CN110742884A - Application of costunolide, dehydrocostunolide and derivatives thereof in preparing medicines for preventing and treating anoxia and protecting myocardium - Google Patents
Application of costunolide, dehydrocostunolide and derivatives thereof in preparing medicines for preventing and treating anoxia and protecting myocardium Download PDFInfo
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Abstract
The invention provides application of costunolide, dehydrocostunolide and derivatives thereof in preparing medicaments for preventing and treating hypoxia myocardial protection. The invention researches the myocardial hypoxia resistance of costunolide and dehydrocostunolide through a normal pressure closed hypoxia experiment and an acute pressure-reduced hypoxia experiment model, and the result shows that the costunolide and the derivative dehydrocostunolide thereof have better hypoxia resistance, and the action mechanism of the dehydrocostunolide and the derivative thereof can be realized through oxidation stress injury resistance. Costunolide can protect SOD and CAT activity in myocardial tissue of hypoxic mouse, enhance ROS and free radical scavenging ability of brain of hypoxic body, and reduce R after hypoxiaOS-induced MDA, H in mouse myocardium2O2OH level, thereby achieving the effect of resisting myocardial anoxia.
Description
Technical Field
The invention relates to the field of medicines, in particular to a method and application of costunolide, dehydrocostunolide and derivatives thereof in preparing a medicine for preventing and treating hypoxic myocardium protection.
Background
The common treatment medicines of myocardial ischemia comprise nitrate coronary vasodilators, β -receptor blockers, calcium ion antagonists and the like, and are particularly widely used as β -receptor blockers, but a plurality of contraindications exist in practical application, including airway blockage, atrioventricular conduction block, abnormal heart failure and the like, so the clinical application is limited.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides the function of costunolide, dehydrocostunolide and derivatives thereof in preparing medicaments for preventing and treating hypoxia myocardial protection.
In order to realize the purpose of the invention, the invention adopts the technical scheme that:
the invention provides application of costunolide, dehydrocostunolide and derivatives thereof in preparing medicaments for preventing and treating hypoxia myocardial protection.
Preferably, the hypoxia is hypoxia at normal pressure or reduced pressure.
Preferably, the hypoxia is acute hypoxia or chronic hypoxia.
Preferably, the hypoxic myocardial protection is to protect the activities of SOD and CAT in the myocardial tissues of hypoxic mice, enhance the ROS and free radical scavenging ability of the brain of the hypoxic organisms, and reduce MDA and H in the myocardial tissues of the mice caused by the ROS after hypoxia2O2OH level.
The invention provides application of costunolide, dehydrocostunolide and derivatives thereof in preparing a medicine for maintaining blood pressure during acute anoxia.
The invention provides a preparation method of costunolide and derivatives thereof, namely dehydrocostunolide, which comprises the following steps:
(1) extracting radix aucklandiae or radix aucklandiae decoction pieces with ethanol, and collecting supernatant to obtain ethanol extractive solution;
(2) extracting the ethanol extract obtained in the step (1) by using petroleum ether, and collecting the lower layer extracted liquid to obtain petroleum ether extract;
(3) extracting the petroleum ether extract liquid in the step (2) by using ethyl acetate, and collecting upper-layer liquid to obtain ethyl acetate extract liquid;
(4) and (4) separating and purifying the ethyl acetate extract obtained in the step (3) by using a high performance liquid chromatograph, and respectively collecting different fractions to obtain costunolide and dehydrocostunolide.
Preferably, in the step (1), the specific method for extracting costus root with ethanol comprises the following steps: pulverizing radix aucklandiae, adding 5-10 times of 50-95% ethanol, soaking for 6-12 hr, ultrasonic treating for 0.5-1 hr, standing for layering, collecting supernatant, extracting the lower layer precipitate with ethanol again, and mixing the supernatants.
Preferably, in the step (4), when the separation and purification are performed by a high performance liquid chromatograph, the chromatographic conditions are as follows: a chromatographic column: waters Atlantis C18 column; flow rate: 1.0-3.0 ml/min; sample introduction volume: 0.1-0.5 ml; detection wavelength: 225 nm; mobile phase: 0-3 min, 15% -25% methanol; 3-10 min, 25% -35% methanol; 10-20 min, 35% -60% methanol; 20-35 min, 60-70% methanol; 35-50 min, 70% methanol, and the flow phase ratio is volume percentage; column temperature: at 25 ℃.
The invention is sealed by normal pressureThe hypoxia experiment and acute decompression hypoxia experiment models research the myocardial hypoxia resisting effect of costunolide and dehydrocostunolide, and the results show that costunolide and derivatives thereof dehydrocostunolide have good hypoxia resisting effect, and the action mechanism of the costunolide and derivatives thereof can be realized through antioxidant stress injury. Costunolide can protect SOD and CAT activity in myocardium tissue of mouse with anoxia, enhance ROS and free radical scavenging ability of brain of mouse with anoxia, and reduce MDA and H in myocardium of mouse caused by ROS after anoxia2O2OH level, thereby achieving the effect of resisting myocardial anoxia.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a HPLC analysis chart of ethyl acetate fraction of Aucklandia lappa.
FIG. 2 is a mass spectrum of costunolide obtained by purification.
FIG. 3 is a mass spectrum of dehydrocostuslactone obtained by purification.
Fig. 4 shows the effect of costunolide on systolic blood pressure in acutely hypoxic rats (mmHg, n 10).
Fig. 5 is a graph of the effect of costunolide on mean arterial pressure in acutely hypoxic rats (mmHg, n 10).
Fig. 6 is a graph of the effect of costunolide on diastolic pressure in acutely hypoxic rats (mmHg, n 10).
FIG. 7 shows the results of HE staining of mouse myocardial tissue (400-fold).
FIG. 8 shows the results of electron microscopy of mouse myocardial tissue (6000-fold).
FIG. 9 shows the effect of costunolide on SOD activity in myocardial tissue of hypoxic mice: (
n＝10)。
FIG. 10 shows the effect of costunolide on myocardial map tissue CAT in hypoxic mice: (
n＝10)。
FIG. 11 shows the application of costunolide in treating myocardial tissue H of hypoxic mice2O2Influence of level (
n＝10)。
FIG. 12 shows the effect of costunolide on myocardial tissue OH clearance in hypoxic mice: (n＝10)。
FIG. 13 is a graph of the effect of costunolide on MDA levels in hypoxic myocardial tissue in mice (
n＝10)。
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Abbreviations are shown in table 1.
Table 1 acronyms
Enrichment, extraction and separation of costunolide and derivatives thereof dehydrocostunolide:
(1) preparation of ethanol extract
100g of costustoot decoction pieces are weighed by an electronic balance, crushed into powder by a crusher and placed in a 1000ml beaker. Adding 50-95% ethanol 5-10 times the mass ratio into radix aucklandiae powder, mixing, soaking for 6-12 hr, ultrasonic treating for 0.5-1 hr, standing for layering, collecting the upper layer of orange red solution, and precipitating the lower layer to obtain insoluble medicinal powder. Separating, collecting supernatant, adding the rest precipitate into 50-95% ethanol with the same volume as the previous step, mixing, soaking for 6-12 hr, ultrasonic treating for 0.5-1 hr, standing for layering, collecting the upper layer of orange red solution, and precipitating the lower layer to obtain insoluble medicinal powder. Separating, collecting supernatant and combining all supernatants. Concentrating all supernatant at 40-65 deg.C by rotary evaporation under vacuum to 50-100ml to obtain solution A.
(2) Petroleum ether extraction
And (3) placing the solution A into a 1000ml separating funnel, adding 50ml of petroleum ether for extraction, uniformly mixing, standing for layering, separating the liquid and removing the petroleum ether layer, wherein the upper layer is a petroleum ether layer, and the lower layer is an extracted layer which is dark red. Adding 50ml petroleum ether into the extracted layer for extraction, and repeating the extraction for 2-5 times until the petroleum ether layer is nearly colorless and the lower layer is orange. The extracted liquid (solution B) is kept orange red, and 50-100ml is totally.
(3) Ethyl acetate fraction preparation
And (3) putting the solution B into a 1000ml separating funnel, adding 50ml of ethyl acetate for extraction, uniformly mixing, standing for layering, separating the upper layer which is orange red of the extracted layer and the lower layer which is orange of the ethyl acetate layer, and keeping the ethyl acetate extract on the upper layer. 50ml of ethyl acetate was further added to the extracted layer and the extraction was repeated 2-5 times until the ethyl acetate layer was pale yellow. The ethyl acetate extracts were combined and orange-yellow, amounting to 100-300 ml.
(4) Preparation of liquid chromatography separation and purification costunolide and dehydrocostunolide monomer
The costunolide and dehydrocostunolide monomers in the ethyl acetate part are separated and purified by a high performance liquid chromatograph. The chromatographic conditions are as follows: a chromatographic column: waters Atlantis C18 column (19X 150mm,10 μm). Flow rate: 1.0-3.0 ml/min; sample introduction volume: 0.1-0.5 ml; detection wavelength: 225 nm. Mobile phase: 0-3 min, 15% -25% methanol; 3-10 min, 25% -35% methanol; 10-20 min, 35% -60% methanol; 20-35 min, 60-70% methanol; 35-50 min, 70% methanol, and the flow phase ratio is volume percentage. Column temperature: at 25 ℃. Collecting 39-40min (costunolide) and 42-43min (dehydrocostunolide) fractions respectively to obtain costunolide and dehydrocostunolide monomers, and determining by HPLC that the retention time is the same as that of the reference substance. The vacuum drying oven was vacuum dried at 55 ℃ to a solid. Standing to cool to room temperature, weighing, sealing, and refrigerating.
(5) And (5) identifying the result by mass spectrum.
The molecular ion peak in costunolide mass spectrum is 233.16(m/z), and the molecular ion peak in dehydrocostunolide mass spectrum is 231.14 (m/z).
Example 1
(1) Preparation of ethanol extract
100g of costustoot decoction pieces are weighed by an electronic balance, crushed into powder by a crusher and placed in a 1000ml beaker. Adding 70% ethanol 8 times the mass ratio into radix aucklandiae powder, mixing, soaking for 12 hr, ultrasonic treating for 45min, standing for layering to obtain orange red upper layer solution, and precipitating the lower layer to obtain insoluble medicinal powder. Separating, collecting supernatant, adding the rest precipitate into 70% ethanol with the same volume as the previous step, mixing, soaking for 12 hr, ultrasonic treating for 45min, standing for layering to obtain upper layer of orange red solution, and lower layer of precipitate as insoluble medicinal material powder. Separating, collecting supernatant and combining all supernatants. Rotary evaporator all supernatants were concentrated to 80ml by rotary evaporation in vacuo at 45 ℃ to give solution A.
(2) Petroleum ether extraction
And (3) placing the solution A into a 1000ml separating funnel, adding 50ml of petroleum ether for extraction, uniformly mixing, standing for layering, separating the liquid and removing the petroleum ether layer, wherein the upper layer is a petroleum ether layer, and the lower layer is an extracted layer which is dark red. Adding 50ml petroleum ether into the extracted layer for extraction, and repeating the extraction for 4 times until the petroleum ether layer is nearly colorless and the lower layer is orange. The extracted solution (solution B) was retained and was orange red, amounting to 80 ml.
(3) Ethyl acetate fraction preparation
And (3) putting the solution B into a 1000ml separating funnel, adding 50ml of ethyl acetate for extraction, uniformly mixing, standing for layering, separating the upper layer which is orange red of the extracted layer and the lower layer which is orange of the ethyl acetate layer, and keeping the ethyl acetate extract on the upper layer. Adding 50ml ethyl acetate into the extracted layer, extracting repeatedly for 3 times until the ethyl acetate layer turns light yellowAnd (4) color. The ethyl acetate extracts were combined and found to be orange yellow, amounting to 200 ml. The ethyl acetate sites were analyzed using Thermo U3000 UPLC. The chromatographic conditions are as follows: a chromatographic column: accucoreTMC18 column (2.1X 100mm,2.6 μm). Flow rate: 0.2 ml/min; detection wavelength: 225 nm. Mobile phase: 0-50 min, 15% -70% of methanol, and v/v. Column temperature: at 25 ℃. The retention time of costunolide is 39-40min, and the retention time of dehydrocostuslactone is 42-43 min.
FIG. 1 is a HPLC chart of ethyl acetate fraction of Aucklandia lappa. Wherein, 1, costunolide; 2. dehydrocostunolide.
(4) Preparation of liquid chromatography separation and purification costunolide and dehydrocostunolide monomer
The costunolide and dehydrocostunolide monomers in the ethyl acetate part are separated and purified by a high performance liquid chromatograph. The chromatographic conditions are as follows: a chromatographic column: waters Atlantis C18 column (19X 150mm,10 μm). Flow rate: 2.5 ml/min; sample introduction volume: 0.5 ml; detection wavelength: 225 nm. Mobile phase: 0-3 min, 20% methanol; 3-10 min, 30% methanol; 10-20 min, 50% methanol; 20-35 min, 65% methanol; 35-50 min, 70% methanol, and the flow phase ratio is volume percentage. Column temperature: at 25 ℃. Collecting 39-40min (costunolide) and 42-43min (dehydrocostunolide) fractions respectively to obtain costunolide and dehydrocostunolide monomers, determining by HPLC that the retention time is consistent with that of a reference substance, identifying the molecular weight by high resolution mass spectrometry, and obtaining molecular ion peak 233.16(m/z) in costunolide mass spectrometry. The molecular ion peak in the dehydrocostuslactone mass spectrum is 231.14 (m/z). The vacuum drying oven was vacuum dried at 55 ℃ to a solid. Standing to cool to room temperature, weighing, sealing, and refrigerating.
FIG. 2 is a mass spectrum of costunolide obtained by purification.
FIG. 3 is a mass spectrum of dehydrocostuslactone obtained by purification.
Example 2 screening of anti-hypoxic agent Effect of active ingredients, Costunolide, dehydroCostunolide
According to the preparation method of example 1, according to the experimental results, 33.0g of costunolide and dehydrocostunolide are obtained from 2kg of medicinal materials, wherein, 17.6g of costunolide and 15.4g of dehydrocostunolide are obtained; the extraction rate of costunolide and dehydrocostunolide is 1.65%.
Drying the obtained parts by rotary evaporation, and drying under reduced pressure to obtain radix aucklandiae ethanol extract, petroleum ether extract, ethyl acetate extract and brown powder. Balb/c mice (female) were administered by gavage according to the dose groups of Table 2, and a normal pressure closed hypoxia experiment was performed. The costunolide and dehydrocostuslactone monomer prepared by separation and purification of ethyl acetate part of costus root in example 1 were subjected to a closed oxygen deficiency test under normal pressure, and the test results are shown in table 3. As shown in Table 2, the main anti-hypoxia active site of the costustoot drug is an ethyl acetate site. As can be seen from Table 3, both costunolide and dehydrocostunolide obtained by separation and purification from ethyl acetate fraction have good anti-hypoxia effect, and the anti-hypoxia drug effect thereof is dose-dependent. The ethyl acetate part of radix aucklandiae is mainly sesquiterpene lactone, and costunolide, dehydrocostuslactone and isomer are used as main active ingredients.
TABLE 2 mouse Normal pressure closed hypoxia experiment (effective active site screening)
Note: in comparison to the set of models,*P<0.05，**P<0.05。
TABLE 3 mouse Normal pressure closed hypoxia experiment (active site effective dose screening)
Note: in comparison to the set of models,*P<0.05，**P<0.05。
example 3 Effect of Costunolide on blood pressure in acutely hypoxic rats
A low-pressure hypoxia experiment cabin (Guizhou torpedo), a BP-2010 intelligent noninvasive sphygmomanometer (Beijing Soft Dragon), an XT-2000i animal whole blood cell analyzer (Japanese Sysmex), a BX-3010 animal full-automatic biochemical analyzer (Japanese Sysmex) and a blood gas analyzer (ABL90 FLEX analyzer).
The total number of SPF Wistar rats is 40, the male and female half of the rats respectively have the body weight of 210 +/-20 g. Groups of 10 Costunolide (CTL) prepared in example 1 were divided into 4 groups by semi-random division according to males and females, and administered at a dose of 250mg/kg according to the results of the previous experiment, and were continuously administered by gavage for 3 days; the positive control group is subjected to gavage administration of 300mg/kg of Acetazolamide (ACETAZOLAMIDE, ACZ) for 3 days continuously, and the administration volume is 20 ml/kg; the Normal control (Normal) group and the hypoxia Model (Model) group were gavaged with 0.9% physiological saline for 3 days. The mice are placed in a large low-pressure cabin feeding cabin 30min after the last administration, and water is not fed. After the cabin door is closed, the pressure is reduced at the speed of 10m/s to simulate the rising altitude, the altitude is maintained for 12 hours (8 percent of oxygen, 92 percent of nitrogen and 0.035MPa) after the rising altitude reaches 8000m, and the altitude is reduced to 4500 m. The experimenter enters the buffer cabin, raises the altitude to 4500m, takes out the rat, measures the blood pressure, simultaneously measures the biochemical index, and takes out the myocardial tissue for freezing storage at minus 80 ℃. The experimental operation is completed in a low-pressure experimental chamber (simulating the plateau condition with the altitude of 4500 m) (the blood biochemical index is measured outside the chamber). In order to reduce the error caused by individual differences of rats, the blood pressure and heart rate of the normal group used in the experiment were the mean values of the basal blood pressure and heart rate of 4 groups of rats before entering the cabin. The experimental data are processed by statistical software, and the experimental results are obtained
And (4) showing.
Acute hypoxia has a great influence on the hemodynamics of rats, and the systolic pressure, the mean arterial pressure and the diastolic pressure of rats are all reduced after 8000m of acute low-pressure hypoxia for 12h, which may be related to the damage of the heart after hypoxia. As can be seen from FIGS. 4-6, the blood pressure of the costunolide group is close to that of the normal group, which is closely related to its effect on the myocardial protection.
Fig. 4 shows the effect of costunolide on systolic blood pressure in acutely hypoxic rats (mmHg, n 10).
Note: in comparison with the normal group,#P<0.05，##P<0.01; andcompared with the model group, the model group is compared,*P<0.05，**P<0.05。
fig. 5 is a graph of the effect of costunolide on mean arterial pressure in acutely hypoxic rats (mmHg, n 10).
Note: in comparison with the normal group,#P<0.05，##P<0.01; in comparison to the set of models,*P<0.05，**P<0.05。
fig. 6 is a graph of the effect of costunolide on diastolic pressure in acutely hypoxic rats (mmHg, n 10).
Note: in comparison with the normal group,#P<0.05，##P<0.01; in comparison to the set of models,*P<0.05，**P<0.05。
example 4 protective Effect of Costunolide on the simulation of plateau hypoxic mice
The present example mainly observes the protective effect of costunolide on hypoxic neurons and its antioxidant effect from the morphological point of view.
(1) Effect of costunolide on the microstructure of myocardial tissue in hypoxic mice.
FIG. 7 shows the results of HE staining of mouse myocardial tissue (400-fold).
As can be seen from FIG. 7, the normal group of cardiomyocytes had normal morphology, intact structure and regular arrangement of the myocardial fibers; the hypoxia model group has thickened muscle fibers, partial cell lysis, increased and lightly stained cell nuclei, homogeneous muscle fibers and unobvious nuclear contraction; vasodilatation congestion in the muscle tissues of the acetazolamide (300mg/kg) group, mild thickness of part of muscle fibers are different, mild edema of muscle cells occurs, the striation structure is not regularly arranged, and a small amount of myocardial cell nuclei are reduced and condensed; the costunolide group myocardial fibers are arranged regularly, cell lysis is slightly seen, and the cell nucleus is less deepened.
(2) Effect of costunolide on the ultrastructure of myocardial tissue in hypoxic mice.
FIG. 8 shows the results of electron microscopy of mouse myocardial tissue (6000-fold).
As can be seen from FIG. 8, the normal group cells were well defined and the myocardial fibers were well aligned; the model group has swelling and adhesion, and the muscle fiber is seriously broken; in the acetazolamide group, part of muscle fibers are broken, and part of mitochondria are swelled and dissolved; costunolide has good myocardial fiber and mitochondrial morphology.
Pathological section HE staining results and transmission electron microscope experiment results show that costunolide has a good protective effect on myocardial cells of hypoxic mice.
Example 5 protective Effect of Costunolide on oxidative stress in hypoxic mice
Hypoxia causes a large increase in mouse hypoxia myocardium ROS, decreases antioxidant enzyme activity, and causes myocardial functional damage.
(1) Protection of simulated plateau anoxic mouse (SOD) superoxide dismutase activity
Mouse brain and myocardial tissue sample treatment: taking 10% of the tissue homogenate to be detected, centrifuging at 4000rpm and 4 ℃ for 5min, and taking the supernatant for determination. Diluting into different times, performing preliminary experiments according to the specification of an SOD activity detection kit (constructed by Nanjing) to ensure that the percentage inhibition rate is between 15 and 55 percent, and determining according to the concentration in the range according to the specification.
FIG. 9 shows the effect of costunolide on SOD activity in myocardial tissue of hypoxic mice: (
n＝10)。
Note: in comparison with the normal group,#P<0.05，##P<0.01; in comparison to the set of models,*P<0.05，**P<0.05。
as can be seen from fig. 9, the anti-oxidation ability of the myocardial tissue of the mouse is significantly reduced due to the oxygen deficiency, the anti-oxidation ability of the myocardial tissue of the mouse is significantly improved due to the administration of the positive drugs acetazolamide and the costunolide, and the activity level of the SOD of the myocardial tissue of the mouse after the oxygen deficiency can be significantly improved due to the costunolide, so that the oxygen free radical clearance of the myocardial tissue of the mouse caused by the oxygen deficiency is increased, and the oxygen deficient myocardial tissue is protected from the active oxygen damage.
(2) Activity measurement of Catalase (CAT) for simulating plateau hypoxia mouse tissue
Catalase decomposes the hydrogen peroxide, and ammonium molybdate is added to react with the hydrogen peroxide to form a pale yellow complex, which is measured at 405nm, as described.
FIG. 10 shows the effect of costunolide on myocardial map tissue CAT in hypoxic mice: (
n＝10)。
Note: in comparison with the normal group,#P<0.05，##P<0.01; in comparison to the set of models,*P<0.05，**P<0.05。
as can be seen from FIG. 10, the CAT activity in the hypoxic state tends to increase, the acetazolamide group has no significant difference from the normal group and the model group, and the myocardial CAT activity of the hypoxic mice can be significantly increased after the costunolide is administered.
(3) Content determination of tissue hydrogen peroxide for simulating plateau anoxic mice
FIG. 11 shows the application of costunolide in treating myocardial tissue H of hypoxic mice2O2Influence of level (n＝10)。
Note: in comparison with the normal group,#P<0.05，##P<0.01; in comparison to the set of models,*P<0.05，**P<0.05。
as can be seen from FIG. 11, hydrogen peroxide (H) was observed in the mouse myocardial tissue in the anoxic state2O2) The content is obviously increased, which indicates that the body generates a large amount of oxygen free radicals due to oxygen deficiency. Administration of Positive drug acetazolamide H2O2The level of the compound is lower than that of the model group, and H is obtained after the costunolide is administered2O2The level was very significantly lower than the model group.
(4) Determination of hydroxy radical OH scavenging capacity of myocardial tissue of plateau hypoxia simulation mouse
A red substance is produced by Fenton reaction, and the absorbance is in direct proportion to the amount of OH produced. Simulating the measurement of tissue OH content of plateau anoxic mice, H in the tissue2O2The amount reflects how much ROS the body generates. The reaction principle is as follows: h2O2Reacting with molybdic acid to generate complex. 10% of the tissue samples were centrifuged at 15000g for 5min to prepare a reaction solution, which was then measured at 405 nm.
FIG. 12 shows the effect of costunolide on myocardial tissue OH clearance in hypoxic mice: (
n＝10)。
Note: in comparison with the normal group,#P<0.05，##P<0.01; in comparison to the set of models,*P<0.05，**P<0.05。
as can be seen from FIG. 12, the hydroxy radical (. OH) scavenging ability of myocardial tissue of mice in the model group in the anoxic state is significantly lower than that of the normal control group, and the OH scavenging ability of the positive drug acetazolamide group is significantly recovered compared with that of the model group, and is close to that of the normal group. After costunolide is given, the free radical (OH) scavenging capacity is obviously increased compared with that of a model group, which shows that costunolide and sesquiterpene lactone derivative thereof have a protective effect on myocardial OH scavenging capacity of an anoxic organism.
(5) Content determination of Malondialdehyde (MDA) of tissue of mouse simulating altitude anoxia
The reaction principle is that Malondialdehyde (MDA) reacts with Thiobarbital (TBA) to generate a red substance, 10% of tissue homogenate is centrifuged at 4500 rpm, the supernatant is taken to prepare a reaction solution according to the instruction, the reaction is carried out, the absorbance is measured at 532nm, and the result is calculated by substituting the formula.
FIG. 13 is a graph of the effect of costunolide on MDA levels in hypoxic myocardial tissue in mice (n＝10)。
Note: in comparison with the normal group,#P<0.05，##P<0.01; in comparison to the set of models,*P<0.05，**P<0.05。
as can be seen from fig. 13, the mouse myocardial tissue MDA level is significantly increased in the hypoxic state, the MDA content is not significantly changed from that in the model group after the administration of the positive drug acetazolamide, and the mouse myocardial MDA content level is significantly lower than that in the model group after the administration of costunolide.
According to the invention, the plateau hypoxia resistance of costunolide and dehydrocostunolide is researched through a normal-pressure closed hypoxia experiment and an acute pressure-relief hypoxia experiment model, and the result shows that costunolide and derivatives thereof, namely dehydrocostunolide, have good pressure-relief hypoxia resistance, and the action mechanism of dehydrocostunolide can be realized through antioxidant stress. Costunolide can protect SOD and CAT activity in myocardium tissue of mouse with anoxia, enhance ROS and free radical scavenging ability of brain of mouse with anoxia, and reduce MDA and H in myocardium of mouse caused by ROS after anoxia2O2OH level, thereby achieving the effect of resisting decompression and anoxia.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. Application of costunolide, dehydrocostuslactone and their derivatives in preparing medicine for preventing and treating anoxia myocardial protection is provided.
2. Use according to claim 1, characterized in that: the oxygen deficiency is oxygen deficiency under normal pressure or reduced pressure.
3. Use according to claim 1, characterized in that: the hypoxia is acute hypoxia or chronic hypoxia.
4. Use according to claim 1, characterized in that: the anoxic myocardium protection is to protect SOD and CAT activity in the anoxic mouse myocardium tissue, enhance ROS and free radical scavenging ability of anoxic organism brain, and reduce MDA and H in mouse myocardium caused by ROS after anoxia2O2OH level.
5. Application of costunolide, dehydrocostuslactone and their derivatives in preparing medicine for maintaining blood pressure in acute anoxia is provided.
6. The preparation method of costunolide and its derivative dehydrocostunolide is characterized by comprising the following steps: the method comprises the following steps:
(1) extracting radix aucklandiae or radix aucklandiae decoction pieces with ethanol, and collecting supernatant to obtain ethanol extractive solution;
(2) extracting the ethanol extract obtained in the step (1) by using petroleum ether, and collecting the lower layer extracted liquid to obtain petroleum ether extract;
(3) extracting the petroleum ether extract liquid in the step (2) by using ethyl acetate, and collecting upper-layer liquid to obtain ethyl acetate extract liquid;
(4) and (4) separating and purifying the ethyl acetate extract obtained in the step (3) by using a high performance liquid chromatograph, and respectively collecting different fractions to obtain costunolide and dehydrocostunolide.
7. The method of claim 6, wherein: in the step (1), the specific method for extracting costustoot by ethanol comprises the following steps: pulverizing radix aucklandiae, adding 5-10 times of 50-95% ethanol, soaking for 6-12 hr, ultrasonic treating for 0.5-1 hr, standing for layering, collecting supernatant, extracting the lower layer precipitate with ethanol again, and mixing the supernatants.
8. The method of claim 6, wherein: in the step (4), when the high performance liquid chromatograph is used for separation and purification, the chromatographic conditions are as follows: a chromatographic column: waters Atlantis C18 column; flow rate: 1.0-3.0 ml/min; sample introduction volume: 0.1-0.5 ml; detection wavelength: 225 nm; mobile phase: 0-3 min, 15% -25% methanol; 3-10 min, 25% -35% methanol; 10-20 min, 35% -60% methanol; 20-35 min, 60-70% methanol; 35-50 min, 70% methanol, and the flow phase ratio is volume percentage; column temperature: at 25 ℃.
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