.

Further preferably, the carrier comprises at least one of a solvent, a polymer and a liposome. Still further preferably, the solvent includes, but is not limited to, water, physiological saline, and other non-aqueous solvents. Still further preferably, the polymer includes, but is not limited to, polylysine, polyethyleneimine and modifications thereof, chitosan, polylactic acid, gelatin. Still further preferably, the liposome may be, but is not limited to, cholesterol, soy lecithin, egg yolk lecithin.

Even more preferably, the carrier further comprises one or more of a diluent and an excipient. Specifically, the diluent includes one or more of starches, sugars, celluloses and inorganic salts. Specifically, the excipient comprises one or more of a binder, a filler, a lubricant in a tablet, a matrix part in a semisolid preparation ointment or cream, a preservative in a liquid preparation, an antioxidant, a flavoring agent, an aromatic agent, a cosolvent, an emulsifier and a coloring agent.

Further preferably, the mass fraction of the compound of formula (I) or the pharmaceutically acceptable salt thereof or the traditional Chinese medicine probiotic fermentation product is 10-90%, 15-85% or 30-80%.

Further preferably, the pharmaceutical or nutraceutical may further comprise a second active ingredient. The second active ingredient is selected according to the use of the drug or health product, and is not limited herein. Specifically, the second active ingredient has a hypolipidemic effect when the composition is used for reducing blood lipids, and optionally, the second active ingredient comprises one or more of vitamin C, vitamin E, coenzyme Q, glutathione, carotene and betaine; when the composition is used for reducing blood fat and resisting inflammation, the second active ingredient has at least anti-inflammatory activity.

More preferably, the mass fraction of the second active ingredient in the composition is 1% to 70%.

Preferably, the health product or the medicine is in the form of tablet, capsule, powder, granule, pill, syrup, solution, suspension or aerosol.

Preferably, the health product is in the form of gel or water.

Further preferably, the health product also comprises an auxiliary material matrix, wherein the auxiliary material matrix comprises monosaccharide, oligosaccharide, polysaccharide, amino acid, preservative, pH regulator and blood fat reducing auxiliary agent.

Has the advantages that: (1) the invention provides a brand-new compound shown in formula (I), a traditional Chinese medicine probiotic fermentation product and a preparation method thereof; the compound shown in the formula (I) and the traditional Chinese medicine probiotic fermentation product have excellent hypolipidemic and anti-inflammatory effects, and can be used for preparing medicines and health-care products. (2) The experimental result shows that in vivo, the traditional Chinese medicine probiotic fermentation product and the monomeric compound with the structure of the formula (I) can obviously reduce the accumulation of serum metabolites caused by hyperlipidemia, and is beneficial to reducing the morphological and functional damage of vascular endothelial cells. In addition, the traditional Chinese medicine probiotic fermentation product and the monomer compound with the structure of the formula (I) can improve the expression of Foxo1/PGC-1 alpha and mitochondrial complex in thoracic vessels. In vitro experiment results show that the traditional Chinese medicine probiotic fermentation product and the monomer compound with the structure of formula (I) activate mitochondrial PGC-1 alpha mainly by enhancing the expression of Foxo 1. In addition, the anti-inflammatory effects of the traditional Chinese medicine probiotic fermentation product and the monomer compound with the structure of the formula (I) are mainly mediated through a p38 MAPK pathway.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only drawings of some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a mass spectrum of a compound of formula (I) according to the present invention; wherein 1a is a positive ion mode mass spectrogram; and 1b is a negative ion mode mass spectrogram.

FIG. 2 shows the NMR spectra of the compounds of formula (I) according to the invention.

FIG. 3 shows the NMR carbon spectrum of the compound of formula (I) according to the invention.

FIG. 4 is a graph showing the therapeutic effects of the fermented product of probiotic bacteria of the present invention and the monomeric compound having the structure of formula (I) on hyperlipidemic mice.

FIG. 5 is a graph showing the effect of the fermentation product of probiotic bacteria of the present invention and the monomeric compound having the structure of formula (I) on the improvement of mitochondrial function.

FIG. 6 is a graph showing the effect of the fermentation product of probiotic bacteria of the present invention and the monomeric compound having the structure of formula (I) on the copy number of mtDNA and the expression of PGC-1. alpha. and FoxO 1.

FIG. 7 is a graph showing the effect of the fermentation product of probiotic bacteria and the monomeric compound having the structure of formula (I) on the mitochondrial function of endothelial cells damaged by PA.

FIG. 8 is an experimental diagram of the anti-inflammatory protective action mechanism of the traditional Chinese medicine probiotic fermentation product and the monomeric compound with the structure of formula (I).

Detailed Description

The technical solution of the present invention will be clearly and completely described with reference to the following examples. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

EXAMPLE 1 preparation of the Compound of formula (I)

(1) Mixing 30g of traditional Chinese medicine raw materials and 10g of probiotic dry powder in a three-dimensional mixer until the color is uniform, adding 50g of water, and fermenting for 24 hours at the temperature of 25 ℃ and the pH value of 5.5 to obtain fermentation liquor;

(2) centrifuging the fermentation liquor at 8000r/min for 30min, removing precipitate, retaining supernatant, and freeze drying the supernatant to obtain crude product;

(3) separating the crude product by chromatography to obtain a compound of formula (I);

the traditional Chinese medicine raw materials in the step (1) are prepared from honey, hawthorn extract, astragalus extract, salvia extract, schisandra extract, ginkgo leaf extract and kudzu root extract according to a weight ratio of 1: 1: 1: 1: 1: 1: 1, preparing a composition; the probiotic dry powder is prepared from lactobacillus acidophilus dry powder, bifidobacterium infantis dry powder and bifidobacterium breve dry powder according to the weight ratio of 1: 1: 1, preparing a composition;

the chromatographic technique in the step (3) is a preparative HPLC technique; the specific conditions of the preparative HPLC technique are as follows: adopting an ODS (oxide dispersion strengthened) reversed-phase chromatographic column; selecting an ultraviolet detector, wherein the detection wavelength is 220 nm; using a 0.1 volume percent trifluoroacetic acid water solution as a mobile phase A, using a 0.1 volume percent trifluoroacetic acid acetonitrile solution as a mobile phase B, wherein the ratio of the mobile phase A: mobile phase B75: 25; and (3) collecting the part corresponding to the chromatographic peak for 9.3-9.9 min, concentrating and drying to obtain the compound shown in the formula (I).

The mass spectrum, hydrogen spectrum and carbon spectrum data of the compound of the formula (I) are as follows (the specific spectrum is shown in figures 1-3): mass spectrometry data showed compound 572.3[ M + Na ]]⁺,548.3[M-H]^-Determining the molecular formula as C₃₀H₃₅N₃O₇。¹H NMR(600MHz,DMSO)δ:8.42(m,2H),7.80(m,2H),7.32(dd,2H),7.24(t,2H),7.16(d,2H),6.96(dd,2H),4.60(m,1H),3.81(s,9H),3.71(s,3H),3.25(m,4H),1.68(m,2H)；¹³H NMR(150MHz,DMSO)δ:171.48,165.75,165.64,161.61,152.56,139.84,138.55,129.78,129.30,129.13,128.05,126.28,126.19,113.34,104.70,60.07,55.98,55.33,55.11,45.90,45.78,39.92,37.35,37.02,36.55,29.34,29.04。

The compound 3,4,5-trimethoxy-N- (3- (2- (4-methoxybenzamido) -3-phenylpropanamido) propyl) benzamide prepared by the method can be confirmed to be obtained by mass spectrometry, hydrogen spectrometry and carbon spectrometry.

EXAMPLE 2 preparation of the Compound of formula (I)

(1) Taking 400g of traditional Chinese medicine raw materials and 100g of probiotic dry powder, mixing in a three-dimensional mixer until the color is uniform, then adding 600g of water, and carrying out ultrasonic extraction for 60 min; then fermenting for 30h under the conditions that the temperature is 25 ℃ and the pH value is 5.5 to obtain fermentation liquor;

(2) centrifuging the fermentation liquor at 8000r/min for 30min, removing precipitate, retaining supernatant, and freeze drying the supernatant to obtain crude product;

(3) separating the crude product by chromatography to obtain a compound of formula (I);

the traditional Chinese medicine raw materials in the step (1) are prepared from honey, hawthorn, astragalus membranaceus, salvia miltiorrhiza, schisandra chinensis, ginkgo leaves and kudzuvine root according to a weight ratio of 1: 1: 1: 1: 1: 1: 1, preparing a composition; the probiotic dry powder is prepared from lactobacillus acidophilus dry powder, bifidobacterium infantis dry powder and bifidobacterium breve dry powder according to the weight ratio of 1: 1: 1, preparing a composition;

the chromatographic technique in the step (3) is a preparative HPLC technique; the specific conditions of the preparative HPLC technique are as follows: adopting an ODS (oxide dispersion strengthened) reversed-phase chromatographic column; selecting an ultraviolet detector, wherein the detection wavelength is 220 nm; using a 0.1 volume percent trifluoroacetic acid aqueous solution as a mobile phase A, using a 0.1 volume percent trifluoroacetic acid acetonitrile solution as a mobile phase B, wherein the ratio of the mobile phase A: mobile phase B75: 25; and (3) collecting the part corresponding to the chromatographic peak for 9.3-9.9 min, concentrating and drying to obtain the compound shown in the formula (I) (the mass spectrum, the hydrogen spectrum and the carbon spectrum data are consistent with those in the example 1).

Example 3 preparation of traditional Chinese medicine probiotic fermentation product

(1) Mixing 30g of traditional Chinese medicine raw materials and 10g of probiotic dry powder in a three-dimensional mixer until the color is uniform, adding 50g of water, and fermenting for 24 hours at the temperature of 25 ℃ and the pH value of 5.5 to obtain fermentation liquor;

(2) centrifuging the fermentation liquor at 8000r/min for 30min, removing precipitate, retaining supernatant, and freeze drying the supernatant to obtain traditional Chinese medicine probiotic fermentation product;

the traditional Chinese medicine raw materials in the step (1) are prepared from honey, hawthorn extract, astragalus extract, salvia extract, schisandra extract, ginkgo leaf extract and kudzu root extract according to a weight ratio of 1: 1: 1: 1: 1: 1: 1, preparing a composition; the probiotic dry powder is prepared from lactobacillus acidophilus dry powder, bifidobacterium infantis dry powder and bifidobacterium breve dry powder according to the weight ratio of 1: 1: 1.

Example 4 preparation of traditional Chinese medicine probiotic fermented product

(1) Taking 400g of traditional Chinese medicine raw materials and 100g of probiotic dry powder, mixing in a three-dimensional mixer until the color is uniform, then adding 600g of water, and carrying out ultrasonic extraction for 60 min; then fermenting for 30h under the conditions that the temperature is 25 ℃ and the pH value is 5.5 to obtain fermentation liquor;

(2) centrifuging the fermentation liquid at 8000r/min for 30min, removing precipitate, retaining supernatant, and freeze drying the supernatant to obtain probiotic fermented product.

The traditional Chinese medicine raw materials in the step (1) are prepared from honey, hawthorn, astragalus membranaceus, salvia miltiorrhiza, schisandra chinensis, ginkgo leaves and kudzuvine root according to a weight ratio of 1: 1: 1: 1: 1: 1: 1, preparing a composition; the probiotic dry powder is prepared from lactobacillus acidophilus dry powder, bifidobacterium infantis dry powder and bifidobacterium breve dry powder according to the weight ratio of 1: 1: 1.

Experimental example 1

In order to evaluate the effects of the traditional Chinese medicine probiotic fermentation product prepared by the method and the monomeric compound with the structure of the formula (I), the following effect experiment is carried out.

Animal experiment design: 6-8 week old C57BL6 male mice were placed in a thermostated (22-28 ℃) and humidified (60%) animal room and were allowed free access to food and water under 12 hour light/dark cycle conditions. The mice were randomly divided into 4 groups: control group (Control) (same volume sterile water gavage 9d), hyperlipemia group (p407) (same volume sterile water gavage 9d), and traditional Chinese medicine probiotic fermented product treatment group (abbreviated as TCMFJ) prepared in example 3 (200mg/kg/d, gavage once a day, gavage for 9 days). The monomer compound treatment group (abbreviated as TCMDT) with the structure of formula (I) (200mg/kg/d, gavage once a day for 9 days) (n is more than or equal to 6). p-407 is a hydrophilic triblock copolymer composed of polyoxyethylene and polyoxypropylene units, used in an intraperitoneal injection induced hyperlipidemia model. After 9 days of gastric lavage, the control group was injected with sterile normal saline, and the other 3 groups were injected intraperitoneally with p407(0.5g/kg) for 24h (day 10) to induce acute hyperlipidemia in mice. Blood samples from mice were collected, centrifuged at 4500rpm for 12min at 4 ℃ and serum frozen at-80 ℃ for subsequent analysis.

Thoracic aorta preparation and isometric tension measurement: the thoracic arteries were isolated in Krebs solution. The thoracic artery was then cut into 2-3mm long rings under a dissecting microscope. The thoracic arteries used for Western blotting experiments were frozen at-80 ℃ and further analyzed. Thoracic aortic tension measurements were made with reference to prior literature methods. Vascular reactivity study: endothelium-dependent relaxation was determined by measuring the diastolic response of the thoracic artery to acetylcholine. Determination of reactive oxygen species: and (3) measuring the content of active oxygen in the cells by adopting an ELISA ROS kit.

The experimental results are as follows:

(1) as shown in fig. 4, after injecting p407 into mice, the contents of triglyceride, cholesterol and non-esterified fatty acid in serum and the content of ROS in thoracic aorta increased significantly, indicating successful modeling of the mouse model for acute hyperlipidemia. Under the treatment of 200 mg/kg/day dosage of traditional Chinese medicine probiotic fermentation product and monomer compound with structure of formula (I), serum metabolism change caused by hyperlipidemia and other parts are recovered to normal. Similarly, 200 mg/kg/day of traditional Chinese medicine probiotic fermentation product and the monomeric compound with the structure of the formula (I) effectively reduce the increase of the active oxygen of the thoracic aorta. The results show that the traditional Chinese medicine probiotic fermentation product and the monomer compound with the structure of the formula (I) have excellent blood fat reducing effect and can reduce the accumulation of serum metabolites caused by hyperlipidemia.

(2) As shown in FIG. 5, there is increasing evidence that mitochondrial dysfunction is a significant cause of endothelial dysfunction, and FoxO1/PGC-1 α plays a key role in mediating mitochondrial biogenesis. The analysis experiment result shows that the function of thoracic artery mitochondria of the p407 hyperlipidemic mouse is damaged by reducing the expression of PGC-1 alpha, FoxO1, complete V and the like. The traditional Chinese medicine probiotic fermentation product and the monomeric compound treatment with the structure of the formula (I) at the dose of 200 mg/kg/day greatly enhance the expression of FoxO1/PGC-1 alpha (figure 5 a). Meanwhile, the treatment of the traditional Chinese medicine probiotic fermentation product and the monomeric compound with the structure of formula (I) at the dose of 200 mg/kg/day remarkably improves the protein levels of thoracic artery mitochondria complex I, complex II, complex III and complex V (figure 5b), which indicates that the traditional Chinese medicine probiotic fermentation product and the monomeric compound with the structure of formula (I) can improve the functions of mitochondria.

(3) As shown in figure 6, further studies on the cellular level show that the traditional Chinese medicine probiotic fermentation product and the monomeric compound with the structure of the formula (I) can enhance PGC-1 alpha by activating FoxO1, thereby promoting the biosynthesis of mitochondria. The traditional Chinese medicine probiotic fermentation product and the monomer compound with the structure of formula (I) can increase the copy number of mtDNA and the expression of PGC-1 alpha and FoxO1 to play a therapeutic role (figure 6a, b).

(4) As shown in FIG. 7, further studies show that the traditional Chinese medicine probiotic fermentation product and the monomeric compound with the structure of formula (I) can reduce the damage of PA to the functions of HAECs mitochondria and enhance the expression of mitochondria complex I, complex II and complex III. The traditional Chinese medicine probiotic fermentation product and the monomer compound with the structure of the formula (I) can promote the expression of key regulatory factors MFN1, MFN2 and OPA1 serving as mitochondrial fusion. The traditional Chinese medicine probiotic fermentation product and the monomer compound with the structure of the formula (I) can normalize the mitochondrial function of endothelial cells destroyed by PA. The traditional Chinese medicine probiotic fermentation product and the monomeric compound with the structure of formula (I) can obviously inhibit the generation of ROS induced by PA, which is probably a key potential mechanism of endothelial protection. The inflammatory response is a major feature of ROS production by endothelial cells.

(5) As shown in figure 8, the probiotic fermentation product of traditional Chinese medicine and the monomeric compound with the structure of formula (I) inhibit the expression of inflammatory factors interleukin 6(IL-6) and matrix metalloproteinase 1(MMP-1) induced by PA. In addition, the traditional Chinese medicine probiotic fermentation product and the monomer compound with the structure of the formula (I) remarkably reduce the expression of P-p38 and p-JNK induced by PA. These results indicate that the p38 MAPK signal pathway plays a key role in the anti-inflammatory protection of traditional Chinese medicine probiotic fermentation products and the monomeric compound with the structure of formula (I).

While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.