CN115844933B - Application of clindamycin total flavone in preparing heart failure resisting medicine - Google Patents
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Abstract
The invention relates to the field of medicine application, in particular to application of clinopodium polycephalum total flavonoids in preparing medicines for resisting heart failure. The Clinopodium polycephalum total flavone is prepared by reflux extracting with ethanol, separating with macroporous adsorbent resin, collecting ethanol eluate, concentrating the eluate, and drying. The invention discovers that the clinopodium total flavonoids can obviously improve cardiac hypertrophy and cardiac function, has protective effect on mice cardiac hypertrophy and cardiac fibrosis induced by Ang II, and can reduce total protein concentration and oxidative stress to inhibit cardiac hypertrophy induced by Ang II. The invention takes the clindamycin total flavone extract as the active ingredient, and the active ingredient is mixed with pharmaceutically acceptable excipient or carrier auxiliary materials to prepare the heart failure resistant medicament.
Description
Technical Field
The invention relates to the field of medicine application, in particular to application of clindamycin total flavonoids in preparing medicines for resisting heart failure.
Background
With the rapid development of social economy, the national lifestyle has changed greatly, and the influence of cardiovascular diseases on the health of residents caused by unhealthy lifestyles of residents is also increasingly important, so that the incidence rate of cardiovascular diseases is continuously increased. Heart failure is the end-stage manifestation of heart disease and is accompanied by structural and electrical remodeling of the heart, which in turn exacerbates heart failure. Reconstruction of the heart structure refers to structural changes in the heart under the influence of various factors. Cardiac electrical remodeling refers to the occurrence of various forms of adaptive electrical function alterations in the various pathological or physiological factors of heart failure. The structural and electrical remodeling of the heart are interactive, and act to compensate for the initial onset of heart failure, which in turn exacerbates heart failure.
One of the manifestations of heart failure structural remodeling is myocardial hypertrophy, which we can affect heart failure structural remodeling by improving myocardial hypertrophy. Commonly used anti-heart failure drugs are mainly angiotensin converting enzyme inhibitors, angiotensin ii receptor antagonists, calcium antagonists, beta-adrenergic receptor antagonists and the like. Although these drugs have a certain therapeutic effect, they eventually do not completely prevent further deterioration of heart failure.
In recent years, natural medicines have been attracting attention in the treatment and prevention of cardiovascular diseases. The plant Clinarch (Clinachus nutansCN), also known as crocodile flower, is a plant of the genus crocodile flower of the family Acanthaceae, which is widely distributed in tropical and subtropical areas such as Malaysia and Vietnam, and in areas such as Hainan, yunnan and Guangdong of China. The whole plant is sweet, pungent, slightly bitter and mild in nature, enters liver and kidney channels, and has the effects of clearing heat and detoxicating, removing stasis and detumescence, diminishing inflammation and dispelling alcohol effects, preventing and resisting cancer and the like. Chemical analysis shows that the clindamiana leaves contain compounds such as sterol, triterpene, flavone, polypeptide and the like, and have pharmacological activities such as anti-inflammatory, antioxidant, anti-tumor, immunity improvement, cardiovascular protection and the like.
At present, few researches on the physiological activity of the beakly herbal medicine are reported at home and abroad, and most of the researches are concentrated on anti-tumor, antioxidation and other aspects. For example, the Chinese patent application with publication number of CN107648289A discloses the preparation of the clinopodium polycephalum extract and the application of the clinopodium polycephalum extract in anti-tumor aspect, and the extract is found to have obvious inhibition effect on human leukemia, liver cancer, lung cancer, breast cancer and colon cancer cells through an in-vitro cytotoxicity test, and has better anti-tumor activity. Also, for example, chinese patent application publication No. CN114989248A discloses a Clinopodium polycephalum polypeptide having anti-inflammatory and antioxidant activities, and its preparation method and application. Aiming at the fresh report of the action of the clindamycin extract on the cardiovascular aspect, the action of the clindamycin extract on the myocardial hypertrophy resistance is not reported. At present, the application of the clinopodium extract in preparing the anti-heart failure medicine is not reported yet. The application finds that the clinopodium polycephalum extract is mainly clinopodium polycephalum total flavone extract with the function of remarkably treating heart failure for the first time, and the clinopodium polycephalum has potential value for developing into an anti-heart failure drug.
Disclosure of Invention
Accordingly, the invention aims to study the efficacy of the clinopodium polycephalum total flavonoids in resisting heart failure and provides an application of the clinopodium polycephalum total flavonoids in preparing medicines for resisting heart failure.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the application of clindamycin total flavone in preparing heart failure resisting medicine is provided.
The preparation method of the clindamycin total flavonoids comprises the following steps:
s1, crushing the dried clinopodium polycephalum leaves, reflux-extracting with 50% -90% ethanol, collecting an extracting solution, recovering ethanol, and drying to obtain a crude extract;
s2, the crude extract is put on macroporous adsorption resin, water and 30% -60% ethanol are sequentially used for eluting, 30% -60% ethanol eluent is collected, ethanol is recovered, and the extract of the total flavonoids of the clinopodium polycephalum is obtained after drying.
Preferably, the preparation of the clindamycin total flavonoids comprises the following steps:
s1, crushing the dried clinopodium polycephalum leaves, extracting with 70% -80% ethanol under reflux for 1-3 times, each time for 1-2 hours, combining and collecting the extracting solution, recovering ethanol, and drying to obtain a crude extract;
s2, the crude extract is put on macroporous adsorption resin, water and 30% -40% ethanol are sequentially used for eluting, 30% -40% ethanol eluent is collected, ethanol is recovered, and the extract of the total flavonoids of the clinopodium polycephalum is obtained after drying.
Ultraviolet spectrophotometry is adopted to measure the content of total flavonoids in the dry leaves of the clinopodium polycephalum, and a standard curve is drawn to obtain a regression equation. Dissolving the clinopodium polycephalum total flavone to obtain a sample, measuring absorbance of the clinopodium polycephalum total flavone solution, and calculating the clinopodium polycephalum total flavone content.
Preferably, the use of the clinopodium total flavonoids in preparing medicines for resisting cardiac insufficiency caused by heart failure and pathological heart remodeling is provided.
Preferably, the use of said clinopodium total flavonoids in the manufacture of a medicament for combating cardiomyocyte hypertrophy caused by heart failure and pathological heart remodeling.
Preferably, the use of the clinopodium total flavonoids in preparing medicines for resisting myocardial interstitial fibrosis caused by heart failure and pathological heart remodeling is provided.
Preferably, the use of said clinopodium total flavonoids in the manufacture of a medicament for combating cardiomyocyte death caused by heart failure and pathological heart remodeling.
Preferably, the heart failure resistant medicament is prepared from clinopodium polycephalum total flavonoids and auxiliary materials, wherein the auxiliary materials are excipients.
Compared with the prior art, the invention has the beneficial effects that:
the invention discovers that the clinopodium total flavonoids can obviously improve cardiac hypertrophy and cardiac function, has protective effect on mice cardiac hypertrophy and cardiac fibrosis induced by Ang II, and can reduce total protein concentration and oxidative stress to inhibit cardiac hypertrophy induced by Ang II. The clindamycin has high application value, high flavone content and simple extraction and preparation method of total flavone, and has potential value of developing into heart failure resistant medicines.
Drawings
FIG. 1 is a graph showing the effect of HE staining on the detection of the total flavonoids of clindamycin on Ang II-induced myocardial hypertrophy of mice in example 3 of the present invention.
FIG. 2 shows the result of Masson staining of AngII-induced myocardial tissue of mice with cardiac hypertrophy by clindamycin total flavonoids in example 4 of the present invention.
FIG. 3 shows the effect of the clindamycin total flavonoids on the activity of H9C2 cells in example 5 of the present invention.
FIG. 4 shows the effect of the total flavonoids of clindamycin on Ang II-induced H9C2 cell protein content in example 6 of the present invention.
FIG. 5 is a graph showing the effect of the clindamycin total flavonoids on the ROS level in AngII-induced H9C2 cells in example 7 of the present invention, wherein the graph (A) is a ROS fluorescence diagram; panel (B) is the effect of varying concentrations of clindamycin total flavonoids (40. Mu.g/mL, 80. Mu.g/mL, 160. Mu.g/mL) on AngII-induced levels of rat cardiomyocyte ROS.
Detailed Description
In order to better understand the technical content of the present invention, the following provides specific examples to further illustrate the present invention. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. Modifications and equivalents will occur to those skilled in the art upon understanding the present teachings without departing from the spirit and scope of the present teachings.
EXAMPLE 1 preparation of Clinopodium polycephalum total flavonoids
Pulverizing the dried Clinopodium polycephalum herba Clinopodii leaf, reflux-extracting with 75% ethanol at 80deg.C for 2 times (feed-liquid ratio 1:20) each for 1 hr, collecting the extractive solutions, rotary evaporating to recover ethanol, and freeze-drying to obtain crude extract. Enriching flavonoid glycoside in crude extract by HPD-100 type macroporous adsorption resin, eluting with water for 10 column volumes to remove strong polar impurities, eluting with 40% ethanol-water solution for 7 column volumes to elute flavonoid glycoside, recovering ethanol by rotary evaporation, and lyophilizing to obtain total flavonoid extract of Clinopodium polycephalum. Measuring the content of total flavonoids in the dry leaves of the clinopodium polycephalum by ultraviolet spectrophotometry, and drawing a standard curve to obtain a regression equation. Dissolving the clinopodium polycephalum total flavone to obtain a sample, measuring absorbance of the clinopodium polycephalum total flavone solution, and calculating the clinopodium polycephalum total flavone content.
Example 2 Effect of Clinopodium polycephalum total flavonoids on cardiac function in mice
1. Grouping and administration of mice
Normal group: the same volume of physiological saline was infused daily for one month, and after a continuous period, the physiological saline was injected subcutaneously for 3 weeks.
Model group: the same volume of physiological saline was infused daily for one month, and then AngII (0.6mg.kg) -1 ·d -1 ) 3 weeks.
Low dose group of clinopodium total flavonoids: 75 mg/kg of stomach is irrigated every day -1 After the clindamycin total flavone solution is continued for one month, angII (0.6mg.kg) -1 ·d -1 ) 3 weeks.
150 mg/kg of the total flavonoids of clindamycin per day -1 After the clindamycin total flavone solution is continued for one month, angII (0.6mg.kg) -1 ·d -1 ) 3 weeks.
High dose group of clinopodium total flavonoids: 300 mg/kg of stomach is irrigated every day -1 After the clindamycin total flavone solution is continued for one month, angII (0.6mg.kg) -1 ·d -1 ) 3 weeks.
2. Determination of cardiac function
AngII (0.6mg.kg) -1 ·d -1 ) After 3 weeks, the heart function of the mice was examined using an ultrasound imaging system after anesthesia and M-mode ultrasound images were recorded. The mice were measured for left ventricular end diastole (LVEDD), left Ventricular End Systole (LVESD), and Left Ventricular Ejection Fraction (LVEF) and left ventricular short axis foreshortening rate (LVFS) were calculated.
After the last subcutaneous injection, the diet was fasted for 24 hours. After 3 weeks of subcutaneous injection of AngII (0.6 mg/kg/d), we assayed the mouse heart function by ultrasound. As shown in table1, we can observe that the model group mice had elevated LVEDD (left end diastole inner diameter), LVESD (left end systole inner diameter) values, while LVEF (left ventricular ejection fraction), lves (left ventricular short axis reduction fraction) were reduced compared to the normal group, indicating that the model group mice exhibited myocardial hypertrophy and reduced cardiac function (P < 0.01). Compared with the model group, the total flavonoids of the clinopodium polycephalum have reduced LVEDD and LVESD values, and increased LVEF and LVFS, which shows that the total flavonoids of the clinopodium polycephalum can obviously improve myocardial hypertrophy and cardiac function (#P < 0.01).
TABLE1 ultrasonic results of total flavonoids of Clinopodium polycephalum on AngII-induced mouse myocardial hypertrophy model Table1. Ultrasusoudreulttsofthe totalflavanonoid softAngII-indducedrouseheaddel
Note that:
n=6, P compared to normal group<0.01. In comparison with the mice of the model group, ## P<0.01。
example 3 influence of Clinopodium polycephalum total flavonoids on various cardiac measurements in mice
AngII (0.6mg.kg) -1 ·d -1 ) After 3 weeks, each group of mice was taken, weighed, and then killed by cervical dislocation, quickly fixed on an operating table, and the chest skin was cut off with surgical scissors, the heart and lung were fully exposed, and the heart was removed by cutting off from the aortic root. The heart was rinsed 3 times, weighed after rinsing, and the heart morphology was observed and photographed. Finally, the length of the tibia of the right hind limb of the mouse is measured. The heart mass index, left ventricle mass index, lung weight to body weight ratio, tibia length to heart ratio increase were calculated.
As shown in Table2, the model groups HW/BW (cardiac volume/weight), LVW/BW (left ventricular weight/weight), LW/BW (lung weight/weight), HW/T were compared with the control groupBoth L (heart weight/tibia length) are markedly increased (×p)<0.01). The model group HW/BW, LVW/BW, LW/BW and HW/TL of the clindamycin total flavone low, medium and high dosage ratio are obviously reduced ## P<0.01)。
TABLE2 Effect of Clinopodium polycephalum total flavonoids on cardiac measurement index of AngII-induced mouse cardiac model Table2. Theefsoft flavonoid from AngII-indducedmouseheat diagnostic on AngII-induced mouse cardiac model
Note that:
n=6; p compared to normal group<0.01; in comparison with the group Ang II, ## P<0.01。
as shown in the HE staining of fig. 1, under a 20-fold microscope, the central myocytes of the control group were normal in morphology, clear in structure, uniform in size, free of obvious mast cells, and orderly arranged among cells; in the model group, the morphology of cardiomyocytes was changed, cardiomyocytes were significantly hypertrophic, and intercellular arrangement was disturbed. Compared with the model group, the clinopodium total flavone group myocardial cells have more uniform morphology, reduced cell hypertrophy degree and more orderly cell arrangement.
EXAMPLE 4 Clinopodium polycephalum total flavonoids inhibit collagen deposition in myocardial tissue of mice
When the cardiac muscle is hypertrophic, the collagen of the cardiac muscle tissue is increased. In Masson staining, red represents muscle fibers and blue represents collagen deposition. As shown in fig. 2, the model group had a significantly blue deposition compared to the blank group, indicating an increase in collagen in the myocardial tissue of the mice in the model group; compared with the model group, the clindamycin total flavonoids can obviously reduce collagen deposition.
Example 5 Effect of different concentrations of Clinopodium polycephalum total flavonoids on cell viability
CCK-8 detection of cell viability
When H9C2 cells grew to cover 80% of the area of the flask, the original culture medium was discarded, washed 2 times with 3mLPBS, digested with 0.25% trypsin, centrifuged, the supernatant discarded, and 3mL of complete medium was added for blow mixing and counting under a microscope. Then at 3X 10 5 The cells were seeded into 96-well plates at a concentration of one per mL, 100 μl of cell fluid was added to each well, and 6 multiplex wells were placed in each group. After 24h, the clindamycin total flavonoids (0, 10, 20, 40, 80, 160, 320 mug/mL) were added at different concentrations. Incubation was continued for 24h. And adding 10 mu LCCK8 solution into each hole of the liquid exchange, and incubating for 0-4 hours in a dark place, wherein the influence of the total flavonoids of the clindamycin on the activity of H9C2 cells is detected by a 490nm wavelength of an enzyme-labeled instrument.
The effect of the total flavonoids of the clinopodium polycephalum on the activity of the cells after 24H of H9C2 intervention is measured by adopting a CCK-8 method, as shown in figure 2, the effect of the total flavonoids of the clinopodium polycephalum (0, 10, 20, 40, 80, 160, 320 mug/mL) on the activity of the H9C2 cells is measured, the activity of the H9C2 cells is improved by the total flavonoids of the clinopodium polycephalum in the range of 0-320 mug/mL, and when the concentration of the clinopodium polycephalum is 40 mug/mL, 80 mug/mL and 160 mug/mL, the activity of the cells is increased to the maximum, so that the concentration of the clinopodium polycephalum adopted in the subsequent cell experiments is 40 mug/mL, 80 mug/mL and 160 mug/mL (P < 0.01).
Example 6 influence of Clinopodium polycephalum total flavonoids on total protein content of cells
The protein concentration of the protein is measured by the BCA kit, as shown in FIG. 4, and the result shows that the AngII (L mu mol/L) incubation for 24 hours can effectively increase the total protein concentration in rat myocardial H9C2 cells, and has statistical significance (P)<0.001 A) is provided; pretreatment of Clinopodium polycephalum total flavone extract (40 μg/mL,80 μg/mL,160 μg/mL) for 24 hr can inhibit increase of H9C2 cell total protein concentration induced by AngII # P<0.05， ## P<0.01， ### P<0.001)。
Example 7 Clinopodium polycephalum total flavonoids reduce cellular ROS levels in cardiac hypertrophy
Detection of cellular ROS levels
Rat cardiomyocytes H9C2 were counted, inoculated in 6-well plates, grouped as above, 3 multiplex wells were set, cultured for 24 hours, and DCFH-DA diluted 1000-fold. The culture broth was discarded and 1 mM CFH-DA was added. The tinfoil paper is wrapped in a dark place and placed in an incubator at 37 ℃ for 15min. The serum-free culture medium was washed to clean the cells, and the cells were photographed in a dark place using a fluorescence microscope.
AngII can induce oxidase activation and excessive reactive oxygen species production, excessive ROS production can trigger cell dysfunction, lipid peroxidation and DNA mutation, and can lead to cell injury or death, inducing myocardial hypertrophy [12-14]. ROS have been considered as one of the key factors in the development of myocardial hypertrophy. The effect of the total flavonoids extract of clindamini on the ROS level of Ang ii-induced H9C2 cells was examined using ROS kit. As shown in fig. 5 (a), the Ang ii group showed significantly enhanced fluorescence intensity (< 0.05) compared to the control group. Pretreatment of the clindamycin total flavonoids (40 mug/mL, 80 mug/mL, 160 mug/mL) for 24 hours can weaken the fluorescence intensity (#P0.01) of AngII induced H9C 2. As shown in FIG. 5 (B), the level of ROS in the Ang II group was significantly increased, and the total flavonoids of Clinopodium polycephalum inhibited the increase of ROS level induced by Ang II. P <0.001 compared to control group; compared to the Ang ii group, #p <0.01.
Claims (7)
1. The application of the clinopodium polycephalum total flavonoids in preparing medicaments for resisting heart failure comprises the following steps of:
s1, crushing the dried clinopodium polycephalum leaves, reflux-extracting with 50% -90% ethanol, collecting an extracting solution, recovering ethanol, and drying to obtain a crude extract;
s2, the crude extract is put on macroporous adsorption resin, water and 30% -60% ethanol are sequentially used for eluting, 30% -60% ethanol eluent is collected, ethanol is recovered, and the extract of the total flavonoids of the clinopodium polycephalum is obtained after drying.
2.The use of the clinopodium polycephalum total flavone in preparing an anti-heart failure medicament according to claim 1, wherein the preparation of the clinopodium polycephalum total flavone comprises the following steps:
s1, crushing the dried clinopodium polycephalum leaves, extracting with 70% -80% ethanol under reflux for 1-3 times, each time for 1-2 hours, combining and collecting the extracting solution, recovering ethanol, and drying to obtain a crude extract;
s2, the crude extract is put on macroporous adsorption resin, water and 30% -40% ethanol are sequentially used for eluting, 30% -40% ethanol eluent is collected, ethanol is recovered, and the extract of the total flavonoids of the clinopodium polycephalum is obtained after drying.
3. The use of the clinopodium polycephalum total flavone in the preparation of an anti-heart failure medicament according to claim 1, wherein the clinopodium polycephalum total flavone is used for preparing a medicament for resisting heart dysfunction caused by heart failure and pathological heart remodeling.
4. The use of the clinopodium polycephalum total flavone in the preparation of an anti-heart failure medicament according to claim 1, wherein the clinopodium polycephalum total flavone is used for preparing a medicament for resisting myocardial cell hypertrophy caused by heart failure and pathological heart remodeling.
5. The use of the clinopodium total flavonoids according to claim 1 for preparing an anti-heart failure medicament, wherein the use of the clinopodium total flavonoids for preparing a medicament for resisting myocardial interstitial fibrosis caused by heart failure and pathological heart remodeling is provided.
6. The use of the clinopodium polycephalum total flavone in the preparation of an anti-heart failure medicament according to claim 1, wherein the clinopodium polycephalum total flavone is used for preparing a medicament for resisting myocardial cell death caused by heart failure and pathological heart remodeling.
7. The use of the total flavonoids of clinopodium polycephalum in preparing an anti-heart failure medicament according to claim 1, wherein the medicament is prepared from the total flavonoids of clinopodium polycephalum and auxiliary materials, wherein the auxiliary materials are excipients.
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