CN106822071B - Chinese medicinal effective component for treating coronary heart disease and hyperlipidemia, its preparation method and method for separating effective component from the same - Google Patents
Chinese medicinal effective component for treating coronary heart disease and hyperlipidemia, its preparation method and method for separating effective component from the same Download PDFInfo
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Abstract
The invention belongs to the field of medicine science and technology, and particularly relates to a traditional Chinese medicine effective part extracted from rhizomes of curcuma mauritiana for treating coronary heart disease and hyperlipidemia, a preparation method thereof and a method for separating effective components from the effective part. Pharmacological experimental research shows that: the curcumenol, the kaempferol, the curdione, the zedoarondiol and the total extract all have obvious effect of treating myocardial ischemia, can obviously reduce the range of myocardial infarction areas of rats with myocardial injury caused by ischemia, and have obvious protective effect on myocardial ischemia injury of rats with coronary artery ligation. The application is a divisional application of an invention patent application named as 'a traditional Chinese medicine effective part for treating coronary heart disease and hyperlipidemia and a preparation method thereof'.
Description
The application is a divisional application of an invention patent application with the application number of 201410491640.7 filed 9/23 2014 and the invention name of 'a traditional Chinese medicine effective part for treating coronary heart disease and hyperlipidemia and a preparation method thereof'.
Technical Field
The invention belongs to the field of medical science and technology, and particularly relates to a traditional Chinese medicine effective part extracted from rhizomes of curcuma mauritiana and used for treating coronary heart disease and hyperlipidemia, a preparation method thereof and a method for separating effective components from the effective part.
Background
Heart disease, malignant tumor and cerebrovascular disease are 3 diseases which seriously threaten human life at present, the coronary heart disease has been the first name of the health killer board in China since the last 90 th century, the living standard of people is continuously improved along with the rapid development of economy in China and the occurrence of huge changes in living style and dietary structure, and the incidence of the coronary heart disease in China is in a continuously rising trend. Coronary heart disease is the most common heart disease, and is also called Ischemic Heart Disease (IHD), which refers to myocardial dysfunction and organic lesions caused by coronary artery stenosis and insufficient blood supply. The main causes of coronary heart disease are: bad living habits such as hyperlipidemia, hypertension, diabetes, obesity, and smoking. The disturbance of lipid metabolism in vivo is the most important cause of coronary heart disease, modern medical research shows that the level of oxidized low-density lipoprotein cholesterol and apolipoprotein has a close relationship with the risk of coronary heart disease events, and hyperlipidemia is closely related to atherosclerosis and is an important cause of coronary heart disease. Besides death caused by myocardial infarction due to coronary artery obstruction, frequent attack of angina pectoris greatly limits learning and working abilities of patients, and in severe cases, the patients cannot take care of their lives, thus bringing heavy burden to families and society. Therefore, it is a significant task to find a drug for effectively treating coronary heart disease and angina pectoris to reduce the threat of cardiovascular diseases to human health.
The Curcuma wenyujin Y.H.Chen et C.Ling is rhizome of Curcuma wenyujin Y.H.Chen (Curcuma aromatica Salisb. of Zingiberaceae) of dicotyledonous plant, is distributed in Guangxi, Guizhou, etc. of China, and is a local wild medicinal plant resource. The curcuma mauritiana has various medicinal effects, is clinically used for mainly treating chest rib stabbing pain, amenorrhea, rheumatism shoulder and arm pain, traumatic swelling and pain and the like, and has anti-inflammatory and immunosuppressive effects according to modern pharmacological research. However, the research on the effect of the composition on treating myocardial ischemia, coronary heart disease and hyperlipidemia is not reported.
When folk proved formulas are collected, the clinical application practice of compatibility of the Guangxi Nanning area and other medicinal materials for treating chest stuffiness is found, the method is simple and obvious in curative effect, and many of the collected cases are serious patients who can not walk, dizziness, general weakness, chest distress, short breath and insomnia after being bedridden for a long time and long-term patients who can not be obviously improved after taking common cardiovascular medicines for many years. In order to fully develop the clinical application value of the medicinal material, the medicinal material is deeply researched, the clinical proved recipe is researched, different effective parts of the Chinese medicinal material are subjected to pharmacodynamic screening, a large number of accepted animal models are used for pharmacodynamic verification, and target effective parts and effective components are screened out.
Disclosure of Invention
The invention discovers effective parts of curcuma aromatica for treating target coronary heart disease and hyperlipidaemia by screening the drug effects of different effective parts of curcuma aromatica, separates four sesquiterpene monomer effective components such as curzeranol, primitive zedoary alcohol, curdione, zedorondiol and the like by comprehensively and deeply analyzing and researching sesquiterpene components in the effective parts, and has the following structural formulas by physical and chemical constants and spectral analysis:
in a first aspect of the present invention, there is provided a Chinese medicinal effective fraction for treating coronary heart disease and hyperlipidemia, which is characterized in that: the effective part containing sesquiterpene component is obtained by extracting dried rhizome of Curcuma wenyujin Y.H.Chen et C.Ling (Curcuma aromatica Salisb.) of Curcuma as medicinal part with organic solvent, separating and purifying the extract, wherein the content (mass percentage) of sesquiterpene component in the effective part is more than 50%.
Specifically, the main compounds in the sesquiterpene component are curzenediol, curzeranol, curdione and zedorondio, wherein the content of curzeranol in the effective part is 10-40%, the content of curzeranol in the effective part is 15-35%, the content of curzeranone in the effective part is 10-30%, and the content of zedorondio in the effective part is 5-30%.
The second aspect of the invention provides a preparation method of a traditional Chinese medicine effective part, which is characterized by comprising the following steps:
(1) adding 2-12 times (weight ratio) of one or more of ethanol, methanol, n-propanol, isopropanol, n-butanol or ethyl acetate with the concentration of 50-95% into the medicinal part of the curcuma mauritiana for reflux extraction, recovering the solvent from the extract, and concentrating to obtain a crude extract of the curcuma mauritiana; or pulverizing medicinal part of radix Curcumae, sieving, adding ethanol as entrainer, and placing in supercritical CO2Extracting to obtain an extract, namely a crude extract of the curcuma;
(2) extracting the crude extract of the curcuma mauritiana with 2-18 times of water saturated n-butyl alcohol, ethyl acetate or petroleum ether-ethyl acetate, recovering a solvent from an extract liquid, and concentrating to obtain the effective part of the curcuma mauritiana.
The invention also provides another preparation method of the effective part of the traditional Chinese medicine, which is characterized by comprising the following steps:
(1) adding 2-12 times (by weight) of 50-95% ethanol or one or more of methanol, n-propanol, isopropanol, n-butanol and ethyl acetate into the medicinal part of the curcuma mauritiana for reflux extraction, recovering the solvent from the extract, and concentrating to obtain a crude extract of the curcuma mauritiana; or pulverizing medicinal part of radix Curcumae, sieving, adding ethanol as entrainer, and placing in supercritical CO2Extracting to obtain an extract, namely a crude extract of the curcuma;
(2) adsorbing the crude extract of the curcuma mauritiana by using macroporous resin, washing the resin column by using water with the volume being 1-3 times of the column volume and ethanol with the concentration being 10-30% with the volume being 1-3 times of the column volume respectively, discarding water and diluted alcohol eluent, resolving the effective components adsorbed by the resin by using 80-95% of ethanol, collecting 80-95% of ethanol eluent in sections, recovering ethanol, and concentrating to obtain the effective part of the curcuma mauritiana.
Preferably, the macroporous resin is styrene type porous adsorption resin, and the polarity is weak polarity or medium polarity; for example, macroporous resins of D101, HPD-100, HPD-300, HPD-450, HPD-5000 or H103 type can be used.
The third aspect of the invention provides a method for separating effective components from effective parts of curcuma mauritiana, which comprises the following steps:
(1) preparing the effective part of the curcuma;
(2) subjecting the effective part of radix Curcumae to silica gel column chromatography with petroleum ether-ethyl acetate system of eluent 8-20:1, and collecting eluent to obtain curdione crude product; purifying the crude curdione product by preparative high performance liquid chromatography to obtain a curdione refined product;
(3) replacing the eluent with a petroleum ether-ethyl acetate system with a ratio of 4-7:1, collecting the eluent to obtain a zedoardiol crude product, and purifying the zedoardiol crude product by using a preparative high performance liquid chromatography to obtain a zedoardiol refined product;
(4) replacing the eluent with 3-2:1 petroleum ether-ethyl acetate system, collecting eluent to obtain third fraction, collecting the third fraction, and separating and purifying by preparative high performance liquid chromatography to obtain curcumendiol and primitive zedoary alcohol.
Has the advantages that:
the effective parts of the curcumenol, the kaempferol, the curdione, the zedorondiol and the curcuma mauritiana are found to have obvious effect of treating myocardial ischemia for the first time, can obviously reduce the range of myocardial infarction areas of rats with myocardial injury caused by ischemia, has obvious protection effect on myocardial ischemia injury of rats with coronary artery ligation, and has good curative effect on coronary heart disease and hyperlipoidemia.
The specific implementation mode is as follows:
example 1:
the method comprises the steps of removing silt and fibrous roots from rhizomes of the curcuma mauritiana, cleaning, slicing, drying in the sun and manufacturing medicinal parts;
extracting 1800g of curcuma root rhizome decoction pieces, adding 80% ethanol in an amount which is 6 times that of the decoction pieces, refluxing for two times to obtain two extracting solutions, combining the two extracting solutions, filtering, recovering the ethanol from the filtrate, and concentrating under reduced pressure until the relative density is about 1.10(45 ℃) to obtain a curcuma root crude extract;
extracting the crude extract and the third extract with 2 times of water saturated n-butyl alcohol for 6 times, combining n-butyl alcohol extract, recovering n-butyl alcohol under reduced pressure, concentrating, and vacuum drying to obtain 121g of effective components.
Example 2:
the method comprises the steps of removing silt and fibrous roots from rhizomes of the curcuma mauritiana, cleaning, slicing, drying in the sun and manufacturing medicinal parts;
secondly, 2000g of curcuma mauritiana rhizome decoction pieces are taken, 6 times of 75% ethanol is added for backflow to obtain two times, the two extracting solutions are combined, the filtering is carried out, the ethanol is recovered from the filtrate, and the filtrate is decompressed and concentrated until the relative density is about 1.05-1.10 (45 ℃), so that the curcuma mauritiana crude extract is obtained;
extracting the crude extract and the third extract for 6 times by using ethyl acetate in an amount which is 3 times that of the crude extract, combining ethyl acetate extract, recovering ethyl acetate under reduced pressure, concentrating, and drying in vacuum to obtain 116g of the effective part.
Example 3:
the method comprises the steps of removing silt and fibrous roots from rhizomes of the curcuma mauritiana, cleaning, slicing, drying in the sun and manufacturing medicinal parts;
secondly, 2000g of curcuma mauritiana rhizome decoction pieces are taken, 6 times of 75% ethanol is added for backflow to obtain two times, the two extracting solutions are combined, the filtering is carried out, the ethanol is recovered from the filtrate, and the filtrate is decompressed and concentrated until the relative density is about 1.05-1.10 (45 ℃), so that the curcuma mauritiana crude extract is obtained;
the raw extract is prepared by using 3 times of petroleum ether: extracting for 6 times by using a mixed solvent consisting of ethyl acetate (the volume ratio is 3-20: 1), and combining the mixed solvent consisting of petroleum ether: the ethyl acetate extract was subjected to solvent recovery under reduced pressure, concentration and vacuum drying to obtain 105g of the effective fraction.
Example 4
The method comprises the steps of removing silt and fibrous roots from rhizomes of the curcuma mauritiana, cleaning, slicing, drying in the sun and manufacturing medicinal parts;
secondly, 2000g of curcuma root rhizome decoction pieces are taken, 6 times of 75% ethanol is added for reflux to obtain two times, the two extracting solutions are combined, the filtering is carried out, the ethanol is recovered from the filtrate, and the ethanol is concentrated under reduced pressure till the ethanol is exhausted, so that curcuma root ethanol extracting solution is obtained;
subjecting the alcohol extract of the radix curcumae and the radix curcumae to D101 type macroporous resin adsorption, wherein the ratio of the diameter to the height of the resin column is 1: 8, the sample loading amount is medicinal materials: resin 3: 1, washing the resin column with 2 times of column volume of water and 2 times of column volume of 30% ethanol, respectively, and discarding water and diluted alcohol eluent;
fourthly, resolving the effective components adsorbed by the resin by using 4 times of ethanol with the volume of 90% of the column, collecting ethanol eluent in sections, recovering ethanol from the eluent, concentrating, and drying in vacuum to obtain 102g of effective parts.
Example 5:
the method comprises the steps of removing fibrous roots and silt from rhizomes of curcuma mauritiana, cleaning, slicing, drying in the sun, and manufacturing medicinal parts;
crushing root and stem decoction pieces of radix curcumae, sieving with a 10-mesh sieve, weighing 5Kg of powder, and placing in supercritical CO2In an extraction kettle of an extraction device;
the ethanol is used as an entrainer (500g), and the extraction pressure and the supercritical CO are adopted2And the fluid temperature is 35MPa and 50 ℃, the pressure and temperature of the separation kettle I are 8MPa and 50 ℃, the pressure and temperature of the separation kettle II are 5MPa and 35 ℃, supercritical extraction is carried out, circulating extraction is carried out for 1.5-2 hours, and the extract is discharged from the discharge holes of the separation kettle I and the separation kettle II.
And the third step of supercritical CO2The extract is removed to the utmost under reduced pressure of-0.09 MPa at 60 ℃, and 2 times of the ethanol is added into the extract by weight of petroleum ether: extracting for 4 times by using a mixed solvent consisting of ethyl acetate (the volume ratio is 3-20: 1), combining extract liquor, recovering the solvent under reduced pressure, and concentrating to obtain 415g of effective parts.
Example 6: the preparation method of the curcuma aromatica sesquiterpene monomer compound comprises the following steps:
the method comprises the steps of removing silt and fibrous roots from rhizomes of the curcuma mauritiana, cleaning, slicing, drying in the sun and manufacturing medicinal parts;
extracting 3000g of curcuma root rhizome decoction pieces, adding 6 times of 80% ethanol, refluxing for two times, mixing extracting solutions of the two times, filtering, recovering the ethanol from filtrate, and concentrating under reduced pressure until the relative density is about 1.10(45 ℃) to obtain a curcuma root crude extract;
extracting the crude extract and the third extract with 2 times of water saturated n-butyl alcohol for 6 times, combining n-butyl alcohol extract, recovering n-butyl alcohol under reduced pressure, concentrating, and vacuum drying to obtain 204g of effective components.
Taking 204g of an effective part, uniformly stirring with a proper amount of silica gel, loading the effective part on a column by a dry method, carrying out silica gel column chromatography, eluting with a petroleum ether-ethyl acetate system, eluting with petroleum ether-ethyl acetate (according to a ratio of 20:1) to obtain a crude curdione eluent, recovering the solvent from the eluent to the maximum extent, and preparing a liquid phase (a mobile phase is a 50% acetonitrile-water solution) to obtain a curdione refined product 5.7g with the purity of 99.8%. Performing silica gel column chromatography, eluting with petroleum ether-ethyl acetate (at a ratio of 7:1) to obtain zedorondiol crude product eluate, recovering solvent from the eluate, and preparing liquid phase (mobile phase 10% -15% acetonitrile-water solution) to obtain 2.3g of zedorondiol refined product with purity of 98.5%. Eluting with petroleum ether-ethyl acetate (2: 1) to obtain crude curcumenol and crude curcumenol eluate, recovering solvent from eluate, separating with preparative liquid phase (acetonitrile-water solution with mobile phase of 15% -50%) to obtain refined curcumenol 2.5g with purity of 97.8%, mixing the mother liquids after separating curcumenol by HPLC, recovering solvent, and preparing with preparative liquid phase (acetonitrile-water solution with mobile phase of 30%) to obtain refined curcumenol 6.1g with purity of 98.9%.
Example 7
Physicochemical constants and spectroscopy analyses were performed on four compounds: the curdione compound is prepared by adopting LC-MS,1H-NMR、13C-NMR、Dept、1H-1H COSY、13C-1H COSY identification means having a sub-formula C15H24O2As shown in chemical structural formula. The compound zedoarondiol is subjected to LC-MS,1H-NMR、13C-NMR、Dept、1H-1H COSY、13C-1H COSY identification means having a sub-formula C15H24O3As shown in chemical structural formula. The compound curcumenol adopts LC-MS,1H-NMR、13C-NMR、Dept、1H-1H COSY、13C-1H COSY identification means having a sub-formula C15H22O3The structural formula is shown as a chemical structural formula. The compound of the raw zedoary alcohol adopts LC-MS,1H-NMR、13C-NMR、Dept、1H-1H COSY、13C-1H COSY identification means having a sub-formula C15H22O2As shown in chemical structural formula. Wherein the zedoary turmeric diketone13C-NMR is13C NMR(400MHz,CDCl3) δ 16.57,18.54,19.86,21.12,26.41,30.00,34.05,44.24,46.81,53.61,55.92,129.90,131.59,211.14,214.37; of zedoarondiol13C-NMR is13C NMR(400MHz,CDCl3) δ 20.17,21.16,22.06,22.21,22.38,28.47,39.67,51.83,56.15,60.61,71.28,78.49,135.57,139.18,202.55; preparation of curcumenol13C-NMR is13C NMR(400MHz,CDCl3) δ 21.93; 22.00; 22.71; 24.09; 27.19; 36.98, respectively; 37.16; 60.94 parts; 83.19, respectively; 86.44; 127.97, respectively; 132.89, respectively; 143.30, respectively; 151.03, respectively; 194.09, respectively; preparation of Yuanyuangchun13C-NMR is13C NMR(400MHz,CDCl3) Delta 20.35; 21.52; 22.49; 23.39; 26.02, respectively; 27.75; 39.04; 49.62; 53.01, respectively; 79.39; 128.27, respectively; 135.40, respectively; 135.88, respectively; 154.22, respectively; 198.26. the carbon spectrum data was consistent with the compound carbon spectrum recorded in a nuclear magnetic resonance carbon spectrum database (Shanghai micro-spectral information technology, Inc.).
Example 8: content detection and analysis of sesquiterpene component in effective part
(I), instruments and reagents
A Dionex UlltiMate 3000 high-performance liquid chromatograph, an LPG-3400SD high-pressure pump, a WPS-3000(RS) automatic sample injector, a TCC-3x00(RS) column incubator, a VWD-3x00(RS) ultraviolet detector and a Chromeleon 6.8 workstation.
Acetonitrile, TEDIA, chromatographically pure; the water was deionized water (18.2M Ω) made by the Hi-Tech water purification system.
Comparison products: curdione, zedorondiol, curodiol, and kaempferol
(II) preparation of solution
Preparation of control solutions: precisely weighing appropriate amount of curdione, zedorondiol, curadiol and kaempferol, and dissolving in methanol to obtain control solution containing curdione, zedorondiol, curediol and kaempferol about 100 μ g per lmL.
(III) preparing a test solution: precisely weighing appropriate amount of the prepared effective components, and dissolving in methanol to obtain sample solution containing about 2mg per lmL.
(IV) chromatographic conditions:
adopting reverse phase high performance liquid chromatography SymmetryC18(5 μm, 250X 4.6mm), water as mobile phase A, acetonitrile as mobile phase B, flow rate of 1.0mL/min, gradient elution procedure as shown in Table 1, detection wavelength of 230nm, column temperature of 25 ℃.
TABLE 1 gradient elution procedure
(V) determination: precisely sucking the reference solution and the sample solution by 20 mu 1 respectively, injecting into a liquid chromatograph, measuring, and calculating the content of the four sesquiterpene compounds respectively, wherein the sum of the content of the four sesquiterpene compounds is the content of the effective part.
(VI) content measurement result: see Table 2 below
TABLE 2 content of sesquiterpene component in effective fraction obtained by different preparation method
Note: the sesquiterpene components in the effective components mainly comprise curdione, zedorondiol, curendiol and kaempferol.
And (4) conclusion: the detection results show that the process has practical significance.
The drug effect example:
the invention also provides the application of the effective part of the curcuma mauritiana prepared by the 5 methods, curdione, zedorondiol, curdione and kaempferol in preparing medicaments for preventing and treating myocardial ischemia, coronary heart disease and hyperlipidemia.
Pharmacological experiments show that: the effective parts of the curcumenol, the protozedoary turmeric alcohol, the curdione, the zedoarondiol and the curcuma aromatica have obvious treatment effect on an experimental myocardial ischemia model caused by the ligation of the coronary artery of an SD rat, and the medicine can obviously reduce the range of myocardial infarction areas of the rat myocardial injury caused by ischemia and has obvious protection effect on the myocardial ischemia injury of the rat with coronary artery ligation through the determination of the myocardial infarction range of a model animal and CK and LDH.
The effective part of the curcuma mauritiana after gastric lavage can obviously reduce the TG and TC values of the liver and serum of hyperlipidemic rabbits, reduce HDL-C, LDL-C, increase H/L and improve the liver steatosis degree, the liver steatosis degree of each experimental group of animals is obviously reduced, and the lipid-lowering effect of the effective part is basically equivalent to that of simvastatin.
(I) lipid-lowering experiment:
1. and (3) testing the sample: effective component of radix Curcumae (from Shanghai Ba Jia Yi medicine science and technology Co., Ltd.)
2. Positive control i: compound liver-benefiting tablet (from Jiangsu Zhongxing pharmaceutical Co., Ltd.)
Batch number: 130303161, respectively; specification: 0.32 g/tablet, each tablet contains 21mg of silymarin
3. Positive control II: simvastatin tablets (from Hangzhou Moshadong pharmaceutical Co., Ltd.)
Batch number: 120333, respectively; specification: 20 mg/tablet X7 tablets/box
4. Negative control: CMC-Na (national drug group chemical reagent Co., Ltd.)
Batch number: f20091021 (0.5% solution prepared with test water)
5. Laboratory animal
And (2) breeding: new Zealand rabbit (Shanghai Shengwang laboratory animal culture Co., Ltd.)
License number: SCXK (Shanghai) 2012-0007; certificate number: 200700071847
6. Experimental methods
100 male new zealand white rabbits, initial body weight: 2,188.4 + -227.9 g, after 2 weeks of adaptive feeding, except the normal control group fed standard feedstuff, the other animals were dosed with 150g of high fat feedstuff every morning. After continuously feeding for 33 days, fasting is carried out overnight, blood is collected from the ear margin vein, serum is separated, and TG, TC and HDL-C, LDL-C are measured to confirm whether molding is successful or not.
After the model is confirmed to be successful, animals with unqualified blood lipid levels are removed, the model animals are evenly divided into a model control group according to the indexes and the weight, the effective part administration group (equivalent to 10g crude drugs/kg), the compound liver benefiting tablet group (30mg/kg) and the simvastatin group (3mg/kg) prepared by the method 1-5 are divided into groups, administration is started, the blank control group and the model group are subjected to intragastric administration with 4mL/kg of blank solvent, the other groups are subjected to intragastric administration with corresponding samples, once a day, and the continuous administration is carried out until the positive control group has obvious lipid lowering effect. The administration period of the feed is not stopped.
The body weight was weighed once a week during the test period, and the administration dose was adjusted according to the body weight. After 2 weeks of administration, animals are fasted overnight without water supply for 12 hours, blood is collected from the ear vein, serum is centrifugally separated, TG, TC and HDL-C, LDL-C values are detected, and the HDL-C/LDL-C ratio is calculated; after 3 weeks of administration, the above indices were measured by fasting blood collection overnight, and the rabbits were anesthetized with 3% sodium pentobarbital (30mg/kg) in the abdominal cavity the next day after blood collection, 400mg of the liver was dissected and homogenized with 4mL of a lipid extract (n-heptane: isopropanol ═ 2: 3.5), the lipid was extracted by shaking, and the supernatant was centrifuged to measure TG and TC in the liver.
7. Statistical treatment: all the measurement data are as followsShowing, statistical analysis of the data using t-test;
8. results of the experiment
The results of the rabbit liver lipid content experiments in each experimental group are shown in Table 3. The results show that the contents of TG and TC in the liver tissues of the model group are obviously higher than those of the normal control group (p is less than 0.01), which indicates that the modeling is successful. The effective parts of the curcuma mauritiana can obviously reduce the content of TG and TC in liver tissues.
The results of the rabbit serum TG and TC tests of each test group are shown in Table 4. The results show that the serum TG and TC values of each administration group and model group are very obviously higher than those of a normal control group (p <0.01) before administration; and the serum TG and TC values of each administration group are basically consistent with those of the model group, thereby prompting that the model is successfully made and the grouping is uniform.
The results of the rabbit HDL-C, LDL-C content and ratio (H/L) tests in each test group are shown in Table 5. The results show that the serum HDL-C and LDL-C content of each experimental group is very obviously higher than that of a normal control group (p <0.01) before administration; the H/L ratio was very significantly lower than the normal control (p < 0.01); the serum HDL-C, LDL-C and H/L ratios of each administration group were substantially consistent with those of the model group, indicating that the successful animals were evenly grouped. At weeks 2 and 3 post-dose, HDL-C, LDL-C was significantly higher in the model group than in the normal group (p <0.01) and H/L was significantly lower than in the normal group (p < 0.01).
9. Conclusion
The experimental results show that after 3 weeks of administration, the effective parts of the curcuma mauritiana (equivalent to 10g crude drugs/kg) and the compound Yiganling tablets prepared by the five methods can obviously reduce the TG and TC values of the liver and serum of hyperlipidemic rabbits, reduce HDL-C, LDL-C, increase H/L and improve the degree of liver steatosis by gastric administration of 30mg/kg, and the degree of liver steatosis of animals in each test group of the effective parts of the curcuma mauritiana is reduced. The simvastatin group administered by intragastric administration at 3mg/kg can obviously reduce the TG, TC and HDL-C, LDL-C values of the serum of the rabbit with hyperlipidemia, increase H/L and also obviously reduce the TG and TC of the liver. The results indicate that the effective part of the curcuma mauritiana can reduce the blood fat level of animals on the model, and the blood fat reducing effect of the curcuma mauritiana is basically equivalent to that of simvastatin.
TABLE 3 lipid content results of rabbit liver tissue in each experimental group
*p<0.05；**p<0.01, compared to a model set;#p<0.05；##p<0.01, compared with the compound liver-benefiting tablet group;△p<0.05；△△p<0.01, compared to the simvastatin group
TABLE 4 results of rabbit blood lipid content in each experimental group
*p<0.05；**p<0.01, compared to a model set;#p<0.05；##p<0.01, compared with the compound liver-benefiting tablet group;△p<0.05；△△p<0.01, compared to the simvastatin group.
TABLE 5 Rabbit serum HDL cholesterol content and ratio results for each experimental group
*p<0.05；**p<0.01, compared to a model set;#p<0.05；##p<0.01, compared with the compound liver-benefiting tablet group;△p<0.05；△△p<0.01, compared to the simvastatin group.
(II) therapeutic action on myocardial ischemia-reperfusion rats
1. Test article
The effective parts of radix Curcumae prepared by the first, second, third, fourth and fifth methods of the present invention (hereinafter referred to as the first, second, third, fourth and fifth methods) and the four sesquiterpene monomer compounds of curcumenol, kaempferol, curdione and zedorondiol prepared by the sixth method of the present invention are prepared into liquid medicine with required concentration by CMC-Na solution for animal administration.
2. Positive drug
Dioxin xuekang capsule (hereinafter abbreviated as xinxuekang): produced by Olympic group, Chengdu, Inc., with a specification of 100 mg/pellet, lot number 1306022. The CMC-Na solution is prepared into liquid medicine with required concentration for animals to administer at the time of use.
Isosorbide mononitrate tablet (hereinafter abbreviated as mononitrate), which is produced under the trade name of Xinkang, Shannan fibrate pharmaceutical Co., Ltd., specification of 20mg, lot number 07130313. The medicine is ground when in use, and CMC-Na solution is prepared into medicine liquid with corresponding concentration for the administration of the rat by intragastric administration.
3. Medicine box and reagent
Lactate Dehydrogenase (LDH) assay kit, manufactured by Zhongsheng Bei accuse Biotechnology GmbH, Inc., Lot 130521.
Rat creatine kinase isozyme (CK-MB) enzyme-linked immunosorbent assay (ELISA) kit, product of Bio-Swamp, batch No. 201310.
Rat Lactate Dehydrogenase (LDH) enzyme-linked immunosorbent assay (ELISA) kit, product of Bio-Swamp, batch No. 201310.
Chloral hydrate, Tianjin, Kemiou Chemicals, Inc., Lot No. 20120110.
4. Animal(s) production
SD rat, SPF grade, male, 253.9 + -10.4 (230-276) g, provided by Beijing Wintolite laboratory animal technology, Inc., animal production license number SCXK (Jing) 2012-0001.
5. Preparation of the model
Rats were fixed on the operating table in the supine position under the intraperitoneal injection of 360mg/kg of chloral hydrate, 12%. Removing hair in the front and outside of the chest, and sterilizing by conventional method. The skin and muscle layer were incised and purse string sutured, and the fourth intercostal space on the left was opened to the chest, pulling the heart out with a loop hook. Threading the anterior descending coronary branch 3-4 mm below the left atrial appendage with 6/0 atraumatic silk thread, juxtaposing nylon thread with diameter of 1.6mm, ligating together with the anterior descending coronary branch, and leaving the other end in vitro for use. The heart was returned to the chest cavity, the air in the chest cavity was evacuated, and the chest cavity was closed (sham animals threaded only at the corresponding coronary locations, no ligation was performed, and the other procedures were the same as those for ligating the animals). The nylon thread was carefully removed for reperfusion after 30min of ligation. The muscle layer and skin were sutured and the penicillin was injected once daily for 3 consecutive days.
Compared with a normal II-lead electrocardiogram (ECG-II), the ischemia success is determined when ST segment of the ECG-II is raised by more than 0.2mV 10min after coronary artery ligation; reperfusion was successful 10min after reperfusion, with a more than 30% decrease in the elevated ST segment in ECG-II. Animals that did not meet the above criteria for ischemia or ischemia-reperfusion, indicating that molding was unsuccessful, were excluded from the experiment.
6. Grouping and administration of drugs
Animals successfully shaped were randomly selected and divided into 13 groups of 10 animals each. Respectively a pseudo-operation group, a model control group, a tested medicine group, a traditional Chinese medicine positive medicine group and a western medicine positive medicine group.
The administration by gavage is started 30min after the molding is successful, 1 time per day and 7 days continuously; the volume of each administration is 10 ml/kg. The sham operation group and the model control group were given a solvent control solution (CMC-Na solution); the effective part of the curcuma mauritiana prepared by the first method, the second method, the third method, the fourth method and the fifth method, the four sesquiterpene monomer compounds of the curcuma glycol, the curcuma zedoary alcohol, the curdione and the zedorondiol prepared by the sixth method are respectively administered to the tested medicine components, the dosage (equivalent crude drug quantity according to the yield of the effective part and the monomer effective components) is 9g crude drug/kg, and the western medicine isosorbide mononitrate tablet is 0.0062g (tablet weight)/kg (equivalent to the clinical equivalent amount); xinxuekang 0.144g/kg (equivalent to a clinically equivalent amount).
7. Infarct size measurement
After the experiment, rat hearts were taken and washed with physiological saline and then frozen at-20 ℃. The heart was evenly cut into 2mm thick sections under the ligature, stained at 37 ℃ for 10min with 2% TTC, and the percentage of the infarct area in the left ventricle was determined by a pathological image analysis system.
8. Serum myocardial enzyme assay
After the experiment, the rat was bled from the abdominal aorta, centrifuged at 4 ℃ and 3000 rpm. times.10 min, and the supernatant was collected and assayed for LDH and CK-MB by the Elisa method.
9. Statistical treatment
All data are expressed as mean ± sdRepresenting student's t-test, P for pre-and post-drug pairings or comparisons between groups<A difference of 0.05 is statistically significant.
TABLE 6 myocardial infarction Range after ischemia reperfusion injury to rat myocardiumInfluence of (A), (B)n＝10)
Note: 1. comparison with sham group:△△△P<0.001; 2. comparison with model groups:*P<0.05; 3. comparing with the positive medicine group: p>0.05.
10. Effects on serum myocardial enzymes
The serum LDH activity of the model group is obviously increased 7 days after ischemia reperfusion. After the 5 effective parts of the curcuma mauritiana prepared by the method and four separated monomer sesquiterpenoids (equivalent to 9g crude drugs/kg) are treated for 7 days, compared with a model group, the LDH (P is less than 0.05) can be remarkably reduced. The results are shown in Table 7.
TABLE 7 Effect on myocardial enzymes after myocardial ischemia reperfusion injury in rats: (n＝10)
The experimental results show that: the medicine can obviously reduce the myocardial infarction area range of rat myocardial injury caused by ischemia, and has obvious protective effect on myocardial ischemia injury of coronary artery ligation rat.
Claims (3)
1. The application of curdione as the only effective component in preparing the medicine for preventing and treating myocardial ischemia reperfusion injury.
2. The use of claim 1, wherein the myocardial ischemia reperfusion injury is post-myocardial infarction myocardial ischemia reperfusion injury.
3. The use as claimed in claim 1, wherein the curdione has a significantly reduced lactate dehydrogenase effect.
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