CN110693876A

CN110693876A - Application of andrographolide in preparing medicine or health-care product for preventing or/and treating muscle injury

Info

Publication number: CN110693876A
Application number: CN201910999328.1A
Authority: CN
Inventors: 胡元会; 崔翰明; 师帅; 张秋燕; 杜柏; 褚瑜光; 王欢; 邱志凌
Original assignee: Guanganmen Hospital of CACMS
Current assignee: Guanganmen Hospital of CACMS
Priority date: 2019-10-21
Filing date: 2019-10-21
Publication date: 2020-01-17
Anticipated expiration: 2039-10-21
Also published as: CN110693876B

Abstract

The invention discloses an application of andrographolide in preparing a medicine or a health-care product for preventing or/and treating muscle injury, and experiments prove that andrographolide has the effects of overcoming muscle injury such as muscle endurance, muscle strength reduction and the like caused by hyperlipidemia, and avoiding the muscle injury caused by statin treatment, particularly the action of muscle sequence caused by adverse reactions such as rhabdomyolysis and the like in the treatment process of hyperlipidemia, so that the muscle endurance and instant holding power are enhanced, and the muscle is protected; in addition, andrographolide also has effects of regulating blood lipid, reducing cholesterol, triglyceride and low density lipoprotein cholesterol in serum, increasing high density lipoprotein cholesterol, reducing intracellular cholesterol content, inhibiting cholesterol accumulation between collagen and elastic fiber of smooth muscle cell, preventing smooth muscle cell hyperplasia, and resisting atherosclerosis.

Description

Application of andrographolide in preparing medicine or health-care product for preventing or/and treating muscle injury

Technical Field

The invention relates to the field of medicines, relates to a medicine or health-care food for treating hyperlipidemia, and particularly relates to application of an extract component of traditional Chinese medicine andrographis paniculata in preparation of a medicine for resisting adverse reactions of rhabdomyolysis in treatment of hyperlipidemia.

Background

With the development of socio-economic and changes of living standard and lifestyle, the dietary structure of people gradually appears as High Fat Diet (HFD), which is the increasing intake of High calorie and High fat food in ready-to-drink diet, and the common changes of working style, such as sedentary, low physical activity, etc., lead to the imbalance of energy intake and metabolic balance of the body.

High fat diet is considered as one of the important factors harming human health, and is recognized as an important environmental factor for the onset of diseases such as obesity, type 2 diabetes, and metabolic syndrome. High fat diet can not only damage vascular endothelium and aggravate atherosclerosis, but also increase cardiovascular and cerebrovascular diseases, induce cardiac hypertrophy and cardiac dysfunction; the research finds that the muscle strength of obese people with high fat diet is obviously reduced compared with normal people of the same age, the high fat diet is an important risk factor of sarcopenia, and the research on skeletal muscle biopsy of high fat diet people finds that the composition, mitochondria, extracellular matrix, capillary vessel density surrounding muscle fibers and the like of skeletal muscle fibers of the high fat group are changed, mainly manifested as skeletal muscle atrophy and the reduction of the cross-sectional area of skeletal muscle mainly comprising fast muscle (type II) fibers, which is consistent with the atrophy of aged muscle; the study by Sang _ Rok Lee et al found that fat accumulation, loss of grip, and concomitant loss of muscle mass among obese mice on a high fat diet; zhaoyong Hu et al found that high-fat mice not only had decreased muscle regeneration capacity of stem cells, but also impaired myocyte differentiation into myotubes.

Although the most effective lipid-lowering statins for treating hyperlipidemia can effectively lower blood lipid, statins also have other non-lipid-lowering effects, such as: inhibiting atherosclerosis and thrombosis, relieving rejection after organ transplantation, treating osteoporosis, resisting tumor, and resisting senile dementia, but statins also have obvious adverse reaction, mainly manifested by myopathy and liver adverse reaction. Clinical manifestations of statin-related myopathies include myalgia, myositis, and rhabdomyolysis. Rhabdomyolysis is often manifested by a significant increase in CK (phosphokinase) (more than 10 times above the upper limit of normal), can be accompanied by an increase in blood creatinine, and often by myosin urine and myosin blood, and can cause acute renal failure, with rosuvastatin being more adverse than other drugs.

Rhabdomyolysis refers to the necrosis of the muscle cells of wide striations caused by any reason, and the direct consequence of the rhabdomyolysis is that the muscle cell contents leak to extracellular fluid and blood circulation, and can cause a series of complications such as acute renal failure, electrolyte disorder and the like, sometimes with serious illness and poor prognosis. The mechanism of dissolution of striated muscle caused by statin lipid regulating drugs is not completely clear at present, but research finds that the mechanism may be related to the inhibition of drug metabolism and the increase of blood concentration. For example, statins blood lipid regulating drugs cause coenzyme Q10 deficiency, cause cell mitochondrial dysfunction, inhibit energy generation and finally cause cell energy exhaustion and death; or the synthesis of intermediate metabolites for cholesterol synthesis is reduced, and the synthesis of certain important proteins is influenced; or the synthesis of cholesterol is reduced, so that the cholesterol supplied to the cell membrane is reduced, and the permeability and instability of the cell membrane are increased; or the intracellular calcium concentration is increased due to the statin blood lipid regulating drug, so that the intracellular calcium concentration is overloaded, thereby causing cell death.

Adverse reactions in the muscle may occur during the occurrence and development of hyperlipidemia diseases and the use of statins. In addition, the long-term application of statins in the treatment of arteriosclerotic diseases is required to stabilize the plaque, further increasing the risk of adverse reactions. The andrographolide disclosed by the invention can be used for regulating blood fat and reducing the risk of arteriosclerosis, and meanwhile, the action of damaging muscles is avoided to a certain extent.

Disclosure of Invention

The invention aims to solve the technical problems of myopathy existing in the treatment process of the existing hyperlipidemia diseases and hyperlipidemia diseases, in particular to the technical problems of adverse reactions of myopathy (rhabdomyolysis and the like) easily caused by taking statin treatment medicines in the treatment process of the hyperlipidemia diseases, and provides an application of andrographolide in preparing a medicine or a health-care product for treating adverse reactions of muscle injury (rhabdomyolysis) in the hyperlipidemia diseases, namely a new application of andrographolide in preparing a medicine for treating adverse reactions of muscle injury (rhabdomyolysis) in the hyperlipidemia diseases, a new application of andrographolide in preparing a medicine or a health-care product for avoiding muscle injury in the hyperlipidemia diseases, and in the treatment process of the hyperlipidemia diseases, andrographolide has the effects of regulating blood fat, reducing cholesterol, triglyceride and low-density lipoprotein cholesterol, increasing high-density lipoprotein cholesterol, reducing the content of cholesterol in cells, inhibiting the accumulation of cholesterol between collagen and elastic fibers of smooth muscle cells, avoiding the proliferation of the smooth muscle cells, resisting atherosclerosis, overcoming the conditions of muscle endurance and muscle strength reduction caused by hyperlipidemia, overcoming the adverse reaction of muscle injury (rhabdomyolysis) in the treatment process of hyperlipidemia, enhancing the endurance and instantaneous holding power of muscles, and playing a role in protecting the muscles.

Andrographolide (3- [2- [ hydroxy-6-hydroxy-5- (hydroxy-methyl) -5,8 a-dimethyl-2-methyenphthalanyl)]ethylidene]dihydro-4-hydroxyfuran-2(3H) -one) with molecular formula of C₂₀H₃₀O₅(ii) a Molecular weight M350.44; the structural formula is as follows:

in order to achieve the purpose of the invention, the invention provides an application of andrographolide in preparing a medicine or health-care product for preventing or/and treating muscle injury.

In the process of treating hyperlipidemia, the application of andrographolide in preparing a medicament or health-care product for preventing or/and treating muscle injury, namely the application of andrographolide in preparing a medicament or health-care product for resisting muscle injury in treating hyperlipidemia.

Wherein the muscle injury is myalgia, myositis, sarcopenia or rhabdomyolysis, preferably sarcopenia or rhabdomyolysis.

In particular, the muscle damage is mainly manifested by a decrease in muscle strength and endurance and a decrease in muscle mass.

In particular, the muscle damage is muscle damage caused by a hyperlipidemic condition; the muscle damage caused by hyperlipidemia is muscle damage caused by reduction of muscle fibers or/and increase of muscle gaps.

In particular, the muscle damage is myalgia, myositis, sarcopenia or rhabdomyolysis caused by a hyperlipidemic condition.

In particular, the muscle injury is preferably a muscle injury disorder which is caused by hyperlipidemia and mainly manifested by myalgia, myositis, sarcopenia or rhabdomyolysis, such as myofiber contraction and myointerstitial enlargement.

Particularly, the muscle damage is the muscle damage caused by the adverse reaction of rhabdomyolysis in the process of treating the hyperlipidemia by adopting statins.

The application of andrographolide in preparing medicine or health product for treating rhabdomyolysis adverse reaction in hyperlipemia disease is provided.

Wherein the hyperlipidemia condition is one or more of hypercholesterolemia, hypertriglyceridemia, atherosclerosis or coronary heart disease, preferably hypercholesterolemia.

In particular, the hyperlipemia disease is a disease taking atherosclerosis as a pathological basis or/and is used for treating a disease taking hyperlipemia as a pathological basis and obesity secondary to hyperlipemia.

In particular, the atherosclerosis-based pathological conditions are one or more of the associated lesions of cerebral artery, coronary artery, renal artery, mesenteric artery, carotid atherosclerosis, and associated secondary diseases.

In particular, the medicine consists of andrographolide and a pharmaceutically acceptable carrier.

In particular, the medicament is in the form of tablets, capsules, pills, powders, granules, syrups, solutions, emulsions, injections, sprays, aerosols, gels, creams, tinctures, cataplasms, rubber plasters or plasters.

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 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, perfumes, and the like;

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 parenteral medicament is selected from injection, spray, aerosol, patch, etc.

In particular, the preparation for gastrointestinal administration is selected from tablets, capsules, powders, granules, pills, solutions, syrups and the like.

On the other hand, the invention provides the application of andrographolide in preparing a medicine for protecting striated muscle (preventing striated muscle from being damaged and dissolved) in the treatment of hyperlipidemia diseases.

Application of andrographolide in preparing medicine for protecting striated muscle (preventing striated muscle from damage and dissolving) during treatment of hyperlipidemia is provided.

In another aspect, the invention provides an application of andrographolide in preparing a medicament for muscle protection in treating hyperlipidemia diseases.

The use of andrographolide for preparing muscle protecting medicine for treating hyperlipidemia is provided.

The hyperlipidemic condition is one or more of hypercholesterolemia, hypertriglyceridemia, atherosclerosis or coronary heart disease, preferably hypercholesterolemia.

The invention provides a method for treating hyperlipidemia, enhancing muscle strength and protecting muscles, which comprises the step of administering 1-600 mg/kg-d, preferably 10-300 mg/kg-d, and more preferably 60-90 mg/kg-d of andrographolide to a subject.

As used herein, unless otherwise indicated, the term "therapeutically effective amount" is the amount of a drug required to produce an effective effect; the "therapeutically effective amount" is adjustable and variable 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 andrographolide, and the andrographolide is used for preparing a medicament or a health-care product for resisting muscle injury in the treatment of hyperlipidemia, preparing a medicament or a health-care product for treating and preventing muscle injury, particularly striated muscle injury and dissolution in the treatment process of hyperlipidemia, and preparing a medicament or a health-care product for preventing and treating hyperlipidemia and adverse reactions of muscle injury in the treatment process of hyperlipidemia, thereby developing a new field for the application of andrographolide.

2. A series of experimental researches prove that the andrographolide has the obvious functions of preventing and treating the hyperlipidemia or the muscle injury in the treatment process of the hyperlipidemia, particularly the adverse reaction of rhabdomyoid injury and dissolution, and the liver and kidney function influence and injury are not found in the process of treating the hyperlipidemia by the andrographolide.

3. The andrographolide also has the effects of reducing fat and losing weight, has the effect of losing weight, and is used for preventing and treating obesity and hyperlipidemia caused by obesity.

4. The andrographolide disclosed by the invention is used for treating hyperlipidemia, and not only is the blood fat reducing effect of the andrographolide obvious, but also the andrographolide has a remarkable inhibiting effect on the hyperlipidemia; but also improve the muscle strength and strengthen the endurance of the muscle; furthermore, the andrographolide has no side effect of muscle injury of a drug statin for treating hyperlipidemia, can also enhance endurance and instant holding power of muscle, and has an obvious effect of protecting the muscle. The andrographolide is shown to be capable of remarkably reducing adverse reactions in treating hyperlipidemia, and can be taken for a long time.

5. The andrographolide has strong pharmacological action and is used for treating hyperlipidemia; the traditional Chinese medicine composition has the advantages of obvious effect of preventing, conditioning and treating cardiovascular diseases, quick response, small toxic and side effect, no adverse reaction of dissolving striated muscle, good safety, long-term taking and good medicinal prospect.

6. The product of the invention has rich raw material sources, low price, safe clinical use, simple preparation process, small dosage and convenient use, can be prepared into various dosage forms, and is easy to popularize.

Drawings

FIG. 1 is a graph showing the results of the staining change of beta-3T 3 adipocytes and their lipid oil Red 0, wherein: MODEL is MODEL group cell; ATOR is atorvastatin-treated cells; aLOW is sodium bisulfite andrographolide LOW dose (10 μmol/L) treated cells; a HIGH is sodium bisulfite andrographolide HIGH dose (50 μmol/L) treated cells; b LOW is andrographolide LOW dose (10 μmol/L) treated cells, B HIGH is andrographolide HIGH dose (50 μmol/L) treated cells;

FIG. 2 is a H & E staining pattern of gastrocnemius muscle of Apoe mice.

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 modifications may be made without departing from the spirit and 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

2. Test reagent

3. Test drug

EXAMPLE 1 preparation of Andrographolide

Pulverizing herba Andrographitis (1kg) 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: 1.

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: 1).

Adding active carbon into the extract-concentrated solution, stirring, decolorizing for 30min (usually 30-60min), filtering, and concentrating the filtrate to obtain decolorized-concentrated solution; wherein, the dosage of the active carbon is 10 percent (usually 5 to 20 percent) of the raw medicinal material; the decoloration 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 (usually 0.5:1 to 2: 1).

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 crystals are just completely dissolved, and then adding activated carbon for decoloring; then filtering, concentrating the filtrate, and recovering ethanol to obtain a crystallization-decoloration-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 crude product;

drying andrographolide crystal under reduced pressure in vacuum to obtain crude dehydroandrographolide product.

2. Andrographolide purification

Dissolving the 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 respectively; 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 prepared in the step 1) is 1:1 (usually 0.5-2: 1) that is, when the weight (dry weight) of the andrographolide crude product is 1kg, the volume of the macroporous adsorbent resin is 0.5-2L, and when the weight (dry weight) of the andrographolide crude product is 1g, the volume of the macroporous adsorbent resin is 0.5-2ml, preferably 1-2: 1. The ratio of each eluent to the column volume of the macroporous resin column in the elution process is 4:1 (usually 3-5: 1), and the macroporous adsorption resin is AB-8 type macroporous adsorption resin (also can be D101, HP 20).

Measuring the content of the prepared andrographolide:

chromatographic conditions are as follows: with acetonitrile-phosphate buffer (5 mmol. L)^-1Dipotassium hydrogen phosphate-5 mmol. L^-1Potassium dihydrogen phosphate 1:2, adjusting pH to 6.80 with phosphoric acid) as a mobile phase, performing gradient elution, detecting the wavelength of 230nm, and controlling the column temperature to be 30 ℃; the flow rate was 1.0 mL/min^-1The injection volume was 10. mu.L. Compared with andrographolide reference substances, the andrographolide prepared by the invention has the content of more than 98.5%.

Test example 1 Effect of Andrographolide on lipid Synthesis of beta-3T 3 Pre-adipocytes

1. Experimental Material

1.1 cells

The 3T3-L1 preadipocyte cell line was purchased from the cell resource center of the kyoto institute of medicine, beijing, 9, 15, 2014 (which is the national infrastructure of cell line resources, headquarters for NSTI). The cell lines were checked for absence of mycoplasma contamination by PCR and culture, their provenance was identified by PCR, and the results of the identification of the cell lines were identified by STR profiling (FBI, CODIS), all of which were reviewed on the website (http:// cell resource. cn).

1.2 medicine

Andrographolide: white crystalline powder (ethanol) with a content of 99.7%, China institute for food and drug testing;

atorvastatin calcium, purchased from Dalian Milan Biotech (Cat. No.: 134523-03-8).

3T3-L1 cell inducing liquid A (differentiation inducing liquid composition: 0.5mmol/L IBMX (sigma: I7018), 1. mu.g/mL insulin (sigma: I9278), 0.25. mu. mol/L dexamethasone (sigma: D1756), the base liquid is complete culture medium)

3T3-L1 cell inducing liquid B, (1 mug/mL insulin, base liquid is complete culture medium)

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; andrographolide low dose group; andrographolide high dose group.

Resuscitating cultured 3T3-L1 preadipocytes at a ratio of 1X 10⁶Inoculating the cells into a 24-well cell culture plate, culturing in a complete culture medium (DMEM medium, 10% FBS (fetal bovine serum)), when the cell growth coverage area reaches above 90%, giving the complete culture medium (DMEM medium, 10% FBS (fetal bovine serum)) to the blank control group of cells, adding 3T3-L1 cell induction liquid A to the other groups of cells for inducing differentiation for 48h, then changing 3T3-L1 cell induction liquid B for continuing inducing differentiation, after differentiation for 48h, sucking out the treatment liquid, adding the complete culture medium and corresponding treatment drugs, and treating the cells in each group as follows:

model group: DMEM medium (10% FBS); atorvastatin calcium group: DMEM medium (10% FBS) +10 μ M atorvastatin calcium; andrographolide low dose group: DMEM medium (10% FBS) +10 μ M andrographolide; andrographolide high dose group: DMEM medium (10% FBS) +50 μ M andrographolide.

2.2 dyeing

After 10 days of culture, 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 to dissolve

2. The plate was washed twice with PBS and discarded by blotting.

3. Fixation with 4% paraformaldehyde for 30 minutes.

4. The oil red stock was mixed with deionized water at a ratio of 3:2, and then the cells were stained for 15 minutes and washed twice with pbs.

2.3 statistics

The images were observed under a microscope and photographed as shown in FIG. 1, wherein: MODEL is MODEL group cell; ATOR is atorvastatin-treated cells; b low is andrographolide low dose (10. mu. mol/L) treated cells; b high is andrographolide high dose (50 mu mol/L) treated cells; the red area was calculated using Image-Pro Plus 6.0 Image recognition software. SPSS 22.0 performs one-way anova. The results are shown in Table 1.

Table 1 effect on adipocyte lipogenesis (%, n ═ 3)

★ P < 0.05, ★★ P < 0.01, ★★★ P < 0.001, compared to the model group

As can be seen from the results of the above cell experiments, andrographolide in this example has a significant effect on lipid synthesis in 3T3-L1 adipocytes. As the dosage increases, the effect of interfering with lipid synthesis increases.

Test example 2 lipid-regulating Effect of Andrographolide on APOE mice

1. Experimental Material

60 male APOE (-/-) mice, 8 weeks old, 10 Wild Type (WT) C57BL/6J mice, weighing 22 + -2 g, purchased from Wintonlifwa, Beijing.

The drug product was the same as in test example 1.

Mouse rotating type fatigue instrument (Shanghai Yuyan scientific instruments Co., Ltd., model: YLS-10B)

Rat and mouse gripe force Meter (IITC Life Science: Grip Strength Meter for Mice and Rats,2200)

2. Grouping and intervention method

All mice were randomized into groups by weight after 1 week of acclimation, as follows:

normal (blank) control group (group C57): 10C 57BL/6J mice were gavaged with normal diet plus saline.

Model control group (Model group): 10 APOE (-/-) mice, high cholesterol diet + saline gavage.

Positive drug control group (ATOR group, Atorvastatin group): 10 APOE (-/-) mice, high cholesterol diet + atorvastatin calcium (10mg/kg/d) were gavaged.

Andrographolide high dose group (B high group): 10 APOE (-/-) mice, high cholesterol diet + andrographolide (100mg/kg/d) were gavaged.

Andrographolide medium dose group (B midle group): 10 APOE (-/-) mice, high cholesterol diet + andrographolide (50mg/kg/d) were gavaged.

Andrographolide low dose group (group B low): 10 APOE (-/-) mice, high cholesterol diet + andrographolide (25mg/kg/d) were gavaged.

The high cholesterol diet adopts a 'western diet' formula and is provided by Huafukang (the product number is H10141, the formula is shown in table 2), the intragastric administration volume of each group of mice is 10ml/kg, each group of mice is continuously intragastric administered for 4 weeks, and the day 1 is the beginning of the formal experiment, and the total day is 28.

TABLE 2 high cholesterol fodder formulation table

Components	g	Kcal
			Casein protein	195.00	780.00
Methionine	3.00	12.00
			Corn starch	50.00	200.00
Maltodextrin	100.00	400.00
			Sucrose	341.00	1364.00
Cellulose, process for producing the same, and process for producing the same	50.00	0
			Corn oil	10.00	90.00
Anhydrous cream	200.00	1800.00
			Mineral mixture M1001	35.00	0
Calcium carbonate	4.00	0
			Vitamin mixture V1001	10.00	40.00
Choline bitartrate	2.00	0
			Cholesterol	1.50	0
Antioxidant TBHQ	0.04	0
			Total of	1000	4686.00

All the mice in the experimental groups are placed in the center of SPF experimental animals, 5 mice are placed in a cage, the temperature of a mouse house 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 had free access to food and water (autoclaved water) during the test period. The weight was once a week, and each group was gavaged as described above for 4 weeks.

3. Test method

Each group of mice was weighed weekly using an electronic balance and the weight of the mice was recorded.

On the 21 st day of continuous administration, respectively adopting a mouse rotation type fatigue instrument and a big and small mouse grip dynamometer to measure the endurance capacity and instantaneous grip capacity of the mouse;

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 anesthetized mouse in a biochemical tube containing separation gel, placing the anesthetized mouse at room temperature for 2 hours, centrifuging the anesthetized mouse at 3500r/min for 15min, taking out upper serum, subpackaging the anesthetized mouse by using a sterile tube, and storing the anesthetized mouse in a refrigerator at the temperature of-80 ℃ to be tested.

The left hind leg gastrocnemius muscle of the mouse was peeled off and fixed with 4% paraformaldehyde.

4. Index measurement

4.1 detection of four items of blood fat, liver and kidney function

Serum Total Cholesterol (TC), serum Triglycerides (TG), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), urea nitrogen (BUN), and creatinine (CREA or CR) levels were measured using a Beckman AU5822 automated biochemical analyzer. The results are shown in tables 3 and 4.

TABLE 3 Effect on blood lipid and liver and kidney function in APOE mouse models

★ P < 0.05, ★★ P < 0.01, ★★★ P < 0.001, compared to the model group

TABLE 4 Effect on blood lipid and liver and kidney function in APOE mouse models (Table continuation)

★ P < 0.05, ★★ P < 0.01, ★★★ P < 0.001, compared to the model group

As can be seen from the test results of tables 3 and 4 above,

the andrographolide of the invention has the inhibition effect on hyperlipemia: reduce total cholesterol, triglyceride, low density lipoprotein cholesterol, and increase high density lipoprotein cholesterol. Can be used for preventing and treating hyperlipidemia and controlling secondary cardiovascular diseases. In the dose range of the experiment using andrographolide for prevention and treatment, no significant statistical significance is found on the influence of 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.

4.2 animal weight changes

Each group of mice was weighed weekly and the results are shown in tables 5 and 6.

TABLE 5APOE mouse body weights

★ P < 0.05, ★★ P < 0.01, ★★★ P < 0.001, compared to the model group

TABLE 6APOE mouse body weight (continue table)

★ P < 0.05, ★★ P < 0.01, ★★★ P < 0.001, compared to the model group

As can be seen from tables 5 and 6 above, the weight of the andrographolide group mice is obviously lower than that of the model group and the positive drug group, which shows that the product has certain weight-reducing effect and can be used for developing obesity-related drugs.

4.3 mouse endurance Change

After 3 weeks of administration, the variation of the endurance capacity of each group of mice was measured using a rotary fatigue meter, and at a speed of 15 revolutions/min, the upper limit was set to 10min, the mice reached the electrodes when they entered the fatigue state, at which time the wheel was stopped for 30s, the mice entered the rest state, and when the wheel was stopped 3 times, the exhaustion of the mice was determined, the channel was stopped. The system automatically records the endurance value (time) and distance. The test results are shown in table 7.

TABLE 7APOE mouse endurance test results

In comparison with the set of models,^★P＜0.05，★★P＜0.01，^★★★p is less than 0.001; in comparison with the atorvastatin group,^#P＜0.05，^##P＜0.01，^###P＜0.001

as can be seen from the test results of table 7:

1. high fat diet has certain influence on muscle endurance, so that the muscle endurance is reduced, and the passive exercise time of a hyperlipidemia model mouse is shortened; the passive movement distance is reduced; the long-term use of atorvastatin calcium (positive drug) has obvious influence on the muscle endurance of mice, and compared with the mice in a model group, the passive exercise time of the mice in the positive drug group is obviously shortened; the passive movement distance is significantly reduced.

2. When the hyperlipemia mouse takes the atorvastatin calcium (positive control medicament) for reducing blood fat, adverse reaction of rhabdomyolysis is easy to occur, the muscle endurance of the mouse is further reduced, the reduction degree is obvious, and the difference is obvious;

3. the andrographolide overcomes the condition that the muscle endurance of a mouse with hyperlipidemia is reduced, remarkably improves the condition of muscle damage caused by hyperlipidemia, remarkably improves the adverse reaction of the existing drug for treating hyperlipidemia on the reduction of muscle strength and muscle endurance, enhances the muscle strength, has obvious protective effect on muscle, has remarkable difference compared with a model group and a positive drug control group, and remarkably improves the muscle endurance; compared with the endurance of the normal group of mice, the endurance of the mice in the low-dose andrographolide group is equivalent to that of the normal group of mice, and the endurance of the mice in the medium-dose group and the high-dose group is obviously improved and enhanced compared with that of the mice in the normal group.

4.4 mouse grip Change

The mice of each experimental group were subjected to grip measurement using a rat and mouse grip meter 3 weeks after the administration. An operator holds the tail root of the mouse by hand, places the mouse on the gripping net, and continuously applies force along the horizontal direction of the gripping net after a mouse claw contacts the grid, so that the grid is reflectively gripped by the mouse. The operator increases the pulling force gradually and the mouse is forced to loosen, and the recorder automatically records the instantaneous maximum pulling force generated in the process. The above operation was repeated 3 times, and the maximum value was taken as the mouse grip value (g). The test results are shown in table 8.

TABLE 8APOE mouse grip test results

Grouping	Holding power (g)
		Blank control group	231.29±31.85^★★###
Model set	189.71±8.36^###
		Positive drug control group	144.86±15.69^★★★
Andrographolide high dose group	245±21.63^★★★###
		Andrographolide middle dose group	235.43±41.87^★★###
Andrographolide low dose group	204.67±16.72^###

In comparison with the set of models,^★P＜0.05，^★★P＜0.01，^★★★p is less than 0.001; in comparison with the atorvastatin group,^#P＜0.05，^##P＜0.01，^###P＜0.001

as can be seen from the test results of table 8:

1. high fat diet has certain influence on the instantaneous muscle holding power, and the instantaneous holding power of the mice in the model group is obviously lower than that of the mice in the normal group;

2. when the hyperlipemia mouse takes the atorvastatin calcium (positive control medicament) for reducing blood fat, due to the adverse reaction of dissolving striated muscle, compared with the normal blank group and model group mice, the instant muscle holding force of the mice is further reduced, the reduction degree is obvious, and the difference is obvious;

3. the andrographolide provided by the invention can overcome the condition that the instantaneous muscle holding power of a mouse with hyperlipidemia is reduced, obviously improves the adverse reaction of the existing drug for treating hyperlipidemia on the reduction of muscle strength and the instantaneous muscle holding power, enhances the muscle strength, has an obvious protective effect on muscle, has obvious difference compared with a model group and a positive drug control group, and obviously improves the muscle holding power; compared with the instantaneous holding power of the normal group of mice, the instantaneous holding power of the mice of the low and medium dose andrographolide groups is equivalent to that of the normal mice, and the instantaneous holding power of the mice of the high dose group is improved and enhanced to a certain extent compared with that of the mice of the normal group.

4.5APOE mice gastrocnemius H & E staining

Stripping the left hind leg gastrocnemius of the mouse, fixing the left hind leg gastrocnemius in paraformaldehyde, embedding paraffin after 48H, taking paraffin sections at the midpoint cross sections of two tendons, and carrying out the following H & E staining (hematoxylin-eosin staining, also called HE staining):

1. dewaxing: sequentially placing the slices into xylene I20 min-xylene II 20 min-absolute ethyl alcohol I5 min-absolute ethyl alcohol II 5 min-75% alcohol 5min, and washing with tap water.

2. Hematoxylin staining: and (3) dyeing the slices in hematoxylin dyeing solution for 3-5min, washing with tap water, differentiating the differentiation solution, washing with tap water, returning blue to the blue solution, and washing with running water.

3. Eosin staining: the slices are dehydrated for 5min respectively by 85 percent and 95 percent gradient alcohol, and are dyed for 5min in eosin dye solution.

4. Dewatering and sealing: placing the slices in anhydrous ethanol I5 min-anhydrous ethanol II 5 min-anhydrous ethanol III 5 min-dimethyl I5 min-xylene II 5min, sealing with neutral gum.

5. Microscopic examination and image acquisition and analysis.

The staining results are shown in fig. 2, the muscle gap of the model group is increased, the muscle fiber is thinned, the statins are further obviously reflected, and the andrographolide administration group can obviously improve the conditions and shows dose gradient change. The andrographolide improves muscle injury caused by hyperlipidemia, especially muscle injury symptoms caused by hyperlipidemia, such as myalgia, myositis or rhabdomyolysis, which are mainly manifested by myofiber reduction, myointerstitial enlargement, etc., and protects muscle injury caused by hyperlipidemia; can replace statins to a certain extent in the treatment of hyperlipidemia diseases, and avoid muscle adverse reactions possibly brought in the process of using statins. In addition, the andrographolide can also obviously improve muscle injury adverse reaction in the process of treating hyperlipidemia by using statins, and prevent and treat muscle injury and rhabdomyolysis caused by hyperlipidemia treated by statins.

The results of the mouse endurance and instant holding force tests show that: the andrographolide is used for treating hyperlipidemia, has an obvious effect of reducing blood fat and has an obvious effect of inhibiting the hyperlipidemia; moreover, no influence on the functions of the liver and the kidney is found; more importantly, the andrographolide has no side effect of muscle injury of statins, can also enhance endurance and instant holding power of muscles, and has obvious muscle protection effect. The andrographolide can be taken for a long time for treating hyperlipidemia.

Test example 3 Effect of Andrographolide on lipid regulation in Kunming mouse model with high lipid level

1. Experimental Material

50 male mice of Kunming species, 8 weeks old, weighing 20 + -2 g, purchased from Beijing Wittingle, Inc.

The drug product was the same as in test example 1.

Fatigue rotating rod instrument (Chengdutai allied science and technology limited, ZB-200)

2. Grouping and intervention method

the normal control group consisted of 10 mice, which were gavaged with normal diet plus normal saline.

Model control group: 10 mice, high cholesterol diet, fat milk intragastric administration and normal saline intragastric administration.

Positive drug control group: 10 mice, high cholesterol diet + fat milk gavage + atorvastatin calcium (10mg/kg/d) gavage.

Andrographolide group: 10 mice, high cholesterol diet, fat milk gavage, andrographolide (50mg/kg/d) gavage.

The formula and the preparation method of the fat emulsion comprise the following steps: prescription: 20g of lard, 1g of propyl thiouracil, 8g of cholesterol, 1g of sodium glutamate, 5g of sucrose, 5g of fructose, 8015 ml of tween, 15ml of propylene glycol and 35ml of water.

The preparation method comprises the following steps: melting lard in a water bath at 70 ℃, and keeping the temperature for later use; grinding propyl thiouracil, adding cholesterol and propylene glycol, homogenizing at 25000rpm for 2min without visible particles, and keeping the temperature for later use; dissolving 1g of sodium glutamate, 5g of sucrose and 5g of fructose in water, adding Tween 80 while stirring, uniformly stirring, heating in a water bath at 60 ℃, slowly adding the oil phase while stirring at 3000-4000rpm, and uniformly stirring to obtain the oil-soluble chitosan.

All mice are placed in the center of an SPF-level experimental animal, 5 mice are placed in a cage, the temperature of a mouse house 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 had free access to food and water (autoclaved water) during the test period. The weight was once a week, and each group was gavaged as described above for 4 weeks.

3. Test method

The rod rotation endurance of each group was measured 3 weeks after the administration, and the endurance values (seconds, S) were recorded.

4. Index measurement

4.1 detection of four items of blood fat, liver and kidney function

Serum levels of TC (CHO), TG, HDL-C, LDL-C, AST, ALT, CREA, BUN were measured using a Beckmann AU5822 automated biochemical analyzer. The results are shown in Table 9 below.

Table 9 effects on high lipid model blood lipid and liver and kidney function in Kunming mice

	Blank control group	Model set	Positive drug control group	Andrographolide
					LDL-C	0.49±0.13^★★★	1.86±0.56	0.95±0.25^★★★	1.25±0.15^★★
TG	0.93±0.14^★★★	1.88±0.47	0.84±0.26^★★★	0.88±0.18^★★★
					HDL-C	2.4±0.25	2.17±0.53	2.07±0.25	2.27±0.2
CHO	3.04±0.39^★★	4.66±0.89	3.02±0.77^★	3.38±0.5^★★★
					ALT	37.4±11.61	59.4±29.47	77.2±41.12	55.4±13.32
CR	13±1.87	13.2±2.95	12.2±1.1	14.2±3.42
					BUN	9.21±0.72	8.55±1.07	6.81±0.94	8.09±1.17
AST	143.2±31.08	152.2±41.34	155.6±19.06	149.6±52.83

★ P < 0.05, ★★ P < 0.01, ★★★ P < 0.001, compared to the model group

As can be seen from Table 9 above, andrographolide showed a significant lowering effect on blood Triglyceride (TG) and Total Cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) in blood of high-fat-fed Kunming mice as compared with the model group. In this experiment, andrographolide had no significant effect on liver and kidney function.

4.2 animal weight changes

Each group of mice was weighed weekly and the results are shown in table 10.

TABLE 10 Effect on mouse hyperlipidemia model body weight

	Day 0	Day 7	Day 14	Day 21	Day 28
						Control group	23±0.5	33.26±2.75	36.71±1.24	37.78±1.43	38.18±2.1^★★★
Model set	22.06±1.08	33.06±2.19	37.36±1.65	40.18±2.1	43.78±1.65
						Positive group	23.55±1.14	32.78±1.39	37.36±0.86	37.93±0.91	39.16±0.78^★★★
Andrographolide group	23.09±1.32	31.86±3.33	35.53±3.06	36.19±2.55	37.36±1.97^★★★

★ P < 0.05, ★★ P < 0.01, ★★★ P < 0.001, compared to the model group

As can be seen from Table 10, andrographolide has a significant weight loss effect in Kunming mice. And is superior to the trend of the positive control drug. Shows that the andrographolide can inhibit triglyceride from accumulating and precipitating in cells, and can be used for preventing and treating obesity.

4.3 determination of animal endurance

After 3 weeks of administration, the upper limit is set to 10min at the speed of 15 r/min, the mouse falls off by the rotating rod when entering the fatigue state, and the timing termination system is triggered to automatically record the endurance value (time). The results are shown in Table 11.

TABLE 11 Effect of Andrographolide on muscle endurance in Kunming mouse model (S)

1. high fat diet has certain influence on muscle endurance, so that the muscle endurance is reduced, and the passive exercise time of the hyperlipidemia model Kunming mouse is obviously shortened; the long-term use of atorvastatin calcium (positive drug) has a significant effect on the muscle endurance of mice.

2. When the hyperlipidemia Kunming mouse takes the atorvastatin calcium (positive control drug) for reducing blood fat, the muscle endurance of the mouse is further reduced due to the adverse reaction of dissolving striated muscle, the reduction degree is obvious, and compared with a model group, the model group has obvious difference;

3. the andrographolide provided by the invention can overcome the condition that the muscle endurance of a Kunming mouse with hyperlipidemia is reduced, obviously improves the adverse reaction of the existing drug for treating hyperlipidemia on the reduction of muscle strength and muscle endurance, enhances the muscle strength, has an obvious protective effect on muscle, has obvious difference compared with a model group and a positive drug control group, and obviously improves the muscle endurance; compared with the endurance capacity of the normal group of mice, the endurance capacity of the mice in the medium-dose andrographolide group is equivalent to that of the normal mice, and the endurance capacity of the mice in the high-dose group is obviously improved and enhanced compared with that of the mice in the normal group.

The results of the Kunming mouse endurance test show that: the andrographolide is used for treating hyperlipidemia, has an obvious effect of reducing blood fat and has an obvious effect of inhibiting the hyperlipidemia; moreover, no influence on the functions of the liver and the kidney is found; more importantly, the andrographolide has no side effect of muscle injury of statins, can also enhance the endurance of muscles, and has obvious muscle protection effect. The andrographolide can be taken for a long time for treating hyperlipidemia.

The above-described embodiments of the present invention are merely exemplary 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 modifications may be made without departing from the spirit and scope of the invention.

Claims

1. Application of andrographolide in preparing medicine or health product for preventing and/or treating muscle injury is provided.

2. The use of claim 1, wherein the medicament is comprised of andrographolide and 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, emulsions, injections, sprays, aerosols, gels, creams, tinctures, cataplasms, rubber plasters or plasters.

4. Use according to claim 1 or 2, wherein the muscle damage is myalgia, myositis or rhabdomyolysis, preferably rhabdomyolysis.

5. Use according to claim 1 or 2, wherein the muscle damage is myalgia, myositis or rhabdomyolysis caused by a hyperlipidemic condition, preferably a condition of muscle damage caused by a decrease in muscle fibers and an increase in muscle space caused by a hyperlipidemic condition.

6. Use according to claim 1 or 2, wherein the muscle damage is muscle damage due to adverse effects of rhabdomyolysis during treatment of a hyperlipidemic condition with a statin.

7. Use according to claim 5 or 6, characterized in that the hyperlipidemic condition is one or more of hypercholesterolemia, hypertriglyceridemia, atherosclerosis or coronary heart disease, preferably hypercholesterolemia.

8. The application of andrographolide in preparing medicine for protecting striated muscle in treating hyperlipemia is provided.

9. The application of andrographolide in preparing medicine for protecting muscle in treating hyperlipemia is provided.

10. The application of andrographolide in preparing medicine or health product for treating rhabdomyolysis adverse reaction in hyperlipemia disease is provided.