CN115844933A - Application of clinacanthus nutans total flavonoids in preparation of anti-heart failure drugs - Google Patents
Application of clinacanthus nutans total flavonoids in preparation of anti-heart failure drugs Download PDFInfo
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Abstract
The invention relates to the field of medicament application, in particular to application of clinacanthus nutans total flavonoids in preparing an anti-heart failure medicament. The Clinacanthus nutans total flavone is prepared by reflux extracting with ethanol, separating with macroporous adsorbent resin, collecting ethanol eluate, concentrating the eluate, and drying. The clinacanthus nutans total flavone is found for the first time to be capable of obviously improving myocardial hypertrophy and cardiac function, has a protective effect on myocardial hypertrophy and myocardial fibrosis of mice induced by Ang II, and can reduce total protein concentration and oxidative stress to inhibit myocardial cell hypertrophy induced by Ang II. The invention takes the clinacanthus nutans total flavone extract as an effective component, and the clinacanthus nutans total flavone extract is mixed with a pharmaceutically acceptable excipient or a carrier auxiliary material to prepare the anti-heart failure medicine.
Description
Technical Field
The invention relates to the field of medicament application, in particular to application of clinacanthus nutans total flavonoids in preparing a medicament for resisting heart failure.
Background
With the rapid development of social economy, the life style of the people is greatly changed, the influence of cardiovascular diseases on the health of residents is more and more important due to the unhealthy life style of the residents, and the incidence of the cardiovascular diseases is continuously increased. Heart failure is an end-stage manifestation of heart disease and is accompanied by structural and electrical remodeling of the heart, which in turn exacerbates heart failure. The reconstruction of the heart structure means that the heart has structural changes under the influence of various factors. Electrical remodeling of the heart refers to various forms of adaptive electrical functional changes in various pathological or physiological factors of heart failure. Structural and electrical remodeling of the heart are interdependent, compensating for the early onset of heart failure, which then exacerbates it.
One of the manifestations of structural remodeling in heart failure is myocardial hypertrophy, which we can influence by improving. The commonly used anti-heart failure drugs are mainly angiotensin converting enzyme inhibitors, angiotensin II receptor antagonists, calcium antagonists, beta-adrenoceptor antagonists and the like. Although these drugs have a certain therapeutic effect, eventually, they cannot completely prevent further deterioration of heart failure.
In recent years, natural drugs have been attracting attention for the treatment and prevention of cardiovascular diseases. Clinacanthus nutans (Clinacanthus nutans CN), also known as crocodile flower, is a plant of the genus crocodile of the family Acanthaceae, and is widely distributed in tropical and subtropical areas such as Malaysia and Vietnam, as well as in areas such as Hainan, yunnan and Guangdong in China. The whole plant is used as a medicine, has sweet, pungent, slightly bitter and neutral taste, enters liver and kidney channels, and has the effects of clearing away heat and toxic materials, removing blood stasis, relieving swelling, diminishing inflammation, alleviating hangover, preventing and resisting cancer and the like. Chemical analysis finds that the clinacanthus nutans leaves contain compounds such as sterol, triterpene, flavone, polypeptide and the like, and have pharmacological activities of resisting inflammation, oxidation and tumor, improving immunity, protecting cardiovascular system and the like.
At present, few research reports on the physiological activity of clinacan herbs at home and abroad are reported, and most of the research reports are focused on the aspects of tumor resistance, oxidation resistance and the like. For example, chinese patent application publication No. CN107648289A discloses the preparation of clinacanthus nutans extract and its application in anti-tumor, and the extract has significant inhibitory effect on human leukemia, liver cancer, lung cancer, breast cancer and colon cancer cells through in vitro cytotoxic activity test, and has good anti-tumor activity. Further, as disclosed in chinese patent application publication No. CN114989248A, a clinacanthus hypochondriacus polypeptide having anti-inflammatory and antioxidant activities, which has excellent anti-inflammatory and antioxidant activities, and a preparation method and use thereof are disclosed. The effect of the clinacanthus nutans extract on the cardiovascular aspect is rarely reported, and the effect on the myocardial hypertrophy resistance is not reported. At present, the application of the clinacanthus nutans extract in the preparation of anti-heart failure medicines is not reported. The application discovers for the first time that the clinacanthus nutans extract mainly is a clinacanthus nutans total flavone extract which has the effect of remarkably treating heart failure, and the clinacanthus nutans has the potential value of being developed into an anti-heart failure medicament.
Disclosure of Invention
Therefore, the invention aims to research the efficacy of clinacanthus nutans total flavonoids in resisting heart failure and provides an application of the clinacanthus nutans total flavonoids in preparing a heart failure resisting medicine.
In order to achieve the purpose, the technical scheme of the invention is as follows:
application of Clinacanthus nutans total flavonoids in preparing anti-heart failure medicines is provided.
The preparation of the clinacanthus nutans total flavonoids comprises the following steps:
s1, crushing dried Clinacanthus nutans leaves, performing reflux extraction by using 50-90% ethanol, collecting an extracting solution, recovering the ethanol, and drying to obtain a crude extract;
s2, passing the crude extract through macroporous adsorption resin, eluting with water and 30-60% ethanol in sequence, collecting 30-60% ethanol eluate, recovering ethanol, and drying to obtain the clinacanthus nutans total flavone extract.
Preferably, the preparation of the clinacanthus nutans total flavonoids comprises the following steps:
s1, crushing dried Clinacanthus nutans leaves, performing reflux extraction for 1-3 times with 70-80% ethanol for 1-2 hours each time, combining and collecting extracting solutions, recovering ethanol, and drying to obtain a crude extract;
s2, passing the crude extract through macroporous adsorption resin, eluting with water and 30-40% ethanol in sequence, collecting 30-40% ethanol eluate, recovering ethanol, and drying to obtain the clinacanthus nutans total flavone extract.
Measuring the content of total flavonoids in the dried Clinacanthus nutans leaves by adopting an ultraviolet spectrophotometry, and drawing a standard curve to obtain a regression equation. Dissolving Clinacanthus nutans total flavonoids to prepare a test sample, measuring absorbance of the Clinacanthus nutans total flavonoids solution, and calculating the content of Clinacanthus nutans total flavonoids.
Preferably, the clinacanthus nutans total flavone is used for preparing the medicine for resisting cardiac insufficiency caused by heart failure and pathological cardiac remodeling.
Preferably, the clinacanthus nutans total flavone is used for preparing a medicament for resisting myocardial cell hypertrophy caused by heart failure and pathological heart reconstruction.
Preferably, the clinacanthus nutans total flavonoids are applied to the preparation of the drugs for resisting myocardial interstitial fibrosis caused by heart failure and pathological heart reconstruction.
Preferably, the clinacanthus nutans total flavone is used for preparing the medicine for resisting myocardial cell death caused by heart failure and pathological heart reconstruction.
Preferably, the anti-heart failure drug is a clinically acceptable drug preparation prepared from clinacanthus nutans 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 for the first time that the clinacanthus nutans total flavonoids can obviously improve myocardial hypertrophy and cardiac function, has protective effect on myocardial hypertrophy and myocardial fibrosis of mice induced by Ang II, and can reduce total protein concentration and oxidative stress to inhibit myocardial cell hypertrophy induced by Ang II. The clinacanthus nutans has high application value, high flavone content and simple total flavone extracting and preparing process, and has latent value in developing medicine for treating heart failure.
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FIG. 1 is a graph showing HE staining to examine the effect of total flavonoid extract of Clinacanthus nutans on Ang II-induced myocardial hypertrophy in mice according to example 3 of the present invention.
FIG. 2 shows Masson staining results of myocardial tissue of mouse with hypertrophy of myocardium induced by Ang II with total flavonoids of Clinacanthus nutans in example 4 of the present invention.
FIG. 3 is a graph showing the effect of total flavonoids of Clinacanthus nutans on the viability of H9C2 cells in example 5 of the present invention.
FIG. 4 is a graph showing the effect of total flavonoids of Clinacanthus nutans on the Ang II-induced H9C2 cell protein content in example 6 of the present invention.
FIG. 5 is an effect of Clinacanthus nutans total flavonoids on Ang II-induced ROS levels of H9C2 cells in example 7 of the present invention, wherein FIG. (A) is a ROS fluorescence graph; FIG. B is a graph of the effect of various concentrations of Clinacanthus nutans total flavonoids (40. Mu.g/mL, 80. Mu.g/mL, 160. Mu.g/mL) on Ang II-induced ROS levels in rat cardiomyocytes.
Detailed Description
In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Those skilled in the art should understand that they can make modifications and equivalents without departing from the spirit and scope of the present invention, and all such modifications and equivalents are intended to be included within the scope of the present invention.
Example 1 preparation of total flavonoids from Clinacanthus nutans
Pulverizing dried Clinacanthus nutans leaf, reflux-extracting with 75% ethanol at 80 deg.C for 2 times (material-to-liquid ratio: 1: 20) for 1h each time, collecting the extractive solutions, rotary evaporating to recover ethanol, and freeze-drying to obtain crude extract. The HPD-100 type macroporous adsorption resin is used for enriching the flavonoid glycoside in the crude extract, firstly, 10 column volumes are eluted by water to remove impurities with strong polarity, then, 7 column volumes are eluted by 40% ethanol-water solution to elute the flavonoid glycoside, the ethanol is recovered by rotary evaporation, and the extract of the total flavonoids of clinacanthus nutans is obtained by freeze drying. Measuring the content of total flavonoids in the dried Clinacanthus nutans leaves by using an ultraviolet spectrophotometry, and drawing a standard curve to obtain a regression equation. Dissolving Clinacanthus nutans total flavonoids to prepare a test sample, measuring absorbance of the Clinacanthus nutans total flavonoids solution, and calculating the content of Clinacanthus nutans total flavonoids.
Example 2 Effect of Clinacanthus nutans Total Flavonoids on Heart function in mice
1. Grouping and administration of mice
Normal group: the stomach was perfused with the same volume of normal saline every day, and after one month continuously, normal saline was injected subcutaneously for 3 weeks.
Model group: the stomach was perfused with the same volume of physiological saline every day, and after one month continuously, angII (0.6 mg kg) was injected subcutaneously -1 ·d -1 ) For 3 weeks.
Low dose group of nutclinacanthus nutans total flavonoids: 75 mg/kg per day for intragastric administration -1 After a continuous one month period of the Clinacanthus nutans total flavone solution, angII (0.6 mg kg. Multidot.kg) was injected subcutaneously -1 ·d -1 ) For 3 weeks.
The common Clinacanthus nutans total flavone medium dose group is 150 mg/kg per day by intragastric administration -1 After a continuous one month period of the Clinacanthus nutans total flavone solution, angII (0.6 mg kg. Multidot.kg) was injected subcutaneously -1 ·d -1 ) For 3 weeks.
Clinacanthus nutans Total Flavonoids high dose group: gavage 300 mg/kg per day -1 After a continuous one month period of the Clinacanthus nutans total flavone solution, angII (0.6 mg kg. Multidot.kg) was injected subcutaneously -1 ·d -1 ) For 3 weeks.
2. Determination of cardiac function
Subcutaneous injection of AngII (0.6 mg kg) -1 ·d -1 ) After 3 weeks, the mice were anesthetized and then tested for cardiac function using an ultrasound imaging system and M-mode ultrasound images were recorded. The Left Ventricular End Diastolic Diameter (LVEDD), left Ventricular End Systolic Diameter (LVESD) of the mice were measured, and the Left Ventricular Ejection Fraction (LVEF) and left ventricular short axis shortening rate (LVFS) were calculated.
After the last subcutaneous injection, fasting was performed for 24 hours. After 3 weeks of subcutaneous injection of AngII (0.6 mg/kg/d), we measured the cardiac function of the mice by ultrasound. As shown in table1, we can observe that the values of LVEDD (left ventricular end diastolic diameter), LVEDD (left ventricular end systolic diameter) and LVEF (left ventricular ejection fraction), LVFS (left ventricular short axis shortening fraction) of the model group mice are increased and decreased compared with the normal group, indicating that the model group mice have myocardial hypertrophy and decreased cardiac function (. About.. Mark.p < 0.01). Compared with the model group, the Clinacanthus nutans total flavone group has reduced LVEDD and LVESD values, while LVEF and LVFS are increased, which shows that the Clinacanthus nutans total flavone can obviously improve myocardial hypertrophy and improve cardiac function (# # P < 0.01).
TABLE1 Clinacanthus nutans Total Flavonoids on Ang II induced mouse myocardial hypertrophy model ultrasonic results Table1. Ultrasoundsesultshot anterior of AngII-indexedMouseheerthodel
Note:
n =6, P compared to normal group<0.01. Compared with the mice in the model group, ## P<0.01。
example 3 Effect of Clinacanthus nutans Total Flavonoids on various cardiac measurement indices of mice
Subcutaneous injection of AngII (0.6 mg kg) -1 ·d -1 ) After 3 weeks, each group of mice was taken, weighed, sacrificed by dislocation of cervical vertebrae, quickly fixed on an operating table, and the chest skin was cut with surgical scissors to fully expose the heart and lungs, cut from the aortic root, and the heart was removed. The heart was rinsed 3 times, weighed after rinsing, observed for heart morphology and photographed. Finally, the tibial length of the right hind limb of the mouse was measured. Calculating the heart mass index, the left ventricle mass index, the lung weight to body weight ratio, the tibia length to heart ratio increase.
As shown in Table2, the model groups showed significant increases in HW/BW (total heart volume/body weight), LVW/BW (left ventricle weight/body weight), LW/BW (lung weight/body weight), HW/TL (heart weight/tibia length) compared to the control group (. About.P.)<0.01). While the Clinacanthus nutans total flavone has obvious reduction in low-medium dose group ratio model group HW/BW, LVW/BW, LW/BW and HW/TL ( ## P<0.01)。
TABLE2 Effect of Clinacanthus nutans Total Flavonoids on various cardiac measurements in Ang II-induced mouse Heart model Table2.Theeffect Softotalflavonoids from Ang II-induced mouseloss heart models
Note:
n＝6; p compared to normal group<0.01; compared with the group of Ang II, ## P<0.01。
as shown in FIG. 1, the staining by HE shows that under 20-fold microscope, the myocytes in the control group are normal in shape, clear in structure, uniform in size, free of obvious mast cells and orderly arranged among the cells; in the model group, the morphology of the cardiomyocytes changed, the cardiomyocytes became significantly hypertrophic, and the intercellular arrangement was disturbed. Compared with the model group, the clinacanthus nutans total flavonoid group has the advantages that the myocardial cell morphology is uniform, the cell hypertrophy degree is reduced, and the cell arrangement is regular.
Example 4 Clinacanthus nutans Total Flavonoids inhibit collagen deposition in mouse myocardial tissue
When the myocardium is hypertrophic, the collagen of the myocardial tissue increases. In Masson staining, red represents muscle fibers and blue represents collagen deposition. As shown in fig. 2, the model group had a distinct blue deposit compared to the blank group, indicating increased collagen in the myocardial tissue of the model group mice; the total flavonoids of clinacanthus nutans resulted in a significant reduction in collagen deposition compared to the model group.
Example 5 Effect of various concentrations of Clinacanthus nutans Total Flavonoids on cell viability
CCK-8 detection of cell viability
When the H9C2 cells grew to cover 80% of the area of the flask, the original culture broth was discarded in a clean bench, washed 2 times with 3mL pbs, digested with 0.25% trypsin, centrifuged, the supernatant discarded, 3mL of complete medium was added, blown and mixed well, and counted under a microscope. Then at 3X 10 5 Cells were seeded into 96-well plates at one/mL concentration, 100 μ L of cell fluid was added to each well, and 6 replicate wells were set for each group. After 24h, various concentrations of Clinacanthus nutans total flavonoids (0, 10, 20, 40, 80, 160, 320. Mu.g/mL) were added. Incubation was continued for 24h. And adding 10 mu LCCK8 solution into each hole of the solution, incubating for 0-4H in a dark place, and detecting the influence of the clinacanthus nutans total flavonoids on the activity of the H9C2 cells by using a 490nm wavelength of an enzyme-labeling instrument.
The CCK-8 method was used to measure the effect of clinacanthus nutans total flavonoids on the cell viability after 24H intervention of H9C2, as shown in fig. 2, different concentrations of clinacanthus nutans total flavonoids (0, 10, 20, 40, 80, 160, 320 μ g/mL) on the H9C2 cell viability, the clinacanthus nutans total flavonoids increased the H9C2 cell viability in the range of 0-320 μ g/mL, and when the concentration of clinacanthus nutans was 40 μ g/mL,80 μ g/mL,160 μ g/mL, the cell viability was the most increased, so the concentration of clinacanthus nutans used in the subsequent cell experiments was 40 μ g/mL,80 μ g/mL,160 μ g/mL (P < 0.01).
Example 6 Effect of Clinacanthus nutans Total Flavonoids on Total cellular protein content
The BCA kit is used for measuring the protein concentration, as shown in figure 4, the result shows that the AngII (L mu mol/L) is incubated for 24H and can effectively increase the total protein concentration in the rat myocardial H9C2 cells, and the statistical significance is shown (P)<0.001 ); clinacanthus nutans total flavone extract (40. Mu.g/mL, 80. Mu.g/mL, 160. Mu.g/mL) pre-treated for 24H, capable of inhibiting AngII-induced increase in total protein concentration of H9C2 cells (( # P<0.05， ## P<0.01， ### P<0.001)。
Example 7 Clinacanthus nutans Total Flavonoids decrease cellular ROS levels during myocardial hypertrophy
Detecting cellular ROS levels
Rat cardiomyocytes H9C2 were counted, plated in 6-well plates, grouped as above, 3 duplicate wells were set, cultured for 24 hours, and diluted 1000-fold with DCFH-DA. The culture medium was discarded and 1ml of DCFH-DA was added. Wrapping with tinfoil paper in dark place, and incubating at 37 deg.C for 15min. Serum-free medium was used to wash out cells, and the cells were photographed under a fluorescent microscope in the dark.
Ang ii induces oxidase activation and excess reactive oxygen species production, excessive ROS production can trigger cellular dysfunction, lipid peroxidation and DNA mutation, and may lead to cellular damage or death, inducing myocardial hypertrophy [12-14]. ROS has been recognized as one of the key factors in the development of myocardial hypertrophy. The effect of total flavonoids from clinacanthus nutans on Ang ii-induced ROS levels in H9C2 cells was examined using an ROS kit. As shown in fig. 5 (a), the Ang ii group showed a significant increase in fluorescence intensity compared to the control group (. About.. P < 0.05). Clinacanthus nutans total flavonoids (40. Mu.g/mL, 80. Mu.g/mL, 160. Mu.g/mL) were pretreated for 24H, and were able to attenuate the fluorescent intensity (# # P < 0.01) of Ang II-induced H9C 2. As shown in fig. 5 (B), the Ang ii group ROS level was significantly increased, and the total flavonoids of clinacanthus nutans could inhibit Ang ii-induced ROS level increase. P <0.001 compared to control; compared to the Ang II group, # # P <0.01.
Claims (8)
1. Application of Clinacanthus nutans total flavonoids in preparing anti-heart failure drugs.
2.The use of clinacanthus nutans total flavonoids in the preparation of anti-heart failure drugs according to claim 1, wherein the preparation of the clinacanthus nutans total flavonoids comprises the following steps:
s1, crushing dried Clinacanthus nutans leaves, performing reflux extraction by using 50-90% ethanol, collecting an extracting solution, recovering the ethanol, and drying to obtain a crude extract;
s2, passing the crude extract through macroporous adsorption resin, eluting with water and 30-60% ethanol in sequence, collecting 30-60% ethanol eluate, recovering ethanol, and drying to obtain the clinacanthus nutans total flavone extract.
3. The use of clinacanthus nutans total flavonoids in the preparation of anti-heart failure drugs according to claim 1, wherein the preparation of the clinacanthus nutans total flavonoids comprises the following steps:
s1, crushing dried Clinacanthus nutans leaves, performing reflux extraction for 1-3 times with 70-80% ethanol for 1-2 hours each time, combining and collecting extracting solutions, recovering ethanol, and drying to obtain a crude extract;
s2, passing the crude extract through macroporous adsorption resin, eluting with water and 30-40% ethanol in sequence, collecting 30-40% ethanol eluate, recovering ethanol, and drying to obtain the clinacanthus nutans total flavone extract.
4. The use of clinacanthus nutans total flavonoids in the preparation of a medicament for resisting heart failure according to claim 1, wherein the use of clinacanthus nutans total flavonoids in the preparation of a medicament for resisting cardiac insufficiency caused by heart failure and pathological cardiac remodeling.
5. The use of clinacanthus nutans total flavonoids in the preparation of a medicament for resisting heart failure according to claim 1, wherein the use of the clinacanthus nutans total flavonoids in the preparation of a medicament for resisting myocardial cell hypertrophy caused by heart failure and pathological heart reconstruction.
6. The use of Clinacanthus nutans total flavonoids in the preparation of anti-heart failure drugs according to claim 1, wherein the Clinacanthus nutans total flavonoids is used in the preparation of drugs for resisting myocardial interstitial fibrosis caused by heart failure and pathological heart reconstruction.
7. The use of clinacanthus nutans total flavonoids in the preparation of a medicament for resisting heart failure according to claim 1, wherein the use of the clinacanthus nutans total flavonoids in the preparation of a medicament for resisting myocardial cell death caused by heart failure and pathological heart remodeling.
8. The use of clinacanthus nutans total flavonoids in the preparation of anti-heart failure drugs according to claim 1, wherein the drugs are prepared into clinically acceptable pharmaceutical dosage forms from clinacanthus nutans total flavonoids and excipients, and the excipients are excipients.
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| CN107582977A (en) * | 2017-10-18 | 2018-01-16 | 海南梵思科技有限公司 | A kind of Chinese medicine composition for treating angiocardiopathy and preparation method thereof |
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| CN107582977A (en) * | 2017-10-18 | 2018-01-16 | 海南梵思科技有限公司 | A kind of Chinese medicine composition for treating angiocardiopathy and preparation method thereof |
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