CN104013669A - Method for preparing cyclocarya paliurus total flavonoid with effect of reducing blood glucose - Google Patents

Abstract

The invention relates to a preparation method for obtaining total flavonoids with hypoglycemic effect from leaves of Cyclocarya paliurus. Take the dry leaves of Cyclocarya paliurana, add 8-12 times the amount of 70-85% ethanol aqueous solution, then reflux extraction for 3 times, each time for 2 hours, filter, combine the extracts, and concentrate. Take the concentrated dry extract and dissolve it in 75% ethanol, mix the sample with 30-60 mesh polyamide according to 1:1-3, put it on the column according to 1:10-15, and then use water, 40% ethanol, 45-60 % ethanol, 80-90% ethanol; and each gradient is eluted for 10 column volumes, of which 45-60% ethanol is divided into two sections, that is, 45-60% ethanol (the first 5 column volumes) and 45-60% ethanol Under 60% ethanol (the last 5 column volumes); the part under 45-60% ethanol and the part under 80-90% ethanol are combined to form the total flavonoids, and the content reaches more than 50%. Pharmacological experiments show that the total flavonoids of Cyclocarya paliurus has a good hypoglycemic effect.

Description

Preparation method of total flavonoids of Cyclocarya paliurus with hypoglycemic effect

technical field

The invention relates to a preparation method for obtaining total flavonoids with hypoglycemic effect from Cyclocarya paliurus leaves.

Background technique

Diabetes mellitus is a common multiple chronic disease caused by relative or absolute deficiency of insulin secretion and decreased sensitivity of target tissue cells to insulin, resulting in disturbance of sugar, protein, fat, water and electrolyte metabolism. Together with cancer and cardiovascular disease, it is called the world's three major diseases. The biggest feature is that the course of the disease is long and complicated, and it is difficult to cure. Clinically divided into two types: insulin-dependent (type 1) and non-insulin-dependent (type 2), of which type 2 diabetes accounts for 90% to 95%. Type 2 diabetes must strictly control blood sugar levels and related risk factors in order to improve the quality of life of patients. At present, there are about 360 million diabetic patients in the world. In 1985, there were only 30 million diabetic patients in the world. In 2000, the number increased to 150 million. It is estimated that by 2030, it will go straight to 600 million. With the rapid economic development, changes in lifestyle and prolongation of life expectancy, the prevalence of diabetes in China has increased rapidly. At present, the prevalence of diabetes in adults over the age of 20 in my country has exceeded 10%, and another 16% are on the verge of diabetes. It is estimated that about 3,000 new diabetic patients will be added every day. At present, my country has about 94 million diabetic patients, and has replaced India as the country with the largest number of diabetics in the world. Hyperglycemia is the main sign of diabetes. Hyperglycemia can cause microangiopathy and make the microcirculation of diabetic patients abnormal in varying degrees. With the prolongation of the disease course and untimely treatment, the aggravation and development of microvascular disease will be promoted, causing various acute and chronic complications throughout the body, involving multiple organs and tissues, leading to blindness, renal failure, cardiovascular disease, neuropathy, limb gangrene, etc., and even death. Diabetes has become the third "health killer" that causes human death after cardiovascular disease and malignant tumors. At present, 4 million people in the world die of diabetes or complications of diabetes every year. Diabetic blood sugar levels and related risk factors must be strictly controlled in order to improve the quality of life of patients.

Cyclocarya paliurus (Batal) Ijinskaja, also known as money tree and willow, is a plant of the genus Juglandaceae Cyclocarya paliurus (Batal) Ijinskaja. It is a unique national second-class protected tree species and an endangered plant species under national key protection. In thirteen provinces including Jiangxi, Guangxi, Sichuan, Guizhou, Hubei, etc. Cyclocarya paliurus has rich bioactive ingredients and high medical value. Its leaves, bark, and roots can be used as medicine. It is warm in nature, pungent and slightly bitter, and has the functions of clearing away heat, reducing swelling, detoxifying, and relieving pain (China Medicinal Materials Corporation. China Summary of Chinese Medicine Resources [M]. Beijing: Science Press, 1994); modern pharmacology shows that Cyclocarya paliurus extract has physiological activities such as lowering blood sugar, lowering blood pressure, and lowering blood fat. , anti-cancer and other physiological functions, especially in the prevention and treatment of diabetes (Wang Kequan, Cao Ying. Research progress on chemical constituents and pharmacological effects of Cyclocarya paliurus [J]. Heilongjiang Medicine. 2007, 8(31): 577-579) . Animal experiments and clinical studies have found that Cyclocarya paliurus leaves and their extracts or effective parts have obvious hypoglycemic effects, can effectively reduce postprandial blood sugar in mice, improve glucose tolerance in mice, and reduce the risk of diabetes in mice caused by high sugar and high fat. Blood sugar, cholesterol and triglyceride levels, and can improve insulin resistance.

Contents of the invention

The purpose of the present invention is to provide a preparation method of total flavonoids of Cyclocarya paliurus with hypoglycemic effect; the preparation method of the present invention is simple in process and operation, and can provide total flavonoids of Cyclocarya paliurus with stable performance and good pharmacological activity.

The preparation method of total flavonoids of Cyclocarya paliurus with hypoglycemic effect comprises the following steps:

(1) Extraction: take the dried leaves of Cyclocarya paliurus, soak them in aqueous ethanol solution, extract, obtain the extract, concentrate to obtain dry extract

(2) Polyamide column chromatography: Dissolve the extract obtained in step (1) with ethanol, mix it with polyamide, put it on a polyamide column, and then elute with ethanol solutions of different gradients to obtain eluent , concentrated, and the solvent was evaporated to obtain the total flavonoids of Cyclocarya paliurus.

The concentration of the aqueous ethanol solution used in the above step (1) is 70-85%, adding an extraction solvent equivalent to 8-12 times the weight of the medicinal material each time, heating and refluxing for extraction, keeping a slight boiling state for 2 hours each time, and extracting 3 times.

In the above step (2), the polyamide is 30-60 mesh, the ethanol concentration used for mixing the sample is 75%, the extract and the polyamide are mixed at a ratio of 1:1-3, and the column is loaded at a ratio of 1:10-15.

The ethanol solutions of different gradients described in the above step (2) are water, 40% ethanol, 45-60% ethanol, 80-90% ethanol respectively; The part is divided into two sections, that is, the upper part of 45-60% ethanol (the first 5 column volumes) and the lower part of 45-60% ethanol (the last 5 column volumes); the lower part of 45-60% ethanol and the part of 80-90% ethanol are combined It is the total flavonoids, and the content reaches more than 50%.

The total flavonoid content of Cyclocarya paliurus in the present invention has reached more than 50%, and can be made into any pharmaceutically acceptable preparations, such as oral administration preparations: tablets, pills, capsules, granules, dropping pills, Oral liquid, etc.

The total flavonoids of Cyclocarya paliurus prepared by the preparation method of the present invention, with acarbose as a contrast, set different concentration groups and carried out the in vitro α-glucosidase activity inhibition experiment, the results show that the total flavonoids of Cyclocarya paliurus has an effect on α-glucose Glucosidase has a strong inhibitory effect; the IC50 values of acarbose and total flavonoids of Cyclocarya paliurus are 58.608mg/L and 45.691mg/L, respectively.

The total flavonoids of Cyclocarya paliurus prepared by the preparation method of the present invention is in the experiment (glucose tolerance test) research of lowering the blood sugar of mice, 40 mice are divided into 5 groups, 8 in every group, respectively blank group, sucrose group, Sucrose + total flavonoids group, starch group, starch + total flavonoids group, taking the blood glucose concentration of the mice in each group as an index, taking blood from the orbit to measure the blood glucose concentration of the mice 1h and 2h after gavage, the results showed that the total Flavonoids have a good hypoglycemic effect.

The preparation steps of the total flavonoids of Cyclocarya paliurus in the present invention are less, the operation is simple, the cost is low, and the content of total flavonoids is high; and the total flavonoids of Cyclocarya paliurus has a strong inhibitory effect on α-glucosidase, can effectively lower blood sugar, and can be used It is suitable for the preparation of drugs for the prevention and treatment of diabetes, and points out the direction for the further development and utilization of the useful value of Cyclocarya paliurus.

Description of drawings

Starch and sucrose glucose tolerance test results.

Detailed ways

The content of the present invention will be further described in detail below through specific examples, but these examples should never be construed as limiting the present invention. Any changes made to the implementation of the present invention by those skilled in the art without departing from the above-mentioned idea of the present invention are included in the present invention.

The preparation of embodiment 1 Cyclocarya paliurus total flavonoids

(1) Extraction: Weigh 0.8Kg of dried and thick leaves of Cyclocarya paliurus, and reflux extract with 75% ethanol for 3 times, each time for 2 hours, and the amount of ethanol is 8000ml each time. Combine the extracts and recover ethanol to obtain dry extract of ethanol extract.

(2) Polyamide column chromatography: Weigh 125 g (30-60 mesh) of the treated polyamide, and wet-pack it into a column (column 5 cm×70 cm), with a column volume (BV) of 750 ml. Then weigh 10.0 g of the dry extract of the ethanol extract, dissolve it with 75% ethanol, mix it into 20 g of the treated polyamide, evaporate the ethanol to dryness, and put it into the column. Each gradient eluted 10BV with water, 40% ethanol, 50% ethanol (first 5 column volumes), 50% alcohol (last 5 column volumes), 80% ethanol, respectively. The 50% ethanol and 80% ethanol parts were combined, and the total flavonoid content was determined to be 55.66% by the aluminum nitrate color method.

Example 2 Inhibitory effect of total flavonoids of Cyclocarya paliurus on α-glucosidase activity

The inhibitory effect of total flavonoids of Cyclocarya paliurus on α-glucosidase activity was divided into blank group and different concentration groups of acarbose (final concentrations were 250mg/L, 125mg/L, 62.50mg/L, 31.25mg/L, respectively). , 15.63mg/L, 7.81mg/L, 7.81mg/L, 0mg/L) and different concentration groups of total flavonoids (final concentrations were 250mg/L, 125mg/L, 62.50mg/L, 31.25mg/L, 15.63 mg/L, 7.81mg/L, 7.81mg/L, 0mg/L). The operation procedure is to add 2 mL of phosphate buffer, 0.2 mL of NPG (4-nitrophenol-α-D glucopyranoside) solution, 0.1 mL of acarbose or total flavonoids of Cyclocarya paliurus into the test tube, and blank Add PBS0.3mL to the group, and put it in a constant temperature water bath at 37°C for 10min; then add 0.2mL of α-glucosidase solution (no addition in the blank group), put it in a constant temperature water bath at 37°C for 10min, add 1.5mL of ethanol to terminate the reaction, and use Shimadzu U V- 120-02 UV-Vis spectrophotometer to measure the absorbance at 405nm. The enzyme activity inhibition rate formula is as follows:

(A _{negative control} : the absorbance when the content of each part is 0mg/L)

Attached table 1 Experimental results of the inhibitory effect of total flavonoids of Cyclocarya paliurus on α-glucosidase activity

The IC ₅₀ values of acarbose and total flavonoids of Cyclocarya paliurus were 58.608mg/L and 45.691mg/L, respectively. The experimental data were processed with SPSS17.0 software.

Example 3 Glucose tolerance test of total flavonoids of Cyclocarya paliurus

Experimental animals: ICR male mice, purchased from Nanjing Qinglongshan Experimental Animal Center, weight range: 18-22 g.

experimental method

ICR male mice were adaptively fed for 2-3 days, and randomly divided into blank group, sucrose group, sucrose+total flavonoids group, starch group, starch+total flavonoids group, with 8 mice in each group. Before the experiment, the mice were fasted for 20 hours. Blank group: gavage 1ml of normal saline; sucrose group: gavage of 1ml sucrose solution (prepared with normal saline at a dose of 3g/Kg); sucrose+total flavonoids group: gavage of 0.5ml sucrose solution dosage preparation) and 0.5ml total flavonoids (prepared by 600mg/Kg dose with normal saline); starch group: gavage 1ml starch solution (prepared by 3g/Kg dose with normal saline); ml starch solution (prepared by 3g/Kg dosage with physiological saline) and 0.5ml 80% ethanol part (prepared by 600mg/Kg dosage with physiological saline). Blood was collected from the orbits of the animals in each group at 0, 1, and 2 hours after gavage, and the blood glucose values at each time point were measured with a glucose oxidase method kit. The experimental results are shown in Table 2.

Attached Table 2 Total Flavonoids Glucose Tolerance Test Results

The experimental data was statistically analyzed with SPSS17.0 software, and the results were expressed as Mean±SD, "p#<0.001" indicated that there was a significant difference between the blank group and the model group; "p**<0.01, p***< 0.001", indicating that there is a significant difference between the model group and the treatment group.

The total flavonoids mixed with sucrose and starch were administered to mice, and the blood glucose values after 1h and 2h were lower than those of 1h and 2h after sucrose and starch were administered alone, and the difference was significant (p<0.01), indicating that the total Flavonoids have a good effect on reducing blood sugar in mice.

Claims (8)

1. The preparation method of total flavonoids of Cyclocarya paliurus with hypoglycemic effect, comprising the following steps: (1) Extraction: get the dried leaves of Cyclocarya paliurus, soak with ethanol aqueous solution, extract, obtain extract, concentrate to obtain dry extract; (2) Polyamide column chromatography: Dissolve the extract obtained in step (1) with ethanol, mix it with polyamide, put it on a polyamide column, and then elute with ethanol solutions of different gradients to obtain eluent , concentrated, and the solvent was evaporated to obtain the total flavonoids of Cyclocarya paliurus. 2. The preparation method of total flavonoids of Cyclocarya paliurus according to claim 1, characterized in that, in step (1), the concentration of the aqueous ethanol solution used is 70-85%. 3. the preparation method of Cyclocarya paliurus total flavonoids according to claim 1, is characterized in that, in step (1), add the extraction solvent that is equivalent to 8～12 times weight of medical material weight at every turn, heat reflux extraction, every time The second extraction was kept in a slightly boiling state for 2 hours, and extracted 3 times. 4. the preparation method of Cyclocarya paliurus total flavonoids according to claim 1, is characterized in that, in step (2), polyamide is 30～60 orders, and the used ethanol concentration of mixing sample is 75%, and extract and polyamide Mix the sample according to 1:1~3, and load the column according to 1:10~15. 5. the preparation method of Cyclocarya paliurus total flavonoids according to claim 1 is characterized in that, in step (2), the ethanol solutions of described different gradients are respectively water, 40% ethanol, 45～60% ethanol, 80-90% ethanol. 6. The preparation method of total flavonoids of Cyclocarya paliurus according to claim 1, is characterized in that, the ethanol solutions of different gradients described in step (2) all elute 10 column volumes, wherein 45～60% ethanol parts Divided into two sections, that is, above 45-60% ethanol (the first 5 column volumes) and under 45-60% ethanol (the last 5 column volumes). 7. the preparation method of Cyclocarya paliurus total flavonoids according to claim 1, is characterized in that, in step (2), 45～60% ethanol lower part and 80～90% ethanol part merge and are total flavonoids, and content reaches above 50. 8. The application of total flavonoids of Cyclocarya paliurus in the preparation of hypoglycemic drugs or health care products according to the preparation method described in claims 1-7.