CN110559289B - Application of andrographolide sodium bisulfite in preparing medicine for treating atherosclerosis - Google Patents

Application of andrographolide sodium bisulfite in preparing medicine for treating atherosclerosis Download PDF

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CN110559289B
CN110559289B CN201910736474.5A CN201910736474A CN110559289B CN 110559289 B CN110559289 B CN 110559289B CN 201910736474 A CN201910736474 A CN 201910736474A CN 110559289 B CN110559289 B CN 110559289B
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andrographolide
sodium bisulfite
cells
cholesterol
atherosclerosis
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胡元会
崔翰明
师帅
张秋燕
宋庆桥
吴华芹
魏艺
耿彦婷
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Guanganmen Hospital of CACMS
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Abstract

Experiments prove that the andrographolide sodium bisulfite has obvious curative effect of promoting reverse cholesterol transport in cells and resisting atherosclerosis, has quick response and small toxic and side effects, particularly has no influence on liver and kidney functions, is a safe, efficient and stable medicine or health care product for treating and conditioning cardiovascular diseases, is suitable for industrial production, and is easy to popularize. Therefore, a new field is developed for the application of the andrographolide sodium bisulfite, and a new medicine source is provided for promoting reverse cholesterol transport in cells, reducing cholesterol content in cells, and preventing and treating atherosclerosis and cardiovascular diseases.

Description

Application of andrographolide sodium bisulfite in preparing medicine for treating atherosclerosis
Technical Field
The invention relates to a compound capable of regulating blood fat and preventing development of atherosclerotic heart disease, a preparation method and application thereof, in particular to an anti-atherosclerotic reverse cholesterol transport promoter, a preparation method and application thereof.
Background
Cardiovascular disease (CVD), including unstable angina, stroke, acute myocardial infarction with or without ST elevation, and sudden coronary death, is a leading cause of cardiovascular morbidity and mortality worldwide. Most of the cardiac death cases are due to coronary atherosclerotic heart disease. In Beijing area of China, the annual average increase rate of the incidence rate of acute coronary heart disease events in 1984-1993 is 2.3%. The annual average increase in incidence of acute coronary heart disease events in 1984-1997 across the country is 1.7%.
Coronary heart disease is also called coronary atherosclerotic heart disease, and is a heart disease caused by myocardial ischemia, anoxia or necrosis due to stenosis or obstruction of blood vessel cavity caused by coronary artery angiogenesis atherosclerotic lesion. Most Acute Coronary Syndromes (ACSs) are thought to be triggered by atherosclerotic plaque rupture.
In the pathogenesis of coronary heart disease, increased lipids in the blood invade the arterial wall in the form of low density lipoprotein cholesterol (LDL), very low density lipoprotein cholesterol (VLDL) or remnant thereof, accumulate between smooth muscle cells, collagen and elastic fibers, causing smooth muscle cell proliferation. The latter, like monocytes from blood, phagocytose large amounts of lipids into foam cells. Lipoprotein degradation releases cholesterol, cholesterol esters, triglycerides and other lipids, and LDL also combines with proteoglycans in the arterial wall to produce insoluble deposits that stimulate fibroplasia. The above factors together promote the development of fibrous plaque. High cholesterol levels are the most important factors leading to atherosclerosis and cardiovascular disease, and due to the genetic defect of LDL-C metabolism, patients are in high plasma cholesterol levels for a long time, thus greatly increasing the risk of suffering from coronary heart disease. These findings all indicate that modulating blood lipids, lowering serum cholesterol, triglyceride, LDL and oxLDL levels, promoting antioxidant capacity and inhibiting foam cell formation are effective methods for preventing the progression of atherosclerotic lesions.
Disclosure of Invention
The invention aims to solve the technical problems of atherosclerosis lesion caused by accumulation of hyperlipemia and cholesterol in cells of the existing cardiovascular diseases, and provides a new application of andrographolide sodium bisulfite, namely a new application in preparing a drug for promoting reverse cholesterol transport from an intracellular transport promoter, an anti-atherosclerotic reverse cholesterol transport promoter, an anti-atherosclerosis drug, a drug for treating hyperlipidemia and a drug for treating cardiovascular diseases.
The chemical name of the andrographolide sodium bisulfite is 14-dehydroxy-13-dehydroandrographolide-12-sodium sulfonate; the molecular formula is: c 20 H 29 O 4 SO 3 Na; the molecular weight is: 436.23; the structural formula is as follows:
Figure BDA0002162328890000021
the andrographolide sodium bisulfite is white crystal powder, is bitter in taste, has the effects of clearing away heat and toxic materials, diminishing inflammation and relieving pain, has special curative effects on bacterial and viral upper respiratory tract infection and dysentery, is known as a natural antibiotic drug, and pharmacological research shows that after the andrographolide sodium bisulfite is injected into a vein of a mouse, the spontaneous activity of the mouse is obviously reduced; the time for the pentobarbital sodium to fall asleep can be shortened and the sleep duration can be prolonged by injecting the sodium bisulfite andrographolide into the vein of the mouse and then injecting the pentobarbital sodium into the abdominal cavity; after the andrographolide sodium bisulfite is instilled in the vein of the anesthetized domestic dog, the wave forms of the blood pressure, the heart rate, the heart rhythm and the electrocardiogram have no change with clinical significance; the andrographolide sodium bisulfite has obvious tranquilizing effect on the central nervous system of the mouse; the coordination movement of the mouse is not obviously influenced; has synergistic effect with pentobarbital sodium hypnotic effect; the intravenous drip of sodium bisulfite andrographolide in anesthetized domestic dogs has no influence on cardiovascular system.
In order to achieve the purpose of the invention, the invention provides application of andrographolide sodium bisulfite in preparing a promoter for promoting reverse cholesterol transport.
Wherein, the promoter for promoting reverse cholesterol transport also comprises a pharmaceutically acceptable carrier; the promotion of reverse cholesterol transport is to promote reverse cholesterol transport in foam cells, reverse cholesterol in the foam cells to the outside of the cells, reduce the content of cholesterol in the cells and reduce the formation of atherosclerotic plaques; reducing the area of atherosclerotic plaque.
Application of andrographolide bisulphite in preparing medicine or health product for preventing and/or treating atherosclerosis caused by cholesterol in vascular endothelial deposition.
In particular, the enhancer is in the form of tablets, capsules, pills, powders, granules, syrups, solutions, injections, sprays, aerosols, patches.
Pharmaceutically acceptable carriers are generally accepted by health care professionals for this purpose and as inactive ingredients of medicaments. A compilation of pharmaceutically acceptable carriers can be found in tools such as The Handbook of Pharmaceutical excipients (Handbook of Pharmaceutical excipients, 2 nd edition, edited by A.Wade and P.J.Weller; published by American Pharmaceutical Association, washington and The Pharmaceutical Press, london, 1994).
In particular, the carrier includes excipients such as starch, water, and the like; lubricants, such as magnesium stearate and the like; disintegrants, such as microcrystalline cellulose and the like; fillers, such as lactose and the like; binders such as pregelatinized starch, dextrin, and the like; a sweetener; an antioxidant; preservatives, flavoring agents, spices, and the like;
wherein the promoter is administered by a route selected from the group consisting of gastrointestinal administration and parenteral administration.
In particular, the preparation for gastrointestinal administration is selected from tablets, capsules, powders, granules, pills, solutions, syrups and the like.
Wherein, the accelerant is used for treating or/and preventing atherosclerosis, and preventing or/and treating atherosclerosis caused by high cholesterol level and accumulation and precipitation on vascular endothelium.
In particular, prevention of atherosclerotic plaque formation; reducing the area of atherosclerotic plaque; preventing atherosclerosis plaque caused by high cholesterol level and accumulation and precipitation in vascular endothelium; reducing cholesterol content, and reducing atherosclerotic plaque area caused by high cholesterol level and intravascular deposition.
Wherein, the accelerant is used for treating or/and preventing diseases based on atherosclerosis.
Particularly, the diseases of atherosclerosis as pathological basis are cerebral atherosclerosis, coronary atherosclerosis, renal artery, mesenteric artery, carotid artery and other peripheral blood vessel related lesions, and comprise cerebral ischemia, brain atrophy, cerebral vascular rupture hemorrhage secondary to the pathological basis, angina pectoris, myocardial infarction, arrhythmia, refractory hypertension, renal insufficiency, dyspepsia, constipation, hematochezia, paralytic ileus, intermittent claudication, foot dorsum arterial pulsation disappearance, gangrene and the like, and are preferably cerebral atherosclerosis, coronary atherosclerotic heart disease, cerebral infarction or myocardial infarction and the like.
The invention also provides application of the andrographolide sodium bisulfite in preparing a cholesterol reverse transport promoter for resisting atherosclerosis.
In another aspect, the invention provides an application of andrographolide sodium bisulfite in preparing a medicament for resisting atherosclerosis.
The andrographolide bisulphite can be used for preventing formation of atherosclerotic plaque and reducing atherosclerotic plaque.
The andrographolide bisulphite is used for resisting the accumulation and precipitation of cholesterol in the vascular endothelium to cause the formation of atherosclerotic plaques and reducing the accumulation and precipitation of cholesterol in the vascular endothelium to cause the formation of atherosclerotic plaques.
In another aspect, the invention provides an application of andrographolide sodium bisulfite in preparing a medicament for treating cardiovascular diseases.
Wherein the cardiovascular disease is atherosclerotic cardiovascular disease.
In particular, the cardiovascular disease is coronary atherosclerotic heart disease.
The invention also provides application of the andrographolide sodium bisulfite in preparing a medicine for resisting atherosclerotic heart disease.
Wherein the medicine consists of andrographolide sodium bisulfite and a pharmaceutically acceptable carrier.
In particular, the medicament is in the form of tablets, capsules, pills, powders, granules, syrups, solutions, injections, sprays, aerosols, patches.
Wherein the medicament is administered by the gastrointestinal and parenteral routes of administration.
In particular, the parenteral administration route is selected from injection, respiratory administration, dermal administration, mucosal administration or luminal administration.
Wherein the medicament is in the form of tablets, capsules, pills, powder, granules, syrups, solutions, injections, sprays, aerosols, patches and the like.
Wherein the parenteral medicament is selected from injection, spray, aerosol, patch, etc.
Particularly, the preparation for gastrointestinal administration is selected from tablets, capsules, powder, granules, pills, solutions, syrups and the like.
In another aspect, the present invention provides a cholesterol reverse transport promoter, wherein the cholesterol reverse transport promoter comprises andrographolide sodium bisulfite.
In still another aspect, the present invention provides a reverse cholesterol transport promoter for anti-atherosclerosis, wherein the reverse cholesterol transport promoter comprises andrographolide sodium bisulfite.
Wherein the reverse cholesterol transport promoter further comprises a pharmaceutically acceptable carrier.
The invention also provides a preparation method of the cholesterol reverse transport promoting agent andrographolide sodium bisulfite, which comprises the following steps:
a) Dissolving andrographolide in 95% ethanol, heating, stirring, dissolving, adding sodium bisulfite water solution, heating under reflux, stirring, and performing addition reaction for 1-2 hr;
b) Concentrating the addition reaction mixture, and recovering ethanol until no alcohol smell exists; then adding active carbon, decoloring for 30-60min, and filtering;
c) Concentrating the filtrate, crystallizing at a temperature of less than or equal to 4 deg.C, and centrifuging the crystallized mixture to obtain andrographolide sodium bisulfite.
Wherein the molar ratio of andrographolide to sodium bisulfite in step A) is 1:1-2, preferably 1:1.5. the concentration of the sodium bisulfite aqueous solution is 10-20mol/L; the temperature of the addition reaction is 60-80 ℃.
In particular, in order to obtain high-purity andrographolide sodium bisulfite, the method also comprises the step of recrystallizing the prepared andrographolide sodium bisulfite.
Wherein the andrographolide is prepared by the following steps:
1) Soaking herba Andrographitis in ethanol solution, heating and reflux-extracting for 2-3 times, mixing the reflux extractive solutions, concentrating, and recovering ethanol to obtain extract-concentrated solution;
2) Adding active carbon into the extract-concentrated solution, and decolorizing for 30-60min; then filtering and concentrating the decolorized filtrate to prepare decolorized-concentrated solution;
3) Performing cold storage, standing and precipitation treatment on the decolorized-concentrated solution at the temperature of less than or equal to 10 ℃ (preferably 0-4 ℃), standing for at least 12h, discarding the supernatant, and dissolving the precipitate with absolute ethanol to obtain a crude product compound solution;
4) Crystallizing the crude product complex solution at a temperature of 4 deg.C or less (preferably 0-4 deg.C), and centrifuging the crystallized mixture to obtain andrographolide crude crystal;
5) Dissolving the andrographolide crystal crude product in hot absolute ethyl alcohol, and then adding active carbon for decoloring; then filtering, concentrating the filtrate, recovering ethanol, and preparing a crystallization-decoloration-concentrated solution;
6) Crystallizing the concentrated solution at 4 deg.C (preferably 0-4 deg.C), and centrifuging to obtain andrographolide crude product.
Wherein, the mass percentage concentration of the ethanol solution in the step 1) is 50-95%, preferably 75%.
Particularly, the ratio of the volume of the ethanol solution to the weight of the andrographis paniculata medicinal material in the step 1) is 5-20:1, preferably 5-15, namely when the weight (dry weight) of the andrographis paniculata is 1kg, the volume of the ethanol solution of the extraction solution is 5-20L, and when the weight (dry weight) of the andrographis paniculata is 1g, the volume of the ethanol solution of the extraction solution is 5-20ml.
Wherein the ratio of the weight of the extract-concentrate in the step 1) to the weight of the raw material andrographis paniculata is 1:1-5: 1.
Wherein, the dosage of the active carbon in the step 2) is 5-20 percent of the dosage of the raw medicinal materials, the preferred dosage is 10 percent, and the decoloring treatment time is 30-60min; the decolorization treatment can be carried out until the mixture is clear, and the subsequent filtration can be carried out
Particularly, the ratio of the weight of the decolorized-concentrated solution to the weight of the raw material andrographis paniculata is (0.5): 1.
in particular, it also comprises a step 6A): purifying andrographolide crude product, including dissolving andrographolide crude product with appropriate amount of hot water, passing through macroporous adsorbent resin column, and eluting with pure water, 30%,50%,70% and 95% ethanol as eluent; collecting eluate, concentrating the eluate to appropriate amount, refrigerating, recrystallizing, centrifuging, crystallizing, and vacuum drying to obtain purified andrographolide.
Wherein the ratio of the volume of the macroporous adsorption resin in the macroporous resin column to the weight of the andrographolide crude product prepared in the step 6) is 0.5-2:1, preferably 1-2:1, more preferably 1:1, when the weight (dry weight) of the andrographolide crude product is 1kg, the volume of the macroporous adsorption resin is 0.5-2L, and when the weight (dry weight) of the andrographolide crude product is 1g, the volume of the macroporous adsorption resin is 0.5-2ml.
Particularly, the ratio of the volume of each eluent to the column volume of the macroporous resin column in the elution process is 3-5:1.
in particular, the perforated adsorption resin is AB-8 type macroporous adsorption resin, D101 type macroporous adsorption resin or HP20 type macroporous adsorption resin, and AB-8 type macroporous adsorption resin is preferred.
The invention also provides a method for promoting reverse cholesterol transport, resisting atherosclerosis and treating cardiovascular diseases, which comprises the step of administering a therapeutically effective amount of andrographolide sodium bisulfite to a subject, wherein the therapeutically effective amount is 1-100 mg/kg-d, preferably 10-90 mg/kg-d, and further preferably 60-90 mg/kg-d.
The term "therapeutically effective amount" as used herein, unless otherwise indicated, is the amount of drug required to produce an effective effect; the "therapeutically effective amount" is adjusted and varied and ultimately determined by the medical practitioner, taking into account factors including the route of administration and the nature of the formulation, the general condition of the recipient's weight, age, etc., and the nature and severity of the condition being treated.
Compared with the prior art, the invention has the following obvious advantages:
1. the invention develops a new medicinal value for the known compound, namely the andrographolide sodium bisulfite, is used for resisting atherosclerosis, promoting reverse transport of cholesterol in cells (the content of cholesterol in cells is obviously reduced, and the effect of inhibiting accumulation of cholesterol in cells is obvious), can be prepared into a medicament or health-care food for preventing, conditioning and/or treating the atherosclerosis, can be prepared into a medicament or health-care product for treating and conditioning cardiovascular diseases, and thus develops a new field for the application of the andrographolide sodium bisulfite.
2. The series of cell tests and researches prove that andrographolide sodium bisulfite has the obvious effects of preventing and treating atherosclerosis, and the andrographolide sodium bisulfite reverses cholesterol phagocytosed in cells to be transported out of the cells, so that the cholesterol content in the cells is reduced, the risk of smooth muscle cell proliferation is reduced, the formation of foam cells is inhibited, and the development of atherosclerotic lesions is obviously inhibited.
3. A large number of animal experiments are carried out by using andrographolide sodium bisulfite through an oral administration route, and the experimental results show that: the andrographolide sodium bisulfite can obviously reduce atherosclerotic plaques in arteries, the area of plaques in aorta is obviously reduced, and the formation and the area of atherosclerotic plaques in arteries are obviously inhibited, which shows that the andrographolide sodium bisulfite can obviously reduce the content of cholesterol in cells and obviously promote the effect of reverse transport of cholesterol in cells.
The andrographolide sodium bisulfite of the invention has the functions of regulating blood fat, reducing cholesterol and triglyceride in blood, regulating lipoprotein, inhibiting vascular atherosclerosis and resisting atherosclerotic heart disease;
4. the andrographolide sodium bisulfite has strong pharmacological action and is used for resisting atherosclerosis; the traditional Chinese medicine composition has the advantages of obvious effect of preventing, conditioning and treating cardiovascular diseases, quick response, small toxic and side effects, good safety, capability of being taken for a long time, capability of analyzing the anti-atherosclerosis action mechanism by modern pharmacology and good medicinal prospect.
The cell and animal experiment research proves that the andrographolide sodium bisulfite has the functions of regulating blood fat, reducing cholesterol, triglyceride and low-density lipoprotein cholesterol in serum, increasing high-density lipoprotein cholesterol to a certain extent and preventing the development of atherosclerotic lesions.
5. The product has the advantages of rich raw material sources, low price, safe clinical use, simple preparation process, small dosage and convenient use, and can be prepared into various dosage forms, thereby being easy to popularize.
Drawings
FIG. 1 is a graph showing the results of the staining changes of RAW264.7 cells and lipid oil red 0 thereof, wherein: wherein: NORMAL is NORMAL group cells; MODEL is MODEL group cell; ATOR is atorvastatin calcium treated cells; a LOW is sodium bisulfite andrographolide LOW dose treated cells; a HIGH is sodium bisulfite andrographolide HIGH dose treated cells; b LOW is andrographolide LOW dose treated cells; b HIGH is andrographolide HIGH dose treated cells;
FIG. 2 is a graph of oil red staining of mouse aortic trees.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and substitutions are intended to be within the scope of the invention.
The beneficial effects of the drug of the present invention are further illustrated by the following test examples, which include pharmacodynamic tests of the drug of the present invention.
Instruments and materials for use in embodiments of the invention
1. Testing instrument
Instrument name and instrument model manufacturer
HPLC chromatograph 1200series Agilent technologies
2. Test reagent
Figure BDA0002162328890000081
3. Test drug
Figure BDA0002162328890000082
Figure BDA0002162328890000091
EXAMPLE 1 preparation of Andrographolide sodium bisulfite
1. Preparation of Andrographolide
Pulverizing herba Andrographitis (1 kg) into coarse powder, adding ethanol solution (with mass concentration of 75%, usually 50-75%), heating and reflux-extracting for 2 times (usually 2-3 times), wherein the volume of ethanol solution is 5L (usually 5-20L) per extraction, that is, the volume ratio of ethanol solution to herba Andrographitis is 5-20.
Carrying out reduced pressure concentration treatment on the extracting solution until no alcohol smell exists, and preparing an extraction-concentrated solution, wherein the temperature of reduced pressure concentration is 70 ℃, the relative vacuum degree is-0.09 to-0.075 MPa, and the ratio of the weight of the extraction-concentrated solution to the weight of the raw material drug andrographis paniculata is 1:1 (typically 1-5.
Adding active carbon into the extract-concentrated solution, stirring, decolorizing for 30min (usually 30-60 min), filtering, and concentrating the filtrate to obtain decolorized-concentrated solution; wherein the dosage of the active carbon is 10% (usually 5-20%) of the raw medicinal material; the decolorization treatment can be carried out until the mixture is clear, and the subsequent filtration can be carried out; the ratio of the weight of the decolorized-concentrated solution to the weight of the raw material andrographis paniculata is 0.5:1 (typically 0.5.
Standing the decolorized concentrated solution at a temperature of less than or equal to 10 deg.C (preferably 4-10 deg.C), precipitating, standing for at least 12 hr, removing supernatant, adding small amount of anhydrous ethanol into the precipitate, stirring to dissolve the precipitate completely to obtain crude compound solution;
crystallizing (i.e. refrigerating and crystallizing) the crude compound solution at a temperature of less than or equal to 10 deg.C (preferably 0-4 deg.C), and centrifuging the crystallized mixture to obtain andrographolide crude crystal;
dissolving the andrographolide crude crystals in a proper amount of absolute ethyl alcohol, heating and stirring until the crude andrographolide crystals are just completely dissolved, and then adding activated carbon for decoloring; then filtering, concentrating the filtrate, and recovering ethanol to obtain crystallization-decolorization-concentrated solution;
crystallizing the concentrated solution at a temperature of 4 deg.C or less (preferably 0-4 deg.C), centrifuging the crystallized mixture to obtain andrographolide;
vacuum drying andrographolide crystal under reduced pressure to obtain dehydroandrographolide.
2. Purification of andrographolide
Dissolving dehydroandrographolide in hot water, passing through macroporous adsorbent resin column, and eluting with pure water, 30%,50%,70% and 95% ethanol as eluent; collecting each eluate, concentrating the eluate respectively to appropriate amount, refrigerating, recrystallizing, centrifuging, crystallizing, and vacuum drying to obtain purified andrographolide. Wherein, the ratio of the volume of the macroporous adsorption resin in the macroporous resin column to the weight of the andrographolide crude product is 1 (usually 0.5-5. The ratio of each eluent to the column volume of the macroporous resin column in the elution process is 3-5:1, selecting AB-8 type macroporous adsorption resin (D101 and HP20 can also be selected as the macroporous adsorption resin).
Measuring the content of the prepared andrographolide:
chromatographic conditions are as follows: with acetonitrile-phosphate buffer (5 mmol. L) -1 Dipotassium hydrogen phosphate-5 mmol. L -1 Monopotassium phosphate 1; the flow rate is 1.0 mL/min -1 The injection volume is 10. Mu.L. Compared with andrographolide reference substances, the andrographolide content prepared by the invention is more than 98.5%.
3. Addition reaction
Dissolving the purified andrographolide in 95% ethanol, heating, stirring, dissolving, adding 16mol/L (usually 10-20 mol/L) sodium bisulfite water solution, heating under reflux, stirring, and performing addition reaction for 1-2h (to clear); the molar ratio of andrographolide to sodium bisulfite is 1; concentrating the addition reaction mixture, and recovering ethanol until no alcohol smell exists; then adding active carbon, decolorizing for 30min, and filtering; and then concentrating the filtrate, crystallizing at the temperature of less than or equal to 4 ℃, and centrifuging the crystallized mixture to obtain the andrographolide sodium bisulfite.
In order to obtain high-purity andrographolide sodium bisulfite, the method also comprises the step of recrystallizing the prepared andrographolide sodium bisulfite.
Content determination of the prepared sodium bisulfite adduct:
chromatographic conditions are as follows: with acetonitrile-phosphate buffer (10 mmol. L) -1 Dipotassium hydrogen phosphate-10 mmol. L -1 Monopotassium phosphate 1; the flow rate is 1.0 mL/min -1 The injection volume is 10. Mu.L. The comparison of the control with andrographolide sodium bisulfite shows that the purity of andrographolide sodium bisulfite is more than 98.5%.
Test example 1 Effect of Andrographolide sodium bisulfite on reverse transport of RAW264.7 Cholesterol
1. Experimental materials
1.1 cells
RAW264.7 cell line was purchased from the cell resource center of the institute of japan medical college, beijing, japan (national infrastructure for cell line resources, headquarters for NSTI) on 9/15/2014. Cell lines were checked for mycoplasma contamination by PCR and culture and their provenance was identified by PCR, and the results of cell line identification were identified by STR profiling (FBI, CODIS), all of which were reviewed on the website (http:// cell resource. Cn).
Cell recovery:
taking out RAW264.7 cell strain from liquid nitrogen tank, quickly placing into constant temperature water bath, water bathing at 37 deg.C, repeatedly shaking to melt it quickly, transferring the melted cell suspension into centrifuge tube, adding 1ml DMEM medium containing 10% FBS, mixing, placing into centrifuge at 1000rpm,3min, discarding supernatant, adding culture medium again, repeatedly blowing and beating to obtain cell suspension, inoculating into cell culture dish, placing into 37 deg.C, 5% CO2 incubator, culturing for 12h, observing cell recovery adherence condition, changing culture medium when cell adherence reaches above 80%, and continuing culturing.
And (3) cell culture:
the recovered RAW264.7 cells were cultured at 37 ℃ in a 5% CO2 cell culture chamber, and the fresh medium was replaced every 48 hours. When the cell growth coverage area in the culture dish reaches more than 90%, the cell passage treatment can be carried out. And when the cells are subcultured, gently and repeatedly blowing and uniformly mixing the cells, transferring the cell suspension into a 15ml centrifuge tube, centrifuging the centrifuge tube on the machine at 1000rpm for 3-5min, removing the supernatant, adding a fresh culture medium, repeatedly blowing and beating the cell suspension, inoculating the cell suspension into a cell culture dish, continuously putting the cell culture dish into an incubator for culturing for 24h, and then replacing the fresh culture medium.
1.2 medicine
Sodium bisulfite andrographolide: white crystalline powder (ethanol) with a content of 99.7%, china institute for food and drug testing;
atorvastatin calcium, available from sigma, USA (Cat. No.: PZ 0001).
Oxidized low density lipoprotein (ox-LDL) available from Shanghai leaf biologies.
Oil red O powder, available from sigma company, USA (Cat. No.: O0625).
2. Experimental methods
2.1, grouping and administration
The experiment is respectively provided with a blank control group, a model group and an atorvastatin calcium group; sodium bisulfite andrographolide low and high dose groups; the low and high dose groups of andrographolide.
96-well plates were plated at 5000 cells/well, and a foam cell model was established for each of the remaining groups except for the normal group (i.e., blank control group) to which DMEM medium (10% FBS (fetal bovine serum)) was administered: that is, cells were pretreated with serum-free medium (i.e., serum-free DMEM medium) for 24 hours, then added with ox-LDL at a final concentration of 50. Mu.g.mL-1, incubated in a 5-vol CO2 incubator at 37 ℃ for 24 hours, and the morphology of the cells was observed under an optical microscope; after the treatment solution was aspirated, the culture medium and the corresponding treatment drugs were added to each group of cells, and the following treatments were performed:
blank control group: DMEM medium (10% fbs (fetal bovine serum)); model group: DMEM medium (10% fbs); atorvastatin calcium group: DMEM medium (10% fbs) 10 μ M atorvastatin calcium; sodium bisulfite andrographolide low dose group: DMEM medium (10% fbs) +10 μ M sodium bisulfite andrographolide; sodium bisulfite andrographolide high dose group: DMEM medium (10% fbs) +50 μ M sodium bisulfite andrographolide; andrographolide low dose group: DMEM medium (10% fbs) +10 μ M andrographolide; andrographolide high dose group: DMEM medium (10% FBS) + 50. Mu.M andrographolide.
2.2 dyeing
After 24h incubation, cell oil red O was added to each group of cells for staining:
1. preparing a stock solution: weighing oil red O0.5 g, dissolving in isopropanol 100ml, heating in 60 deg.C water bath for dissolving
2. The plate was washed twice with PBS and discarded by blotting.
3. Fixed with 4% paraformaldehyde for 30 minutes.
4. The oil red stock was mixed with deionized water 3.
2.3 statistics
The images were observed under a microscope and photographed as shown in FIG. 1, wherein: NORMAL is NORMAL group cells; MODEL is MODEL group cell; ATOR is atorvastatin-treated cells; aLOW is sodium bisulfite andrographolide high dose (50 μmol/L) treated cells; a HIGH is Andrographolide sodium bisulfite (10. Mu. Mol/L) treated cells; b HIGH is andrographolide HIGH dose (50. Mu. Mol/L) treated cells; b LOW is andrographolide LOW dose (10. Mu. Mol/L) treated cells and Image-Pro Plus 6.0 Image recognition software was used to calculate the red area. SPSS 22.0 performs one-way anova. The results are shown in Table 1.
TABLE 1RAW264.7 cells Effect of reverse cholesterol transport
Figure BDA0002162328890000121
Figure BDA0002162328890000131
Is compared with the model group,: < 0.05 |, { major |, < 0.01 }: < 0.001 }
According to the experimental results, the following components are obtained: the medicine of the andrographolide sodium bisulfite has obvious influence on the content of cholesterol in RAW264.7 cells, can obviously reduce the content of cholesterol in cells, has high use amount of the andrographolide sodium bisulfite, can obviously reduce the content of cholesterol in cells, and shows that the andrographolide sodium bisulfite can reverse the cholesterol phagocytosed in the cells to be transported out of the cells, promote the reverse transport of the cholesterol, reduce the content of the cholesterol in the cells, reduce and inhibit the formation of foam cells, and prevent and treat the progress of atherosclerotic lesions.
The andrographolide sodium bisulfite of the invention plays a role in the regulation of lipid metabolism of the body, mobilizes the cholesterol abundant in peripheral tissues of the body by promoting Reverse Cholesterol Transport (RCT), is carried and transported to the liver or steroid hormone synthesis tissues through HDL (high density lipoprotein), and is finally utilized or discharged out of the body. In the process, cholesterol phagocytosed by macrophages into cells is promoted to be transported out of the cells by cholesterol efflux protein ABCA1/ABCG1 on cell membranes under the action of andrographolide sodium bisulfite, and then transported back to the liver under the mediation of HDL, so that the cholesterol is metabolized. The andrographolide sodium bisulfite provided by the invention has an inhibiting effect on atherosclerotic lesions by intervening reverse transport of macrophage cholesterol.
Example 2 Effect of sodium bisulfite Andrographolide on APOE mice
1. Experimental materials
100 male APOE (-/-) mice at 8 weeks of age, 10 Wild Type (WT) C57BL/6J mice, weighing 22. + -.2 g, purchased from Wintonlifwa, beijing.
The drug product was the same as in example 2.
2. Grouping and intervention method
All mice were randomized into groups by weight after 1 week of adaptive feeding, as follows:
normal control group (C57 group): 10C 57BL/6J mice were gavaged with normal diet plus normal saline.
Model control group (Model group): 10 APOE (-/-) mice, high cholesterol diet + saline gavage.
Positive control group (ATOR group, atorvastatin group): 10 APOE (-/-) mice, high cholesterol diet + atorvastatin calcium (10 mg/kg/d) were gavaged.
Sodium bisulfite andrographolide high dose group (group a high): 10 APOE (-/-) mice, high cholesterol diet + andrographolide sodium bisulfite (100 mg/kg/d) were gavaged.
Sodium bisulfite andrographolide medium dose group (a middle group): 10 APOE (-/-) mice, high cholesterol diet + andrographolide sodium bisulfite (50 mg/kg/d) were gavaged.
Sodium bisulfite andrographolide low dose group (a low group): 10 APOE (-/-) mice, high cholesterol diet + andrographolide sodium bisulfite (25 mg/kg/d) were gavaged.
Andrographolide high dose group (group B high): 10 APOE (-/-) mice, high cholesterol diet + andrographolide (100 mg/kg/d) were gavaged.
Andrographolide medium dose group (B midle group): 10 APOE (-/-) mice, high cholesterol diet + andrographolide (50 mg/kg/d) were gavaged.
Andrographolide low dose group (group B low): 10 APOE (-/-) mice, high cholesterol diet + andrographolide (25 mg/kg/d) were gavaged.
The high cholesterol diet adopts a 'western diet' formula and is provided by Huafukang company (the code is H10141, and the formula is shown in table 2), the intragastric administration volume of each group of mice is 10ml/kg, the continuous intragastric administration of each group of mice is carried out for 4 weeks, and the day 1 is taken from the beginning of the formal experiment, and the total day is 28.
TABLE 2 high cholesterol feed formulation table
Figure BDA0002162328890000141
Figure BDA0002162328890000151
All experimental groups of mice are placed in the center of SPF experimental animals, 5 mice are placed in a cage, the temperature of a mouse room is kept between 18 and 22 ℃, the humidity is kept constant at about 50 percent, and the mice are illuminated for 12 hours and dark for 12 hours. Animals were allowed free access to food and water (autoclaved water) during the test period. Weighed once a week, groups were gavaged as above for 4 weeks.
3. Test method
After 28 days of continuous administration, mice were fasted for 12h without water deprivation and anesthetized with isoflurane gas at 29 d. The method comprises the steps of taking eyeballs of an anesthetized mouse, taking blood, placing the blood in a biochemical tube containing separation gel, placing the tube at room temperature for 2 hours, centrifuging the tube at 3500r/min for 15 minutes, taking out upper serum, subpackaging the serum by using a sterile tube, storing the serum in a refrigerator at the temperature of 80 ℃ below zero, and detecting.
Mice were placed on ice for manipulation. The abdominal cavity is opened, the heart is exposed, the injector is inserted along the left ventricle, the right atrium is cut, 20ml of precooled normal saline (4 ℃) is injected for perfusion, the whitening of the liver can be seen, and no blood flows out from the heart, which indicates that the perfusion is clean. After the perfusion is completed, the aorta is dissected. Part of the solution was fixed with 4% paraformaldehyde, and part of the solution was stored at-80 ℃.
4. Aortic tree preparation and atherosclerotic plaque area determination
The mouse starts from the left ventricle aorta, 3 branches on the aortic arch can be obviously seen until the iliac aorta branches, and all the aorta is taken out. Fix with 4% paraformaldehyde. The fixed aorta was placed in a glass dish with a pre-cooled glass, handled on ice, and carefully stripped of fat around the periphery of the aorta. The aorta was dissected longitudinally. Soaking in distilled water for 10min. Dehydrating in 60% isopropanol for 5min. Dyeing for 1h in oil red, and sealing in dark. Differentiation was observed to background colorless in 60% isopropanol. The aortic tree was fixed on a black background, observed under a stereoscopic microscope and photographed. The photograph is shown in FIG. 2.
FIG. 2 is a photograph showing the staining of the inner side of the aorta after the aorta has been longitudinally sectioned after oil red O staining, and the red portion is the plaque area after staining.
The aortic tree photographs were taken using image recognition software to calculate the atherosclerotic plaque (red) area, and the results are shown in table 3 below.
Table 3 effect on atherosclerotic plaque in aorta of APOE mouse model (%, n = 3)
Figure BDA0002162328890000152
Figure BDA0002162328890000161
Is compared with the model group,: < 0.05 |, { major |, < 0.01 }: < 0.001 }
As can be seen from table 3 above, after APOE mice were given high-fat diet, the atherosclerotic plaques in the aorta of the model control group mice were significantly more than those of the normal control group; atorvastatin calcium (positive drug) has obvious inhibition effect on the plaque area in the aorta of a model mouse; compared with the model group, the sodium bisulfite andrographolide and andrographolide have the inhibition effect on the area of the arterial plaque, and both have statistical significance. The inhibition effect of the sodium bisulfite andrographolide high-dose group is more obvious, and the reduction is more obvious compared with that of a positive medicament and andrographolide.
5. Determination of liver and kidney function
The contents of glutamic-oxaloacetic transaminase (AST), alanine transaminase (ALT), urea nitrogen (BUN) and Creatinine (CREA) in mouse serum were measured by a Beckman AU5822 full-automatic biochemical analyzer, and the measurement results are shown in tables 4 and 5.
TABLE 4 Effect of sodium bisulfite andrographolide on liver function in APOE mice
Figure BDA0002162328890000162
TABLE 5 Effect of sodium bisulfite Andrographolide on APOE mouse model Kidney function
Figure BDA0002162328890000163
Figure BDA0002162328890000171
From the measurement results of tables 4 and 5, it is found that: the andrographolide sodium bisulfite and the positive drug have no significant influence on the liver and kidney functions, which indicates that the andrographolide sodium bisulfite and the atorvastatin calcium as the positive drug have no obvious liver and kidney toxicity in the dosage range used in the experiment.
From the above experimental results, it can be seen that: the andrographolide sodium bisulfite of the invention has an inhibiting effect on the area of atherosclerotic plaques, and can reduce the atherosclerotic plaques caused by hyperlipidemia; can be used for preventing and treating atherosclerosis, hyperlipidemia, cardiovascular diseases, etc. In the dose range of the experiment using the andrographolide sodium bisulfite for prevention and treatment, no significant statistical significance is found on the influence of the liver and kidney functions. The relative safety of the experimental dosage range of animals is beneficial to the clinical development and transformation of the medicine in the next step.
The above-described embodiments of the present invention are intended to be illustrative only, and are not intended to limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Claims (4)

1. Application of andrographolide sodium bisulfite in preparing medicine for treating and/or preventing atherosclerosis.
2. The use of claim 1, wherein said medicament further comprises a pharmaceutically acceptable carrier.
3. Use according to claim 1 or 2, characterized in that the medicament is in the form of tablets, capsules, pills, powders, granules, syrups, solutions.
4. Use according to claim 1 or 2, wherein the medicament is for preventing the formation of atherosclerotic plaques; reducing the area of atherosclerotic plaque.
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