Method for extracting and purifying total iridoid glycoside compounds in dogwood

Abstract

The invention discloses a method for extracting and purifying total iridoid glycoside compounds in dogwood, and relates to the technical field of medicine extraction and purification, and the method is characterized by comprising the following steps: precisely weighing dogwood powder, adding an ethanol solution with the volume fraction of 50%, carrying out heating reflux extraction for 80 minutes at 80 DEG C according to the solid-liquid ratio of 1: 25, collecting an extracting solution, centrifuging, taking supernate, fixing the volume to 25ml / 1g of crude medicine, and drying to obtain the total iridoid glycoside compounds in dogwood. Determining the content by adopting high performance liquid chromatography; the method comprises the following steps: weighing macroporous adsorption resin, packing the macroporous adsorption resin into a column, weighing dogwood adsorption stock solution with the concentration of 0.1 g crude drug / ml according to the ratio of the amount of the macroporous adsorption resin to the dogwood crude drug being 4: 1, loading the sample, standing, eluting with pure water to remove impurities, and eluting with 30% ethanol water for 9 retention volumes to obtain the dogwood total iridoid glycoside extract with the morroniside content of 371.91 mg / g and the total iridoid glycoside content of 371.91 mg / g. The content of loganin is 216.61 mg / g, and the content is increased by about 22 times.

Description

Technical Field

[0001] The present invention relates to the technical field of drug extraction and purification. More specifically, it relates to a method for extracting and purifying total iridoid glycosides from Cornus officinalis Sieb. et Zucc. Background Art

[0002] Cornus officinalis Sieb. et Zucc. is a Chinese medicinal material that can be used both as medicine and food. It tastes sour and astringent, and is slightly warm in nature. Its functions include tonifying the liver and kidney, astringing essence, and nourishing blood, and it is a commonly used tonic medicine. For example, it is included in the prescriptions of Liuwei Dihuang Pills, Shenqi Pills, Zuogui Pills, Yougui Pills, etc. Liuwei Dihuang Pills with Cornus officinalis Sieb. et Zucc. as the main ingredient have good effects on preventing and treating dependent diabetes. A total of 66 iridoid glycoside components of Cornus officinalis Sieb. et Zucc. have been reported in the existing literature. The protective effects of these components on diabetes and its complications, brain tissue, nervous system, and liver injury, as well as their anti-inflammatory and hair growth-regulating biological activities, are reviewed from multiple aspects such as pharmacological activities, molecular mechanisms, and action pathways, in order to explore new ideas and directions for the in-depth research, development, and utilization of iridoid glycosides from Cornus officinalis Sieb. et Zucc.

[0003] At the same time, Cornus officinalis Sieb. et Zucc. is also one of the main components of the Chinese patent medicine Shensong Yangxin Capsule for treating cardiovascular diseases, and has pharmacological effects such as anti-shock and cardiotonic effects. It can improve myocardial nutrient delivery, increase cardiac output, enhance myocardial contractility, improve myocardial energy metabolic disorders in heart failure rats, treat heart failure and acute myocarditis, and protect myocardial cells damaged by high glucose. It also has the effect of preventing hypertension and hyperlipidemia, preventing atherosclerosis in the human body, and reducing the incidence of coronary heart disease.

[0004] However, in the existing technology, the extraction and purification process of total iridoid glycosides from Cornus officinalis Sieb. et Zucc. is relatively complex, and the purity of the finally obtained total iridoid glycosides is low, which is not conducive to further processing them into corresponding drugs, foods, and health products. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for extracting and purifying total iridoid glycosides from Cornus officinalis Sieb. et Zucc., which can effectively increase the extraction amount of total iridoid glycosides from Cornus officinalis Sieb. et Zucc. and the purity of the extract.

[0006] The above technical purpose of the present invention is achieved through the following technical solutions: A method for extracting and purifying total iridoid glycosides from Cornus officinalis Sieb. et Zucc., the method comprising two steps: extraction and purification:

[0007] The extraction method is as follows: Weigh accurately 1.000 g of Cornus officinalis Sieb. et Zucc. powder, dissolve it in an ethanol solution with a volume fraction of 50%, the material-liquid ratio of Cornus officinalis Sieb. et Zucc. powder to ethanol is 1:25, heat under reflux at a temperature of 80 °C for 80 min, transfer the extract to a centrifuge tube, take out the supernatant after centrifugation, make up the volume to 25 ml, filter through a microporous filter membrane, and determine its content by high performance liquid chromatography to obtain the total iridoid glycoside extract of Cornus officinalis Sieb. et Zucc.

[0008] The purification method is as follows:

[0009] Weigh 50 g of macroporous adsorption resin and load it into a column with a column height of 11 cm and a retention volume of 65 ml. Measure the adsorbed stock solution of Cornus officinalis Sieb. et Zucc. according to the ratio of the amount of macroporous adsorption resin to the crude drug of Cornus officinalis Sieb. et Zucc. of 4:1. The concentration of the adsorbed stock solution of Cornus officinalis Sieb. et Zucc. is 0.1 g of crude drug / ml. The loading flow rate is 1 ml / min, let it stand, the elution flow rate is 5 ml / min, let it stand, wash with pure water to remove impurities, and then elute with 30% ethanol-water for 9 retention volumes.

[0010] The present invention is further configured such that: the macroporous adsorption resin includes but is not limited to D-101 type, AB-8 type, ADS-17 type or HPD-600 type.

[0011] The present invention is further configured such that: the macroporous adsorption resin is preferably AB-8 type.

[0012] The present invention also provides an application of the total iridoid glycoside compounds in Cornus officinalis Sieb. et Zucc. in the preparation of drugs, health products and foods for preventing and treating heart failure.

[0013] To sum up, the present invention has the following beneficial effects: The extraction method of the present invention is simple and easy to operate, suitable for industrial production, and can extract the iridoid glycoside compounds in Cornus officinalis Sieb. et Zucc. to the greatest extent. Its purification method greatly increases the content of the iridoid glycoside compounds in Cornus officinalis Sieb. et Zucc., which is beneficial to the subsequent processing of them into corresponding drugs, health products and foods. Description of the Drawings

[0014] Figure 1 It is the content result diagram of morroniside at different temperatures in Example 1 of the present invention;

[0015] Figure 2 It is the content result diagram of loganin at different temperatures in Example 1 of the present invention;

[0016] Figure 3 It is the content result diagram of morroniside at different ethanol concentrations in Example 1 of the present invention;

[0017] Figure 4 It is the content result diagram of loganin at different ethanol concentrations in Example 1 of the present invention;

[0018] Figure 5It is the content result diagram of morroniside at different extraction times in Example 1 of the present invention;

[0019] Figure 6 It is the content result diagram of loganin at different extraction times in Example 1 of the present invention;

[0020] Figure 7 It is the content result diagram of morroniside at different solid-liquid ratios in Example 1 of the present invention;

[0021] Figure 8 It is the content result diagram of loganin at different solid-liquid ratios in Example 1 of the present invention;

[0022] Figure 9 It is the isogram and contour map of A (temperature) and B (ethanol concentration) in Example 1 of the present invention;

[0023] Figure 10 It is the isogram and contour map of A (temperature) and C (solid-liquid ratio) in Example 1 of the present invention;

[0024] Figure 11 It is the isogram and contour map of B (ethanol concentration) and C (solid-liquid ratio) in Example 1 of the present invention;

[0025] Figure 12 It is the isogram and contour map of A (temperature) and B (ethanol concentration) in Example 1 of the present invention;

[0026] Figure 13 It is the isogram and contour map of A (temperature) and C (solid-liquid ratio) in Example 1 of the present invention;

[0027] Figure 14 It is the isogram and contour map of B (ethanol concentration) and C (solid-liquid ratio) in Example 1 of the present invention;

[0028] Figure 15 It is to analyze the purification of iridoid glycosides from Cornus officinalis by different types of macroporous resins with the contents of morroniside and loganin as indicators in Example 2 of the present invention;

[0029] Figure 16 It is the elution ratio of morroniside in Example 2 of the present invention;

[0030] Figure 17 It is the elution ratio of loganin in Example 2 of the present invention;

[0031] Figure 18 It is the schematic diagram of the component contents of iridoid glycosides from Cornus officinalis in Examples 1 and 2 of the present invention; (among them, the one at 9.181 min is morroniside; the one at 14.080 min is loganin)

[0032] Figure 19It is the result graph of the in vitro heart perfusion experiment of the total extract of Cornus officinalis in SD rats in Example 3 of the present invention (A statistical graph of the heart rate change before and after administration; B statistical graph of the change in the excitation conduction velocity of the left ventricle; C Figure 2 Statistical graph of the change in the QRS wave width of the electrocardiogram lead; D statistical graph of the change in the relative refractory period of the left ventricle; * indicates p < 0.05 compared with the control group; ** indicates p < 0.01 compared with the control group)

[0033] Figure 20 It is the representative graph before and after administration of the in vitro heart perfusion experiment of the total extract of Cornus officinalis in SD rats in Example 3 of the present invention; (A representative graph of the excitation conduction time; B representative graph of the left ventricular field potential; C Figure 2 Representative graph of the change in the QRS wave width of the electrocardiogram lead)

[0034] Figure 21 It is the HE staining result graph of the heart tissue of SD rats in Example 3 of the present invention;

[0035] Figure 22 It is the effect graph of the iridoid glycosides from Cornus officinalis in Example 3 of the present invention. Specific embodiments

[0036] The following is a further detailed description of the present invention in conjunction with the attached Figure 1-22 drawings.

[0037] Example 1: Optimization of the extraction process of Cornus officinalis

[0038] (1) Temperature

[0039] Precisely weigh 1.000 g of Cornus officinalis powder, and set the heating reflux temperatures to 60 °C, 70 °C, 80 °C, 90 °C, and 100 °C respectively. The ethanol volume fraction is 50%, the solid-liquid ratio is 1:20, and the extraction time is 80 min. Transfer the extract to a centrifuge tube, centrifuge, take out the supernatant, make up the volume to 25 ml, filter through a microporous membrane, and determine its content by high performance liquid chromatography. The extraction effect is the best at 80 °C. Therefore, a response surface experiment is designed with 80 °C as the central point of temperature.

[0040] (2) Ethanol concentration

[0041] Precisely weigh 1.000 g of Cornus officinalis powder, and set the heating reflux temperature to 80 °C. The ethanol volume fractions are set to 10%, 30%, 50%, 70%, and 90% respectively. The solid-liquid ratio is 1:20, and the extraction time is 80 min. Transfer the extract to a centrifuge tube, centrifuge, take out the supernatant, make up the volume to 25 ml, filter through a microporous membrane, and determine its content by high performance liquid chromatography. The extraction effect is the best when the ethanol volume fraction is 50%. Therefore, a response surface experiment is designed with 50% ethanol concentration as the central point.

[0042] (3) Extraction time

[0043] Accurately weigh 1.000 g of Cornus officinalis Sieb. et Zucc. powder, heat under reflux at 80 °C, with an ethanol volume fraction of 50%, a solid-liquid ratio of 1:20, and extraction times of 30, 60, 80, 100, and 120 min. Transfer the extract to a centrifuge tube, centrifuge, and take out the supernatant. Dilute to 25 ml, filter through a microporous membrane, and determine its content by high-performance liquid chromatography. The extraction time has no significant effect. Therefore, the extraction time is not considered as a factor.

[0044] (4) Solid-liquid ratio

[0045] Accurately weigh 1.000 g of Cornus officinalis Sieb. et Zucc. powder, heat under reflux at 80 °C, with an ethanol volume fraction of 50%, and set the solid-liquid ratios to 1:5, 10, 15, 20, and 25 respectively. The extraction time is 80 min. Transfer the extract to a centrifuge tube, centrifuge, and take out the supernatant. Dilute to 25 ml, filter through a microporous membrane, and determine its content by high-performance liquid chromatography. The extraction efficiency is the highest at 1:20. Therefore, a response surface experiment is designed with a solid-liquid ratio of 1:20 as the central point.

[0046] Based on the above test results, comprehensively considering, using the Box-Behnken design principle, a three-factor and three-level experiment is designed.

[0047] Table 1 Three-factor and three-level experiment

[0048]

[0049] According to Design-Expert 13, input the response surface factor level table to obtain the following experimental design table. Taking temperature, ethanol concentration, and solid-liquid ratio as independent variables, the extraction time is 80 min, and taking loganin and morroniside as response values, the extraction rate Y and the regression model variance analysis table are obtained.

[0050] Since loganin and morroniside are the main components of the total iridoid glycosides in Cornus officinalis Sieb. et Zucc., loganin and morroniside are used as detection indicators in this method.

[0051] Table 2 Box-Behnken experimental design and its results

[0052]

[0053]

[0054] Table 3 Data analysis table of loganin response surface

[0055]

[0056] Using Design Expert software, perform multiple regression fitting on the table to obtain the binary multiple regression equation for the content of morroniside (Y). The regression model equation for temperature (A), ethanol concentration (B), and solid-liquid ratio (C) is as follows:

[0057] Y = -36.2558 + 1.22356A + 0.193682B - 0.000074C - 0.0000000478A*B - 0.000032A*C + 0.004278B*C - 0.007615A 2 - 0.00259B 2 + 0.001757C 2

[0058] As can be seen from the table, the solid-liquid ratio (C) has the greatest impact on the extraction rate of morroniside, followed by the ethanol concentration (B), and the temperature (A) has the least impact on morroniside. Figure 9 、 10 Figures 11 represent the contour plots and surface plots of each factor, reflecting the influence of each factor on the result.

[0059] Table 4 Analysis table of loganin response surface data

[0060]

[0061] Using Design Expert software, perform multiple regression fitting on the table to obtain the binary multiple regression equation for the content of loganin (Y). The regression model equation for temperature (A), ethanol concentration (B), and solid-liquid ratio (C) is as follows:

[0062] Y = 10.27 + 0.0704A + 0.0997B + 0.44C + 0.5501A*B - 0.0337A*C - 0.0067B*C - 0.7398A 2 -- 0.392B 2 + 0.2814C 2

[0063] The contour plot of loganin is similar to that of morroniside. The temperature (A) has the greatest impact on the extraction rate of loganin, followed by the ethanol concentration (B), and the solid-liquid ratio (C) has the least impact on loganin. Figure 12 、 13 Figures 14 represent the contour plots and surface plots of each factor, reflecting the influence of each factor on the result.

[0064] Optimal extraction process of Cornus officinalis, using Design Expert to select the conditions with the maximum contents of morroniside and loganin, i.e., temperature of 78.4136 °C, ethanol concentration of 48.4115%, and solid-liquid ratio of 24.1551, as shown in Table 5. Considering the actual production conditions, a temperature of 80 °C, ethanol concentration of 50%, and solid-liquid ratio of 1:25 are adopted. At this time, the content of morroniside is 17.61 mg / g and the content of loganin is 10.38 mg / g, which is close to the expected values of 18.5416 and 10.8016.

[0065] Table 5 Optimal extraction process of Cornus officinalis

[0066]

[0067] Example 2: Optimization of the process for purifying iridoid glycosides from Cornus officinalis by macroporous resin

[0068] Preparation of the original solution for adsorption of Cornus officinalis: Weigh 30 g of Cornus officinalis powder, soak it in 20 times the amount of 50% ethanol overnight, heat under reflux at 80 °C for 60 min, filter, concentrate the filtrate until the alcohol smell disappears, and make up the volume to a solution of 0.1 g of crude drug / ml with pure water. Let it stand and filter to obtain the original solution for adsorption of Cornus officinalis, which is stored at 4 °C for later use. HPLC analysis was used to determine that the concentration of morroniside in the original solution for adsorption of Cornus officinalis was 1.65 mg / ml, the content of morroniside was 16.5 mg / g, the concentration of loganin was 0.94 mg / ml, and the content of loganin was 9.4 mg / g.

[0069] Static adsorption and desorption: Take 10 ml of the original solution for adsorption of Cornus officinalis, 4 portions. Weigh 1 g of each of the four different types of macroporous resins, namely D-101 type (non-polar), AB-8 type (weakly polar), ADS-17 type (medium polar), and HPD-600 type (polar), into 50 ml conical flasks, add 10 ml of the medicinal liquid to each, shake well, and place in a constant temperature shaker for shaking for 12 h (25 °C, 100 r / min). After filtration, take 20 μl of the supernatant from each for HPLC analysis to determine the adsorption rate. Then add 10 ml of 50% ethanol to each for desorption, shake well, and place in a shaker for shaking for 5 h (25 °C, 100 r / min). After filtration, take 20 μl of the supernatant from each for HPLC analysis to determine the desorption rate.

[0070] Adsorption rate = (original solution concentration × 10 ml - adsorbed solution concentration × 10 ml) / (original solution concentration × 10 ml)

[0071] Desorption rate = (desorbed solution concentration × 10 ml) / (original solution concentration × 10 ml - adsorbed solution concentration × 10 ml) Table 6 Adsorption rates of four different types of macroporous adsorption resins

[0072]

[0073] Table 7 Desorption Rates of Four Different Types of Macroporous Adsorption Resins

[0074]

[0075] After calculation, the adsorption rates of iridoid glycosides from Cornus officinalis Sieb. et Zucc. on the four macroporous adsorption resins were AB-8 type > HPD-600 type > ADS-17 type > D-101 type, and the desorption rates were ADS-17 type > AB-8 type > D-101 type > HPD-600 type.

[0076] Investigation of the amount of eluent and macroporous resin types: Four different types of macroporous resins were packed into columns (70 g each), with a column height of 14.5 cm and a retention volume of approximately 90 ml. After washing with water until the alcohol smell disappeared, four portions of 50 ml of the original adsorption solution of Cornus officinalis Sieb. et Zucc. were respectively loaded onto the columns at a flow rate of 1 ml / min, and left standing overnight after loading. They were successively eluted with 0% (10 BV), 10% (10 BV), 30% (10 BV), 50% (10 BV), 70% (10 BV), and 90% (5 BV) ethanol at an elution flow rate of 5 ml / min. The eluents were collected, concentrated under reduced pressure, and made up to 25 ml with water. 5 ml of each eluent was placed in a weighing dish, evaporated to dryness, and the mass of each elution sample was measured. Another portion of each eluent was determined by high performance liquid chromatography to calculate the contents of loganin and morroniside. According to the chart, it was determined that AB-8 type macroporous adsorption resin and 30% ethanol as the eluent gave the highest contents of loganin and morroniside.

[0077] Table 8 Determination Results of Loganin and Morroniside - I

[0078]

[0079]

[0080] Investigation of the amount of eluent: 50 g of AB-8 type macroporous resin was weighed and packed into a column with a column height of 11 cm and a retention volume of 65 ml. According to the ratio of macroporous resin amount to crude drug of approximately 4:1, 120 ml of the original adsorption solution of Cornus officinalis Sieb. et Zucc. with a concentration of 0.1 g crude drug / ml was measured, and the flow rate was 1 ml / min, then left standing. It was successively eluted with 0% (3 BV), 10% (10 BV), 30% (15 BV), and 50% (8 BV) ethanol concentrations at an elution flow rate of 5 ml / min. Each 1 BV was taken as one fraction, and there were a total of 38 fractions. It was determined by high performance liquid chromatography to calculate the contents of loganin and morroniside, and an elution curve was plotted. It was determined that the elution method was to remove other impurities such as polysaccharides by pure water elution first, and then elute with 30% ethanol aqueous solution for 9 retention volumes.

[0081] Table 9 Determination Results of Loganin and Morroniside - II

[0082]

[0083]

[0084] The process for purifying iridoid glycosides from Cornus officinalis Sieb. et Zucc. using macroporous resin was determined as follows: AB-8 macroporous adsorption resin was selected for the enrichment of iridoid glycosides from the extract of Cornus officinalis Sieb. et Zucc. The sample was loaded at a ratio of macroporous resin to crude drug of 4:1. After removing impurities by washing with water, it was eluted with 30% ethanol-water for 9 retention volumes. After purification, the content of morroniside was 371.91 mg / g, and the content of loganin was 216.61 mg / g. The contents of morroniside and loganin were increased by about 22 times

[0085] Example 3: Pharmacological activity study of total iridoid glycosides

[0086] The effects of the total extract of Cornus officinalis Sieb. et Zucc. on the hearts of normal SD rats were studied by using the Langendorff perfusion experiment of isolated hearts of SD rats in vitro; the preventive and therapeutic effects of the total extract of Cornus officinalis Sieb. et Zucc. on heart failure were studied by inducing heart failure in SD rats with adriamycin in vivo; in vitro cell experiments found that total iridoid glycosides from Cornus officinalis Sieb. et Zucc. had a protective effect on cardiomyocytes damaged by H2O2 oxidation

[0087] (1) Langendorff perfusion electrocardiogram measurement experiment of the total extract of Cornus officinalis Sieb. et Zucc. on isolated hearts

[0088] Main experimental instruments: MappingLab electrocardiogram measurement system, Langendorff perfusion system, stimulator, constant temperature water bath, peristaltic pump

[0089] Instruments: 2 pairs of surgical scissors, 1 pair of ophthalmic scissors, 2 pairs of ophthalmic curved forceps, 1 pair of hemostatic forceps, surgical sutures, 1 1000 ml beaker, 1 100 ml measuring cylinder, 1 250 ml measuring cylinder, 1 1000 ml volumetric flask, 1 2000 ml volumetric flask, 2 1 ml syringes, 1 20 ml syringe, 1 vascular cannula, 1 10 cm glass culture dish, 1 3.5 cm culture dish, 1 1000 μl pipette, 1 5000 μl pipette, 0.45 μm filter membrane, 0.22 μm filter membrane

[0090] Solution preparation:

[0091] KH solution (perfusate):

[0092] C (mM) <![CDATA[MW(g.mol -1 )]]> g / L NaCl 119 58.44 6.9544 <![CDATA[NaHCO3]]> 25 84.01 2.1003 KCl 4 74.55 0.2982 <![CDATA[KH2PO4]]> 1.2 136.08 0.1633 <![CDATA[MgCl2]]> 1 95.2 0.0952 <![CDATA[CaCl2.2H2O]]> 1.8 146.98 0.2646 D-glucose 10 180.16 1.8016

[0093] 10×KH stock solution:

[0094] C (mM) <![CDATA[MW(g.mol -1 )]]> g / L NaCl 119 58.44 69.544 <![CDATA[NaHCO3]]> 25 84.01 21.003 KCl 4 74.55 2.982 <![CDATA[KH2PO4]]> 1.2 136.08 1.633 <![CDATA[MgCl2]]> 1 95.2 0.952

[0095] Heparin sodium: Diluted with normal saline to 1000 U / ml

[0096] Dogwood mother liquor: Take 12 g of dogwood extract (about 2 g of crude drug per 1 g of extract), add 120 ml of KH solution, dissolve it, centrifuge at 4000 rpm for 3 min, and take the supernatant. Filter it successively with 0.45 m and 0.22 m filter membranes to obtain 120 ml of dogwood mother liquor (SZY 200 mg / ml). Store it at 4 °C for later use and filter it with a 0.22 m filter membrane before each use.

[0097] Experimental procedure:

[0098] Weigh the rats (SD rats, male, 6 - 8 weeks old), intraperitoneally inject sodium heparin (3125 U / kg), and decapitate them 15 min later.

[0099] Then place them on the experimental table for fixation, make a "T"-shaped thoracotomy to expose the heart. Use forceps to pick up the lung to lift the heart, and quickly cut off the heart along the back of the lung. Place the heart in a glass petri dish containing pre-cooled Tyrode's solution.

[0100] Quickly find the aorta, cut off the excess tissue, carefully put the aorta on the bottom of the cannula, tie it tightly with surgical sutures, and gently push the pre-prepared KH solution in the syringe into the heart to pump out the residual blood in the heart for Langendorff perfusion. The perfusion rate is 10 mL / min, and the perfusion temperature is 37 ± 0.5 °C.

[0101] Insert a stimulating electrode at the apex of the heart (near the recording electrode), contact the MappingLab matrix multi-channel electrode on the epicardium of LA (left atrium) and LV (left ventricle), and place ECG electrodes on the right atrium and left ventricle of the heart.

[0102] After the heart is stable, the MappingLab matrix multi-channel electrophysiological mapping system records the signal (baseline).

[0103] Determine the diastolic pacing threshold: Perform train stimulation, slowly increase the current intensity (pulse duration is 2 ms), and observe the electrical signal at the same time; when there is obvious 1:1 conduction according to the set stimulation frequency, it is the diastolic threshold current intensity; set the pacing current intensity to 2 × the diastolic threshold current intensity.

[0104] Under the 6 Hz, S1S2 stimulation mode, the MappingLab matrix multi-channel electrophysiological mapping system records the signal

[0105] Administer SZY 5 mg / ml for the first time, circulate the administration, and record the spontaneous electrical signal 1 - 10 min after administration. Then give 6 Hz and S1S2 stimulation (parameters are the same as above) and record the data.

[0106] Administer SZY 10 mg / ml for the second time, circulate the administration, and record the spontaneous electrical signal 1 - 10 min after administration. Then give 6 Hz and S1S2 stimulation (parameters are the same as above) and record the data.

[0107] Administer Iso at 1M for the third time in a cyclic manner, and record the spontaneous electrical signals from 1 to 10 minutes after administration. Then give 6Hz and S1S2 stimulations (parameters as above) and record the data.

[0108] Perfuse the heart with normal KH for 20 minutes and record the spontaneous electrical signals. Then give 6Hz and S1S2 stimulations (parameters as above) and record the data.

[0109] Data analysis:

[0110] Use a commercially available analysis program (EMapScope 5.0, MappingLab, UK) for data analysis. It can analyze the heart rate under spontaneous rhythm, the conduction time, conduction velocity, and conduction dispersion of the atrium and ventricle; the conduction time and conduction velocity of the ventricle under 6hz stimulation; data such as the effective refractory period (ERP), ECG QRS wave width, and atrioventricular delay.

[0111] Experimental results:

[0112] Determine whether the anti-arrhythmic mechanism of the extract of Cornus officinalis is related to changes in cardiac electrical conduction and other characteristics. First, use multi-channel cardiac electrophysiological mapping technology to evaluate the conduction and other parameters of the extract of Cornus officinalis (SZY) on the normal heart. The experimental results show that low-concentration (5mg / ml) SZY and high-concentration (10mg / ml) SZY reduce the heart rate of isolated rat hearts, have a tendency to prolong the conduction time of the left atrium and left ventricle, prolong the relative refractory period and atrioventricular delay time, increase the conduction inhomogeneity of the left atrium and left ventricle, and the above effects are more significant with higher SZY concentrations.

[0113] (2) Experiment on the prevention and treatment of adriamycin-induced heart failure in SD rats with the total extract of Cornus officinalis

[0114] Model establishment, grouping, and administration:

[0115] Purchase 24 SD rats. After 1 week of adaptive feeding, randomly divide them into groups of 8 rats each. Rats in the model group were intraperitoneally injected with adriamycin at 3mg / kg once a week for a total of 4 weeks to prepare a heart failure model. Meanwhile, rats in the blank group were intraperitoneally injected with an equal volume of normal saline. Rats in the administration group were intragastrically administered the total extract of Cornus officinalis (100mg crude drug / kg) every day. Rats in the blank group and the model group were intragastrically administered an equal volume of normal saline once a day for 5 consecutive weeks.

[0116] Observe the pathological morphology of myocardial tissue by HE staining:

[0117] After taking blood from the abdominal aorta, quickly remove the heart, take the myocardial tissue near the auricle and fix it in 4% paraformaldehyde, embed it in paraffin routinely, cut into continuous 5μm sections, bake, dewax, and perform HE staining. Observe the tissue morphology under a microscope and randomly take images for evaluation.

[0118] Normal group: The myocardial cells were arranged neatly, densely, and with clear structure, and no inflammatory necrosis was seen. Model group: The myocardial cells were arranged disorderly, the cell gaps were enlarged, the cell morphology was blurred and irregular, the fibroblasts increased, and there was infiltration of inflammatory cells. Cornus officinalis group: Compared with the model group, the morphology of rat myocardial cells was slightly better, but the cell morphology was also slightly irregular, the myocardial cells were lightly stained, and the cell morphology was enlarged. In conclusion, the total extract of Cornus officinalis had obvious improvement in cardiac staining compared with the model group, indicating that the total extract of Cornus officinalis had a certain repairing and improving effect on the cardiac function of rats with adriamycin-induced heart failure.

[0119] (3) Experiment on the in vitro protection of myocardial cells by the total iridoid glycosides of Cornus officinalis

[0120] Screening of drug administration concentration:

[0121] H9C2 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) + 1% double antibody at 37 °C in a 5% CO2 incubator with saturated humidity. When the cell growth range covered 70% - 80% of the medium, 1 mL of 0.05% trypsin was added to digest the cells, and then the adherent cells were blown off the medium with a sterile pipette tip to prepare a single-cell suspension. A small amount of the remaining cell suspension was taken, diluted, and counted to determine the cell concentration. The diluted cell concentration was 8×10 4 / mL and inoculated into 96-well plates and incubated overnight in the incubator. After 24 h, drugs were added, and the drug concentrations were 100, 50, 25, 12.5, 6.25, 0 μg / mL. After incubating in the incubator for 24 h, CCK-8 was added, 8 μL / well. After incubating in the incubator for 3 h, the OD value was measured at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and the cell viability was calculated to determine that the drug administration concentrations of the total iridoid glycosides of Cornus officinalis were 50, 25, 12.5 μM.

[0122] Determination of SOD activity:

[0123] The diluted cell concentration was 8×10 4 / mL, inoculated into 6-well plates and incubated overnight in an incubator. After 24 h, drugs were added at concentrations of 50, 25, 12.5 μM. After incubating in the incubator for 24 h, H2O2 was added. The cells were starved for 2 h before adding H2O2 (incubated in a serum-free medium in the incubator for 2 h), then H2O2 was added at 200 μM / well, and the model establishment was terminated after 2 h. The experiment was operated according to the instructions of the SOD kit. The results showed that compared with the normal control group, the SOD activity in the oxidative damage group was significantly decreased, and the difference was statistically significant (P < 0.05); the SOD activity of the positive drug (Y, 20 μg / l vitamin C) was statistically significant compared with that in the oxidative damage group (P < 0.05); compared with the oxidative damage group, the middle and low doses in the drug administration group significantly increased the SOD activity of cardiomyocytes (P < 0.05). Total iridoid glycosides from Cornus officinalis have an obvious protective effect on cardiomyocytes damaged by H2O2.

[0124] This specific embodiment is only an explanation of the present invention, and it is not a limitation of the present invention. After reading this specification, those skilled in the art can make modifications to this embodiment without creative contributions as needed, but as long as it is within the scope of the claims of the present invention, it is protected by the patent law.

Claims

1. A method for extracting and purifying total iridoid glycosides in Cornus officinalis, characterized in that: The method comprises two steps of extraction and purification: The extraction method is as follows: accurately weigh 1.000g of cornus officinalis powder, dissolve it in an ethanol solution with a volume fraction of 50%, the solid-liquid ratio of cornus officinalis powder to ethanol is 1:25, heat and reflux at a temperature of 80°C, extract for 80 minutes, transfer the extract to a centrifuge tube, centrifuge, take out the supernatant, fix the volume to 25ml, filter with a microporous filter membrane, and determine the content of the supernatant by high performance liquid chromatography to obtain the cornus officinalis total iridoid glycosides extract; The purification method is as follows: Weigh 50g of macroporous adsorption resin and load it into the column. The column height is 11cm and the retention volume is 65ml. The ratio of macroporous adsorption resin to Cornus officinalis raw medicinal material is 4:

1. Measure the Cornus officinalis adsorption stock solution. The concentration of Cornus officinalis adsorption stock solution is 0.1g raw medicinal material / ml. The loading flow rate is 1ml / min. Let it stand. The elution flow rate is 5ml / min. After eluting with pure water to remove impurities, elute 9 retention volumes with 30% ethanol water.

2. The method for extracting and purifying total iridoid glycosides in Cornus officinalis according to claim 1, characterized in that: The macroporous adsorption resin includes but is not limited to D-101 type, AB-8 type, ADS-17 type or HPD-600 type.

3. The method for extracting and purifying total iridoid glycosides in Cornus officinalis according to claim 2, characterized in that: The macroporous adsorption resin is preferably AB-8 type.

4. Use of the total iridoid glycosides in Cornus officinalis according to claim 1 in the preparation of medicines, health products and foods for preventing and treating heart failure.

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