CN105920152B - A kind of effective part of Huajuhong and its application in the preparation of medicine for treating diabetic cardiomyopathy - Google Patents

Abstract

The invention relates to the technical field of natural medicines, and specifically discloses an effective fraction of citrus red and its application in the preparation of medicines for treating diabetic cardiomyopathy. The effective part of citrus red is prepared by a method comprising the following steps: S1. first extract citrus red with an organic solvent to obtain an organic solvent extract; S2. put the organic solvent extract on a macroporous adsorption resin column, first use a volume The ethanol with a fraction of 10% to 30% is eluted to remove impurities, and then eluted with ethanol with a volume fraction of 50% to 70%, and the ethanol elution site with a volume fraction of 50% to 70% is collected; S3. Obtained in step S2 50%~70% of the ethanol eluted fractions are enriched by silica gel column chromatography to obtain the effective fractions of Huajuhong. The effective fraction can significantly reduce the content of myocardial NF-κB protein in rats, and can be used for treating diabetic cardiomyopathy.

Description

A kind of effective part of citrus red and its use in the preparation of medicine for treating diabetic cardiomyopathy Applications

technical field

The invention relates to the technical field of natural medicines, in particular to an effective fraction of citrus red and its application in the preparation of medicines for treating diabetic cardiomyopathy.

Background technique

Diabetes is a common metabolic disorder that seriously threatens human life. The latest research shows that there are as many as 92.4 million diabetic patients in China, and 148 million pre-diabetic patients. Cardiovascular disease is the main cause of death for diabetic patients. Epidemiological studies have confirmed that more than 70% of diabetic patients die of cardiovascular disease. It is 2-3 times the mortality rate of cardiovascular system diseases in non-diabetic population. Among them, diabetic cardiomyopathy (diabetic cardiomyopathy, DCM) is one of the independent complications of diabetes, which has a high incidence and high risk, and is closely related to the high incidence and high mortality of cardiovascular diseases in diabetic patients. Currently

There is no effective treatment drug in clinical practice.

In recent years, a large number of studies have found that myocardial tissue inflammation is an important mechanism of diabetic cardiomyopathy, with massive infiltration of inflammatory cells in myocardial tissue and abnormal expression of inflammatory factors in local tissues; NF-κB is a central regulatory transcription factor for immune and inflammatory responses. Its activation is closely related to the occurrence and development of diabetic cardiomyopathy, and plays an important role in the changes of myocardial structure and function under hyperglycemia. After NF-κB activation, it can regulate a variety of inflammatory factors and cytokines (such as TNF-α , IL-6, IL-1β, etc.), thus participating in myocardial inflammatory injury.

Our country is rich in natural medicines and Chinese herbal medicines, and it is of great significance to develop a medicine that can treat diabetic cardiomyopathy from natural medicines and Chinese herbal medicines.

Contents of the invention

The technical problem to be solved by the present invention is to provide an effective part of citrus red in order to overcome the above-mentioned deficiencies of the prior art.

The above-mentioned technical problems to be solved by the present invention are realized through the following technical solutions:

An effective part of tangerine red, prepared by a method comprising the following steps:

S1. First, the orange red is extracted with an organic solvent to obtain an organic solvent extract;

S2. Put the organic solvent extract on a macroporous adsorption resin column, first use ethanol with a volume fraction of 10% to 30% to remove impurities, and then elute with ethanol with a volume fraction of 50% to 70%, and collect a volume fraction of 50%~70% ethanol elution site;

S3. The 50%~70% ethanol eluted fraction obtained in step S2 is enriched with effective components through silica gel column chromatography to obtain the effective fraction of Huajuhong.

The huajuhong is the immature or near-ripe dry outer pericarp of the Rutaceae plant Huazhou pomelo.

Preferably, the organic solvent in step S1 is ethanol or methanol.

Preferably, in step S2, the organic solvent extract is placed on a macroporous adsorption resin column, firstly eluted with 25% ethanol to remove impurities, and then eluted with 65% ethanol, and the collected volume fraction is 65% ethanol elution site.

More preferably, the volume fraction of 25% ethanol is 2 to 5 times the volume of the macroporous resin column used; the volume fraction of 65% ethanol is 3 to 6 times the volume of the macroporous resin column used. times.

Most preferably, the amount of ethanol with a volume fraction of 25% is 3 times the volume of the macroporous resin column used; the amount of ethanol with a volume fraction of 65% is 5 times the volume of the macroporous resin column used.

The column volume of the macroporous resin column refers to the volume from the bottom of the column to the deposition surface of the macroporous resin after the column is packed with the macroporous resin.

Preferably, the macroporous resin is a non-polar macroporous resin.

More preferably, the model of the macroporous resin is D101 macroporous resin.

Preferably, the amount of the macroporous resin used is 1 g of the dried organic solvent extract using a macroporous resin column with a column volume of 40-60 ml.

Most preferably, the amount of the macroporous resin used is 1 g of the dried organic solvent extract using a macroporous resin column with a column volume of 50 ml.

Preferably, the specific method of silica gel column chromatography enrichment in step S3 is: put the ethanol elution part prepared by step S2 on the silica gel column, first use solvent A with a volume ratio of 95:5~90:10: The mixed solvent system composed of solvent B is used for elution and removal of impurities; and then the mixed solvent system composed of solvent A:solvent B with a volume ratio of 85:15~80:20 is used for elution and the eluted parts are collected to obtain Huatangerine Effective parts; the solvent A is dichloromethane, and the solvent B is acetone.

Preferably, the weight of the silica gel used is 20 to 40 times the dry weight of the ethanol eluted part.

More preferably, the weight of the silica gel used is 30 times the dry weight of the ethanol eluted part.

More preferably, the specific method of silica gel column chromatography enrichment in step S3 is: put the ethanol elution part prepared by step S2 on the silica gel column, and firstly use solvent A:solvent B with a volume ratio of 90:10 to form The mixed solvent system is used for elution and removal of impurities; then the mixed solvent system with a volume ratio of 85:15 solvent A: solvent B is used for elution and the elution site is collected to obtain the effective site of orange red; the solvent A is dichloromethane, solvent B is acetone.

Further preferably, the solvent A with a volume ratio of 95:5 to 90:10: the amount of the mixed solvent system composed of solvent B is 1 to 3 times the volume of the silica gel column; the solvent with a volume ratio of 85:15 to 80:20 A: The amount of the mixed solvent system composed of solvent B is 3 to 5 times the volume of the silica gel column.

Most preferably, the volume ratio is 90:10 solvent A: the amount of the mixed solvent system composed of solvent B is 2 times the volume of the silica gel column column; the volume ratio is 85:15 solvent A: the mixed solvent system composed of solvent B The amount used is 5 times the volume of the silica gel column.

The column volume of the silica gel column refers to the volume from the column bottom to the silica gel deposition surface after packing the column with silica gel.

The application of the effective part of the above-mentioned citrus red in the preparation of a drug for treating diabetic cardiomyopathy.

The application of the above-mentioned effective part of citrus red as a regulator of NF-κB protein.

Beneficial effects: (1) The effective part of Huajuhong described in the present invention can significantly reduce the content of myocardial NF-κB protein in rats, and can be used to treat diabetic cardiomyopathy; (2) The preparation steps of the effective part of Huajuhong are simple and convenient, Suitable for mass production.

Detailed ways

The present invention is further explained below in conjunction with specific examples, but the examples do not limit the present invention in any form.

Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. Among them, the reagents or instruments used, those whose manufacturers are not indicated, are all commercially available conventional products.

Example 1

An effective part of tangerine red, prepared by a method comprising the following steps:

S1. At first, the citrus red is extracted with ethanol with a volume fraction of 95%, to obtain an ethanol extract;

S2. Put the ethanol extract on a macroporous adsorption resin column (the amount of macroporous resin used is: 1g of dry ethanol extract, use a macroporous resin column with a column volume of 50ml), first use a volume fraction of 3 times the column volume to 25 % ethanol to remove impurities, then elute with 65% ethanol by volume fraction, and collect the ethanol elution site with 65% volume fraction;

S3. The 65% ethanol elution part obtained in step S2 is enriched with active ingredients through silica gel column chromatography to obtain the effective part of Huatangerine; the enrichment method of the silica gel column chromatography is: the ethanol prepared through step S2 The eluted part is concentrated and dried, and then put on a silica gel column (the amount of silica gel is 30 times the weight of the ethanol eluted part after drying, and the specification of silica gel is 200 mesh). The mixed solvent system (amount of 2 times the column volume) was used for elution and impurity removal; and then the mixed solvent system (amount of 5 times the column volume) composed of solvent A: solvent B with a volume ratio of 85:15 was used for elution and Collect the eluted parts to obtain the effective parts of tangerine; the solvent A is dichloromethane, and the solvent B is acetone.

Example 2

An effective part of tangerine red, prepared by a method comprising the following steps:

S1. At first, the citrus red is extracted with ethanol with a volume fraction of 95%, to obtain an ethanol extract;

S2. Put the ethanol extract on a macroporous adsorption resin column (the amount of macroporous resin used is: 1g of dry ethanol extract, use a macroporous resin column with a column volume of 50ml), first use a volume fraction of 3 times the column volume to 10 % ethanol to remove impurities, then elute with 70% ethanol by volume fraction, and collect the ethanol elution site with 70% volume fraction;

S3. The 70% ethanol elution part obtained in step S2 is enriched by silica gel column chromatography to obtain the active ingredient of Huatangerine; the enrichment method of the silica gel column chromatography is: the ethanol prepared by step S2 The eluted part is concentrated and dried, and then put on a silica gel column (the amount of silica gel is 30 times the weight of the ethanol eluted part after drying, and the specification of silica gel is 200 mesh). The mixed solvent system (amount of 2 times the column volume) was used for elution and impurity removal; and then the mixed solvent system (amount of 5 times the column volume) composed of solvent A: solvent B with a volume ratio of 80:20 was used for elution and Collect the eluted parts to obtain the effective parts of tangerine; the solvent A is dichloromethane, and the solvent B is acetone.

Example 3

An effective part of tangerine red, prepared by a method comprising the following steps:

S1. At first, the citrus red is extracted with ethanol with a volume fraction of 95%, to obtain an ethanol extract;

S2. Put the ethanol extract on a macroporous adsorption resin column (the amount of macroporous resin used is: 1g of dry ethanol extract, use a macroporous resin column with a column volume of 50ml), first use a volume fraction of 3 times the column volume to 30 % ethanol to remove impurities, then elute with 70% ethanol by volume fraction, and collect the ethanol elution site with 50% volume fraction;

S3. Enrich the 50% ethanol eluted part obtained in step S2 through silica gel column chromatography to enrich the active ingredient to obtain the effective part of tangerine red; The eluted part is concentrated and dried, and then put on a silica gel column (the amount of silica gel is 30 times the weight of the ethanol eluted part after drying, and the specification of silica gel is 200 mesh). The mixed solvent system (amount of 2 times the column volume) was used for elution and impurity removal; and then the mixed solvent system (amount of 5 times the column volume) composed of solvent A: solvent B with a volume ratio of 85:15 was used for elution and Collect the eluted parts to obtain the effective parts of tangerine; the solvent A is dichloromethane, and the solvent B is acetone.

Example 4

Regulatory effect of the effective parts of Huajuhong of the present invention on cardiac inflammation in rats with diabetic cardiomyopathy

Main test materials: Wistar rats, male, body weight (160±20) g, SPF grade, qualification number SCXK Guangdong 2006-0015, provided by the Experimental Animal Center of Southern Medical University. They were reared in the Animal Laboratory of Guangdong Medical College under constant temperature (24±2)°C and relative humidity of 50% to 70%, and the feeding environment met the requirements for experimental animal environment facilities. Streptozotocin (STZ, Sigma, USA); NF-κB polyclonal antibody kit (Beijing Boaosen Biotechnology Co., Ltd.).

Animal modeling test method: Rats were fed with high-sugar and high-fat feed for 6 weeks to induce insulin resistance, and then intraperitoneally injected 1% streptozotocin 30 mg/kg at one time. After 72 hours, the fasting blood glucose ≥ 1 for 2 consecutive times. 1.1 mmol/L was determined as the diabetic model, and they were continuously fed with high-sugar and high-fat diet for 6 weeks.

Experimental method: Rats were divided into normal control group (10 rats fed with basal feed), model group (10 modeled rats), and experimental groups 1, 2 and 3 (10 modeled rats in each group). Rats were injected with the effective parts of tangerine prepared in Examples 1, 2 and 3 respectively, and the dosage was 20 mg/kg). At the end of the experiment, after fasting for 12 hours, anesthetized, the heart was quickly taken out after blood collection from the abdominal aorta, and the expression of myocardial NF-κB and the content of peripheral blood inflammatory factors were detected by immunohistochemistry. The results are shown in Table 1:

It can be seen from the data in Table 1 that compared with the normal group, myocardial NF-κB protein and peripheral blood TNF-α, IL-6, and IL-1β secretion in the model group increased, indicating that the modeling was successful. The effective parts of citrus red prepared by injecting Examples 1, 2 and 3 can significantly reduce the contents of rat myocardial NF-κB protein and peripheral blood TNF-α, IL-6, IL-1β, which shows that the present invention The effective part of Huajuhong can effectively reduce the content of NF-κB protein and peripheral blood TNF-α, IL-6, IL-1β in rats with diabetic cardiomyopathy, which further shows that it has a certain therapeutic effect on diabetic cardiomyopathy. From the above experimental data, it can also be seen that the effective fraction of citrus red prepared in Example 1 can reduce the content of rat myocardial NF-κB protein and peripheral blood TNF-α, IL-6, and IL-1β more significantly. , which illustrates that the effective fraction of tangerine red prepared under the conditions of Example 1 has a better effect.

Claims (7)

1. a kind of Exocarpium Citri Grandis active component, which is characterized in that be prepared by the inclusion of the method for following steps:

S1. Exocarpium Citri Grandis is extracted first with organic solvent, obtains extractive with organic solvent；

S2. it by large pore resin absorption column on extractive with organic solvent, is first cleaned with the ethanol elution that volume fraction is 10% ~ 30%, The ethanol elution for being again 50% ~ 70% with volume fraction, the alcohol elution that collected volume score is 50% ~ 70%；

S3. 50% ~ 70% alcohol elution step S2 obtained is active constituent-enriched up to Exocarpium Citri Grandis through silica gel column chromatography Active component；

Silica gel column chromatography is enriched in step S3 method particularly includes: by silica gel on the alcohol elution being prepared through step S2 Column, the solvent A for being first 90:10 with volume ratio: the mixed solvent system of solvent B composition carries out elution removal of impurities；Then body is used again Product is than the solvent A for 85:15: the mixed solvent system of solvent B composition is eluted and collects elution position to be had to get Exocarpium Citri Grandis Imitate position；The solvent A is methylene chloride, and solvent B is acetone；The weight of used silica gel is the 20 of alcohol elution dry weight ~ 40 times；Organic solvent described in step S1 is ethyl alcohol.

2. Exocarpium Citri Grandis active component according to claim 1, which is characterized in that will be organic in step S2

Large pore resin absorption column on solvent extractable matter is first cleaned with the ethanol elution that volume fraction is 25%, then is with volume fraction 65% ethanol elution, the alcohol elution that collected volume score is 65%；The dosage of the macroreticular resin, which is that 1g is dry, to be had The macroporous resin column that solvent extract is 40 ~ 60ml using column volume；The macroreticular resin is non-polar macroporous resin.

3. Exocarpium Citri Grandis active component according to claim 2, which is characterized in that the dosage for the ethyl alcohol that volume fraction is 25% It is 2 ~ 5 times of macroporous resin column column volume used；The dosage for the ethyl alcohol that volume fraction is 65% is macroporous resin column column used 3 ~ 6 times of volume.

4. Exocarpium Citri Grandis active component according to claim 3, which is characterized in that the dosage for the ethyl alcohol that volume fraction is 25% It is 3 times of macroporous resin column column volume used；The dosage for the ethyl alcohol that volume fraction is 65% is macroporous resin column cylinder used Long-pending 5 times；The macroreticular resin is non-polar macroporous resin.

5. Exocarpium Citri Grandis active component according to claim 1, which is characterized in that volume ratio is the solvent A of 90:10: solvent B The dosage of the mixed solvent system of composition is 1 ~ 3 times of silicagel column column volume；Volume ratio is the solvent A of 85:15: solvent B composition The dosage of mixed solvent system be 3 ~ 5 times of silicagel column column volume.

6. the described in any item Exocarpium Citri Grandis active components of claim 1 ~ 5 answering in the drug of preparation treatment diabetic cardiomyopathy With.

7. the described in any item Exocarpium Citri Grandis active components of claim 1 ~ 5 are as the application for preparing NF- kB protein regulator.