CN115068467A

CN115068467A - Medical application of andrographolide as LTB4 receptor inhibitor

Info

Publication number: CN115068467A
Application number: CN202110275769.4A
Authority: CN
Inventors: 吉腾飞; 李平平; 万彦军; 崔冰; 赵其锦; 王洪建; 王佳佳
Original assignee: Institute of Materia Medica of CAMS and PUMC
Current assignee: Institute of Materia Medica of CAMS and PUMC
Priority date: 2021-03-15
Filing date: 2021-03-15
Publication date: 2022-09-20

Abstract

The invention relates to the technical field of medicines, and discloses a medical application of andrographolide as an LTB4 receptor inhibitor. The andrographolide is subjected to action target research, and is found to have a remarkable inhibition effect in an LTB4R inhibitor model. Therefore, the invention discloses the application of andrographolide and pharmaceutically acceptable salts and pharmaceutical compositions thereof in preparing LTB4 receptor inhibitors and in preparing medicaments for treating obesity, insulin resistance, abnormal glucose tolerance, hyperlipidemia, diabetes and complications thereof and the like, wherein the diabetic complications comprise diabetic nephropathy, diabetic cardiovascular complications, diabetic cerebrovascular diseases, diabetic eye complications and diabetic foot.

Description

Medical application of andrographolide as LTB4 receptor inhibitor

Technical Field

The invention belongs to the technical field of medicines, and relates to a method for extracting, separating and purifying andrographolide from andrographis paniculata and application of andrographolide in preparing an LTB4 receptor inhibitor; and the application in the preparation of medicaments for treating obesity, insulin resistance, abnormal glucose tolerance, hyperlipidemia, diabetes, complications thereof and the like, wherein the diabetic complications comprise diabetic nephropathy, diabetic cardiovascular complications, diabetic cerebrovascular diseases, diabetic eye complications and diabetic feet.

Background

Diabetes is a group of endocrine-metabolic diseases characterized primarily by chronic hyperglycemia, which can lead to complications in various parts of the body and increase the risk of premature death. In recent years, with the rapid development of economy in China, the aging of the population society, environmental pollution, bad life style, over-high mental stress and other factors, diabetes mellitus is increasingly worsened in China. Diabetes mellitus can hardly be completely cured, complications of diabetes mellitus can bring physiological damage to patients, mental pain, reduction in quality of life and the like, and high and continuous medical expenses can bring huge economic burden to patients and families. Therefore, research on the treatment of diabetes is imminent.

Impaired Glucose Tolerance (IGT) and Impaired Fasting Glucose (IFG) refer to blood glucose levels above the normal range but below the diagnostic criteria, collectively referred to as impaired glucose regulation, also commonly referred to as pre-diabetes. The implications of the emergence of IGT and IFG are three-fold: (1) high risk of developing type 2 diabetes; (2) increased risk of cardiovascular disease; (3) reminding to prevent disease development by intervention measures. The risk of developing type 2 diabetes from IGT and IFG is influenced by factors such as age, weight, etc., and the cumulative incidence of developing type 2 diabetes five years after diagnosing IGT or IFG is approximately 26% and 50%, respectively. Obesity, less physical activity, increased blood lipid, family history of diabetes and hyperinsulinemia are the most risk factors for inducing impaired glucose tolerance.

All type 2 diabetes patients almost pass through the stage of impaired glucose tolerance, and thus, impaired glucose tolerance is considered as a high risk group of onset of diabetes, or "pre-diabetes". Impaired glucose tolerance is often accompanied by obesity, hypertension, hyperlipidemia, and the like. Compared with normal people, the prevalence rate of hypertension and obesity is 2 times higher, and the prevalence rate of coronary heart disease is 8 times higher.

To prevent diabetes, the prevention and treatment of Impaired Glucose Tolerance (IGT) must be performed. Lifestyle changes including diet control, exercise gain, weight loss, smoking cessation, etc. are the most basic interventions. The related research reports that after the IGT patients are intervened by a life style, the incidence rate of diabetes is reduced by 58 percent after 3 years, meanwhile, the blood pressure and the body weight are obviously reduced compared with the prior art, and the incidence rate of diabetes of the IGT patients without any intervention measures is up to 68 percent. Recent intervention tests on IGT (insulin-dependent diabetes mellitus) populations show that the IGT populations subjected to drug intervention not only remarkably reduce the incidence rate of diabetes, but also remarkably reduce the risk of Cerebrovascular diseases (CVD), wherein the newly-generated hypertension is reduced by 34%, the myocardial infarction is reduced by 9%, and any cardiovascular event is reduced by 49%. Therefore, after the diagnosis of the IGT patient is definite, the physician should take a medical initiative to select an appropriate treatment scheme and drugs according to the condition of the disease, and actively treat hyperglycemia and related risk factors.

Therefore, targeted IGT intervention is taken early, helping to reverse IGT, reducing the risk of IGT progressing to diabetes, and reducing the occurrence of CVD.

Diabetes is clinically divided into 3 types: type 1, type 2 and gestational diabetes, with type 2 diabetes being the most common, accounting for about 85% -95%, and even higher. Type 2 diabetes is primarily characterized by insulin resistance. Insulin resistance is a chronic subclinical inflammatory process resulting from decreased glucose uptake and utilization efficiency due to decreased sensitivity of peripheral target tissue cells such as liver, muscle and adipose tissue to insulin. Intracellular inflammatory factors secrete factors which can weaken insulin signals, so that the transmission of the insulin signals is blocked, and insulin resistance is induced. Experimental studies of inflammatory factor and insulin resistance adiponectin levels in type 2 diabetic patients indicate that chronic inflammatory states exist in type 2 diabetic patients. Therefore, improving inflammation-induced insulin resistance and increasing insulin sensitivity has become a significant problem for the treatment of type 2 diabetes patients.

Inflammatory factor leukotriene B4(LTB4) is a leukotriene-like substance associated with inflammatory response, and is produced by sequential catalysis of arachidonic acid by 5-lipoxygenase, 5-lipoxygenase activating protein, leukotriene A4 hydrolase. LTB4 has potent chemotaxis towards macrophages, neutrophils and T cells. LTB4 can promote the development of inflammation by activating and recruiting macrophages, eosinophils, effector T cells, etc. And simultaneously can induce the formation and release of lysosomes and reactive oxygen species in the neutrophils.

LTB4 plays a role in chemotaxis by binding with G protein-coupled receptors Ltb4r1 or Ltb4r2, and regulates biological functions such as proinflammatory factor release. Ltb4r1 (also called Blt1) is a high affinity receptor specific for Ltb4, and is widely expressed in inflammatory and immune cells, including granulocytes, eosinophils, macrophages, Th1 cells, Th2 cells, Th17 cells, CD8 effector T cells, dendritic cells, osteoclasts, and the like. Ltb4r2 (also known as Blt2) is a low affinity receptor and is more widely expressed. The LTB4-Ltb4r1 system has important defense function in the acute infection stage of the host and reduces inflammatory reaction. Chronic activation of the LTB4-Ltb4r1 system can cause chronic inflammation, including atherosclerosis and arthritis, among others. A recent study showed that the Ltb4r1 gene knockout could reduce the inflammatory response and improve insulin resistance in mice. This indicates that the insulin sensitive phenotype is based on the deletion of the Ltb4r1 gene. The hyperinsulinemic-euglycemic clamp experiment also demonstrated that most mice exhibited greater insulin sensitivity after treatment with an inhibitor of Ltb4r 1.

Obesity is an important factor that leads to insulin resistance. Epidemiological studies have also shown that obesity is a significant risk factor for diabetes. Over-obesity, macrophages are activated by the over-accumulated fat, and activated macrophages release LTB4 and other inflammatory molecules to attract more macrophages. This positive feedback adjustment mechanism will produce more LTB 4. Excess LTB4 activates cells other than macrophages, such as hepatocytes, adipocytes, muscle cells, and the like, which are stimulated to develop insulin resistance. In visceral fat of obese subjects, macrophages may account for 40% of the total cell number and secrete a variety of factors that may impair insulin signaling. By using macrophage tracking technology, it was found that the degree of labeling in adipose tissue was substantially lower in mice macrophages treated with the Ltb4r1 inhibitor. LTB4 promotes monocyte migration into adipose tissue, stimulates macrophage chemotaxis in a dose-dependent manner, and promotes insulin resistance. In addition, other immune cell types, such as lymphocytes, eosinophils, and neutrophils also contribute to the inflammatory state of obese tissues. This cytokine-independent mechanism of insulin resistance provides a new mechanism for the relationship between inflammation and decreased insulin sensitivity.

From the molecular mechanism of insulin resistance, energy metabolism disorder, endoplasmic reticulum stress, oxidative stress, mitochondria function impairment, silencing information regulator signal channel down regulation, inflammatory reaction, central regulation disorder and the like are all involved in the generation of insulin resistance. The impairment of the insulin signaling pathway is essential for insulin resistance. An important function of glucose transporter 4(GLUT4) is to transport and transport glucose in adipose and muscle tissue. Studies have shown that LTB4 causes insulin resistance through inhibition of GLUT 4. LTB4 can enhance synthesis of hepatic Diacylglycerol (DAG) and ceramide, induce endoplasmic reticulum stress and increased mitochondrial membrane permeability, inhibit insulin signaling pathway, reduce insulin sensitivity, and induce insulin resistance. At the same time, increased DAG increases the risk of lipotoxicity, and is one of the important mechanisms leading to insulin resistance.

Therefore, inhibition of the action of LTB4 may be an effective treatment for insulin resistant diseases. And pharmaceutically acceptable salts and pharmaceutical compositions thereof in the treatment of obesity, impaired glucose tolerance, hyperlipidemia, diabetes and complications thereof; and the use of chronic inflammation, insulin resistance, cerebrovascular disease and atherosclerosis in relation to the above mentioned diseases.

The herba Andrographitis is dry aerial part of herba Andrographitis (Burm.F.) Nees of Acanthaceae, and is also known as herba Andrographitis, WANGBIXIANCAO, herba Swertiae Dilutae, etc. The functional indications are as follows: clear heat and remove toxicity, cool blood and relieve swelling. Can be used for treating common cold, fever, sore throat, aphtha of the mouth and tongue, cough due to cough, diarrhea, dysentery, pyretic stranguria, pain, carbuncle, swelling, sore, and snake bite.

The andrographis paniculata leaves contain diterpenes: andrographolide (andographolide) 0.6%, 14-deoxyandrographolide (14-deoxyandrographolide) 0.15%, neoandrographolide (neoandrographolide) 0.05%, 14-deoxyandrographolide-19- β -D-glucoside (14-deoxyandrographolide-19- β -D-glucoside) 0.03%, 14-deoxy-12-methoxy-andrographolide (14-deoxy-12-methoxy-andrographolide) 0.001%, and andrographolide (andrographolide) 0.03%; and (3) flavonoids: oroxylin a, wogonin (wogonin); polyphenols: caffeic acid (caffeic acid), chlorogenic acid (chlorogenic acid) and mixtures of dicaffeoylsilicic acids (mix of dicaf-femoylquinic acids).

The lotus root contains flavonoids: andrographolide (andrographin), 5, 2-dihydroxy-7, 8-dimethoxyflavone (panicolin), 3-O-methyl-widmanoflavone, i.e., 5-hydroxy-7,8,2, 3-tetramethylflavone (3-O-methoxylightning, 5-hydroxy-7,8,2, 3-tetramethyflavone), apigenin-4,7-dimethyl ether (apigenin-4, 7-dimethylflavone), 5-hydroxy-7,8-dimethoxyflavanone (5-hydroxy-7,8-dimethoxyflavanone), 5-hydroxy-3,7,8, 2-tetramethoxyflavone (5-hydroxy-3,7,8, 2-tetramethoxyflavone), 5-hydroxy-7, 8-dimethoxyflavone (5-hydroxy-7,8-dimethoxy flavanone), andrographolide A, B, C, D, E (andro-grapholide), F, and optionally alpha-sitosterol.

The andrographolide and its derivatives have wide bioactivity, and research shows that the andrographolide and its derivatives have antipyretic, anti-inflammatory, antiviral, antibacterial, liver protecting, gallbladder promoting, immunity regulating, cardiovascular disease resisting, and anti-tumor effects.

The antipyretic effect is as follows: andrographolide and andrographolide have the effects of inhibiting and delaying body temperature rise caused by Diplococcus pneumoniae and hemolytic streptococcus B, and the action strength of the latter is not as strong as that of the former. The composition has certain antipyretic effect on rabbits with fever caused by paratyphoid bacterin or rats with fever caused by 2, 4-dinitrophenol, and the dehydroandrographolide, andrographolide and dehydroandrographolide have the strongest effect.

Anti-inflammatory action: the permeability of the capillary vessels of the skin or the abdominal cavity of the mouse is increased by adopting dimethylbenzene and acetic acid, and the lavage of deoxyandrographolide or dehydroandrographolide can reduce the exudation of the capillary vessels, and compared with a control, P is less than 0.05 or P is less than 0.01. Has obvious inhibiting effect on hemorrhagic necrotic exudation caused by croton oil in rats and deoxyandrographolide.

Impact on immune function: the herba Andrographitis decoction can improve phagocytosis of Staphylococcus aureus by peripheral blood leukocyte in vitro. The andrographolide injection can also enhance phagocyte function. The in vitro experiment of 3H-thymidine infiltration into lymphocytes shows that the injection of water-soluble derivatives of Andrographis paniculata Nees has inhibitory effect on PHA-promoted 3H-thymidine infiltration.

Effects on pituitary-adrenal cortex system function: the first, second and third andrographis herbs, 4g/kg of third and fourth andrographis herbs and 1g/kg of butin are administrated to the young mouse by intragastric administration, 63-250mg/kg of dehydroandrographolide succinate (DAS) is injected into the abdominal cavity, 1 time per day, 3 days continuously, the thymus can be atrophied, which indicates that the function of adrenal cortex is strengthened.

Effects on the cardiovascular and blood systems: the experimental coronary artery thrombosis myocardial infarction dog is injected with 4g/kg of total flavone (equivalent to 4g of crude drug) by vein, and can remarkably improve epicardial electrocardiogram. Pathological examination shows that no thrombus is formed in the coronary artery, only intimal drop and little platelet aggregation and red and white blood cell adhesion are seen at the damaged coronary artery intima, and most animal myocardial sections are normal. The andrographis paniculata root total flavonoids can also obviously reduce the myocardial ytterbium uptake rate of rats and have a certain protection effect on myocardial injury caused by isoproterenol and experimental myocardial infarction ischemic injury.

Liver protection and gallbladder function: the herba Andrographitis has gallbladder benefiting effect and can increase liver weight of rat. The intraperitoneal injection of andrographolide can obviously increase the bile flow of rats. The physical properties of the bile secreted by it are also changed. Andrographolide also resisted hepatotoxic effects of CCl4, D-galactosamine (800mg/kg) and acetaminophen (3g/kg, orally), significantly reduced levels of SGPT, SGOT, SALP, HTG.

The anti-tumor effect is as follows: the dehydroandrographolide succinate has certain inhibition effect on W256 transplanted tumor. Arginine double salt (OASKARG) prepared from potassium dehydroandrographolide succinate has inhibitory effect on DNA synthesis of cultured breast cancer cells.

Disclosure of Invention

The invention provides a method for extracting, separating and purifying andrographolide from andrographis paniculata, and the application of the compound, the pharmaceutically acceptable salt and the pharmaceutical composition thereof in preparing medicaments for treating obesity, abnormal glucose tolerance, hyperlipidemia, diabetes, complications thereof and other diseases, and the application in preparing medicaments for preventing or treating chronic inflammation, insulin resistance, cerebrovascular diseases and atherosclerosis related to the diseases; also provides the application of andrographolide in preparing LTB4 receptor inhibitor.

In order to solve the technical problems, the invention provides the following technical scheme:

the first aspect of the technical scheme of the invention provides an application of andrographolide and pharmaceutically acceptable salts thereof in preparing an LTB4 receptor inhibitor. The andrographolide is characterized by having the following structural formula:

the second aspect of the technical scheme of the invention provides the application of andrographolide shown as the following formula and pharmaceutically acceptable salts thereof in preparing medicines for preventing or treating obesity, insulin resistance, abnormal glucose tolerance, hyperlipidemia, diabetes and complications thereof;

the diabetic complications comprise diabetic nephropathy, diabetic cardiovascular complications, diabetic cerebrovascular diseases, diabetic eye complications or diabetic feet.

The screening of the antidiabetic effect target of the andrographolide is carried out, and the compound is found to have remarkable activity in an LTB4R inhibitor model, so that the effect target of the compound is an LTB4 receptor.

The third aspect of the technical scheme of the invention provides a method for separating and purifying andrographolide from andrographis paniculata, which comprises the following steps:

(1) pulverizing the above-ground medicinal materials of the common andrographis herb, leaching for 3 times with 65% ethanol at 50 ℃, each time for 2 hours, and decompressing and recovering a solvent to 1/10 from an extracting solution at 40-50 ℃ to obtain the crude extract of the common andrographis herb.

(2) Standing the crude extract of herba Andrographitis, separating out crystal, and filtering; further dissolving, decolorizing and recrystallizing to obtain andrographolide.

(3) Performing MCI column chromatography on the crude extract of the andrographis paniculata from which the andrographis paniculata lactone is separated, eluting with 30% ethanol and 50% ethanol, and eluting two column volumes in each gradient, wherein 1/10 in terms of the column volume is used as one flow portion, and 40 flow portions are connected together.

(4) Mixing the fractions eluted with 50% ethanol, concentrating, standing, and separating to obtain andrographolide.

Preferably, the ratio of the mass of the andrographis paniculata medicinal material in the step (1) to the 65% ethanol used each time is 1: 3.5.

Preferably, the solvent is removed in step (1) by rotary evaporation under reduced pressure.

The fourth aspect of the technical scheme of the invention provides application of a pharmaceutical composition in preparing an LTB4 receptor inhibitor, wherein the pharmaceutical composition contains andrographolide with a therapeutically effective dose, pharmaceutically acceptable salts of andrographolide and a pharmaceutical carrier.

The invention also relates to a pharmaceutical composition comprising a pharmaceutically effective amount of said compound and a pharmaceutically acceptable carrier. For this purpose, if desired, in combination with one or more solid or liquid pharmaceutical excipients and/or adjuvants, suitable administration forms or dosage forms for human use are prepared.

According to the present invention, the compounds of the present invention may exist in the form of isomers, and generally, the term "compounds of the present invention" includes isomers of the compounds.

The pharmaceutical compositions of the present invention may be administered in unit dosage form, either enterally or parenterally, for example orally, intramuscularly, subcutaneously, nasally, oromucosally, dermally, peritoneally or rectally, and the like.

The route of administration of the pharmaceutical composition of the present invention may be administration by injection. The injection includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, acupoint injection, etc. The administration dosage form can be liquid dosage form or solid dosage form. For example, the liquid dosage form can be true solution, colloid, microparticle, emulsion, or suspension. Other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, etc.

The composition can be prepared into common preparations, sustained release preparations, controlled release preparations, targeting preparations and various microparticle drug delivery systems.

In order to prepare the unit dosage form into tablets, various carriers well known in the art can be widely used. As examples of the carrier, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate and the like; wetting agents and binders such as water, ethylparaben, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrating agents such as dried starch, alginates, agar powder, brown algae starch, sodium hydrogen carbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid esters, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cacao butter, hydrogenated oil and the like; absorption accelerators such as quaternary ammonium salts, sodium lauryl sulfate and the like; lubricants, for example, talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated as coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layered and multi-layered tablets.

For making the administration units into pills, a wide variety of carriers well known in the art can be used. Examples of the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc and the like; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecylsulfate, methylcellulose, ethylcellulose, etc.

For making the administration unit into a suppository, various carriers well known in the art can be widely used. As examples of the carrier, there are, for example, polyethylene glycol, lecithin, cacao butter, higher alcohols, higher alcohol enzymes, gelatin, semisynthetic glycerase and the like.

To encapsulate the administration units, the active ingredient is mixed with the various carriers described above, and the mixture thus obtained is placed in hard gelatin capsules or soft gelatin capsules. Or making into microcapsule, suspending in aqueous medium to form suspension, or making into hard capsule or injection.

For example, the composition of the present invention is formulated into injectable preparations, such as solutions, suspensions, emulsions, lyophilized powders, which may be aqueous or non-aqueous, and may contain one or more pharmaceutically acceptable carriers, diluents, binders, lubricants, preservatives, surfactants or dispersants. For example, the diluent may be selected from water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid enzyme, etc. In addition, for the preparation of isotonic injection, sodium chloride, glucose or glycerol may be added in an appropriate amount to the preparation for injection, and conventional cosolvents, buffers, pH adjusters and the like may also be added. These adjuvants are commonly used in the art.

In addition, if desired, colorants, preservatives, flavors, flavorings, sweeteners, or other materials may also be added to the pharmaceutical preparation.

The dose of the pharmaceutical composition of the present invention to be administered depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, body weight, character and individual response of the patient or animal, the administration route, the number of administrations, etc., and thus the therapeutic dose of the present invention can be widely varied. Generally, the dosage of the compounds of the present invention used is well known to those skilled in the art. The actual effective amount of the drug contained in the final preparation of the medicinal composition can be properly adjusted according to the requirement of the effective amount of the drug so as to achieve the treatment of obesity, abnormal glucose tolerance, hyperlipidemia, diabetes, complications thereof and other diseases; and the use of chronic inflammation, insulin resistance, cerebrovascular disease and atherosclerosis in relation to the above mentioned diseases.

In general, for a patient weighing about 75 kg, the compounds of the present invention are administered in a daily dose of 0.001mg/kg body weight to 200mg/kg body weight, preferably 1mg/kg body weight to 100mg/kg body weight. The above-mentioned dosage may be administered in a single dosage form or divided into several, e.g., two, three or four dosage forms, which is limited by the clinical experience of the administering physician and the dosage regimen. The compounds or compositions of the present invention may be administered alone or in combination with other therapeutic or symptomatic agents.

The invention has the beneficial technical effects

The invention discovers that andrographolide separated and purified from andrographis paniculata has an action target of LTB4 receptor for the first time. The invention discovers that andrographolide plays a significant role in reducing blood sugar by regulating LTB4 receptors for the first time. The compound has good application prospect in preparing antidiabetic drugs.

Drawings

The drawings in the present application are intended to provide further explanation of the application, and the illustrative embodiments and description of the application are intended to explain the application and are not to be construed as limiting the application.

FIG. 1 preparation of andrographolide ¹ H NMR

FIG. 2 shows the preparation of andrographolide ¹³ C NMR

FIG. 3 MS of Andrographolide

Figure 4. Andrographolide administration for 2 weeks significantly improved ob/ob mouse glucose tolerance. (A) IPGTT (B) area under the blood glucose curve (C) fasting blood glucose (D) body weight. P < 0.05, P < 0.01, P < 0.001VS control group

Figure 5 andrographolide administration improved ob/ob mouse insulin tolerance for 3 weeks. (A) ITT (B) area under the blood glucose curve. P < 0.05, P < 0.01VS control group

FIG. 6 andrographolide improves ob/ob mouse insulin resistance. (A) Blood glycosylated hemoglobin (HbA1C) (B) blood insulin (C) insulin resistance index. P < 0.05, P < 0.01VS control group

Figure 7 andrographolide administration improved lipid disorders for 4 weeks. (A) Total blood cholesterol (B) low density lipoprotein cholesterol (C) blood triglyceride (D) liver index. P < 0.05, P < 0.01VS control group

Figure 8 andrographolide administration for 4 weeks was shown to improve kidney function in ob/ob mice. (A) Cardiac index (B) blood creatinine. P < 0.05, P < 0.01VS control group

Detailed Description

To further illustrate the present invention, the following detailed description of the invention is given by way of specific examples, which are purely illustrative and intended to describe the invention in detail and not intended to limit the exemplary embodiments according to the application.

Example 1

The preparation method of andrographolide comprises the following steps:

(1) pulverizing 10.0kg of herba Andrographitis, extracting with 65% ethanol at 50 deg.C for 3 times (100L × 3 times) each for 2 hr, and recovering solvent from extractive solution at 40-50 deg.C under reduced pressure to 1/10 (30L) to obtain herba Andrographitis crude extract.

(2) Standing 60L of crude extract of Andrographis paniculata Nees, separating out crystals, and filtering to obtain andrographolide; further dissolving, decolorizing and recrystallizing to obtain 120 g of andrographolide.

(3) Subjecting 5L of crude extract of herba Andrographitis with andrographolide separated out to MCI column chromatography (diameter 15 cm, length 70 cm), eluting with 30% and 50% ethanol, and eluting two column volumes per gradient at flow rate of 120 ml/min; 1/10(1.2 liters) was added in one portion by volume to the column for a total of 40 portions.

(4) Mixing the fractions 26-62 eluted by 50% ethanol, concentrating, standing, and separating out 21 g of andrographolide.

And (3) structural identification: the structure of the separated compound is determined by methods such as nuclear magnetic resonance hydrogen spectrum, nuclear magnetic resonance carbon spectrum, mass spectrum and the like.

Andrographolide: white crystals, HRESI-MS M/z [ M + H ]] ⁺ 351.21616, it is suggested that its molecular formula is C ₂₀ H ₃₀ O ₅ 。 ¹ H NMR(600MHz,CD ₃ OD)δ _H 6.83(1H,t,J＝6.8Hz,H-12),4.99(1H,d,J＝5.8Hz,H-14),4.88(1H,s,H-17a),4.65(1H,s,H-17b),4.45(1H,dd,J＝10.5,6.1Hz,H-15a),4.14(1H,d,J＝10.5,2.3Hz,H-15b),4.10(1H,d,J＝11.4Hz,H-19a),3.39(1H,m,H-3),3.35(1H,d,H-19b),2.59(2H,m,H-11),2.41(1H,t,J＝13.1Hz H-7a),2.02(1H,m,H-7b),1.91(1H,d,H-1a),1.81(1H,m,H-2),1.36(1H,m,H-6a),1.29(2H,m,H-9),1.20(3H,s,CH ₃ -18),0.74(3H,s,CH ₃ -20). ¹³ C NMR(150MHz CD ₃ OD)δ _C 37.9(C-1),28.8(C-2),80.7(C-3),43.5(C-4),56.1(C-5),25.0(C-6),38.8(C-7),149.1(C-8),57.2(C-9),39.8(C-10),25.5(C-11),148.6(C-12),129.6(C-13),66.5(C-14),75.9(C-15),172.4(C-16),109.0(C-17),23.2(C-18),64.8(C-19),15.3 (C-20). And documents [ Xiao-HuaWei, Sheng-Jie Yang, Na Liang. chemical Constitutes of Caesalpinia decapetala (Roth) Alston. molecules 2013,18, 1325-; 10.3390/molecules18011325.]The reported data are consistent, and the compound is determined to be andrographolide.

Example 2

Pharmacological experiment: the effect of andrographolide on a LTB4R inhibitor model was determined.

LTB4R is a receptor for leukotriene B4(LTB4) and belongs to the GPCR family. When LTB4 binds to LTB4R, LTB4R recruits the corresponding G-protein G α 16-mediated downstream signaling pathway, resulting in the release of calcium ions from the endoplasmic reticulum into the cytoplasm. The activation of the LTB4R signaling pathway can be determined by detecting a fluorescence signal at 488/525nm, which is emitted by Fluo-8 calcium ion fluorescent chelating agent after binding calcium ions in cytoplasm. When an inhibitor of LTB4R was added, the signaling pathway was not activated and no change in the fluorescence signal was seen.

We treated hamster ovary cancer Cells (CHO) overexpressing LTB4R and G α 16 with DMSO and compound. The degree of calcium signal increase was compared after stimulation with LTB4 by incubating Fluo-8 into the cytoplasm. If the ratio is lower than that of DMSO-treated cells, the compound has a certain inhibitory effect. If it is close to DMSO, no inhibitory effect is indicated. And determining that the compound has no toxicity to cells by using a cell-titer kit, namely proving that the compound has an inhibition effect on LTB 4R.

Experimental sample

Preparation of a tested sample solution: the test sample was andrographolide. An appropriate amount of sample was accurately weighed and prepared into 0.1M stock solution using DMSO for pharmacological activity testing.

Cell line: hamster ovary Carcinoma (CHO) cell

[ test materials and reagents ]:

reagent: fetal bovine serum, DMED medium, DMSO, Fluo-8 NW assay kit, Compound, LTB4, CP-105956

Materials: black 96-well cell culture plate with transparent bottom and 6cm cell culture dish

[ Experimental apparatus ]

CO ₂ Incubator, enzyme mark instrument (with titration function)

1. Experimental methods

(1) LTB4R inhibitor modeling:

1) adherent CHO cells in logarithmic growth phase are selected, trypsinized and inoculated in a 6cm cell culture dish by a DMEM culture solution containing 10% fetal bovine serum. When the degree of cell confluence reached 70-80%, 3ug of LTB4R-pReceiver and 1.5ug of G.alpha.16-pReceiver plasmid were transfected into the cells.

2) 36 hours after transfection, cells were harvested by trypsinization and plated in 96-well black clear-bottomed cell culture plates with 50,000 cells per well resuspended in DMEM medium containing 10% fetal bovine serum at 37 ℃ with 5% CO ₂ Culturing for 12 h.

3) The next day, the culture medium was replaced with DMEM medium containing 0.5% fetal bovine serum, and DMSO, andrographolide at different concentrations, andrographolide, and positive control were added according to design. 37 ℃ and 5% CO ₂ And culturing for 2 h.

4) Add Fluo-8 and incubate for 30 min. Setting an enzyme-labeling instrument, reading a calcium signal in a 6s resting state at 488/525nM, then dropwise adding LTB4 into a cell hole, dropwise adding LTB4 to a final concentration of 400nM, and continuously reading the change of the calcium ion signal after 45s stimulation at 488/525 nM.

(2) Grouping experiments: the experimental group is added with andrographolide with different concentrations, the control group is added with DMSO with the same volume of solvent, and the proportion of the DMSO is not more than 1%.

(3) Detection method of cytotoxicity:

1) a CHO cell line co-expressing LTB4R and ga 16 that can respond to LTB4 was established by the method of step 1) of LTB4R inhibitor modeling.

2) 36 hours after transfection, cells were harvested by trypsinization and plated into 96-well cell culture plates with white transparent bottom at 50,000 cells per well by resuspension in DMEM medium containing 10% fetal bovine serum.

3) Adding DMSO and andrographolide. The final concentration of andrographolide was 10uM and DMSO was added in the same volume. 37 ℃ and 5% CO ₂ The culture was carried out overnight.

4) The freshly prepared Cell-titer chemiluminescent detection reagent was added and the fluorescence value was rapidly read by the microplate reader. If the cell dies, the fluorescence value decreases

2. Results of the experiment

[ calculation of results ]

1) Calcium signal increase coefficient (Ca) after 45s calcium signal maximum/first 6s calcium signal average

LTB4R inhibition ratio (%) (Ca) _DMSO -Ca _{Compound (I)} )/(Ca _DMSO -Ca _{LTB-free 4} )X 100％

2) Compound toxicity calculation:

toxicity of the compound 1-LUM _{Compound (I)} /LUM _DMSO

Through calculation, andrographolide has low toxicity or no toxicity to CHO cells under the concentration of 10 μm, and has obvious inhibiting effect.

The above description is only a preferred example of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement and the like made to the present application within the central idea and principle of the present application should be included in the protection scope of the present application.

TABLE 1 Effect of Compounds on CHO cells in the LTB4R inhibitor model

Example 3

1. The experimental method comprises the following steps:

1.1 animal grouping and administration

30 ob/ob mice are randomly divided into 3 groups according to 3 indexes such as fasting blood sugar, body weight, the percentage of blood sugar reduction of insulin tolerance 40min and the like: control group, positive medicine rosiglitazone group (3mg/kg, oral administration, administration volume 0.1ml/10g) and andrographolide (20mg/kg, 0.2ml/10g body weight intraperitoneal injection administration), 10 of each group were administered once a day, and after 2 weeks of continuous administration, intraperitoneal injection glucose tolerance test (IPGTT) was performed. An Insulin Tolerance Test (ITT) was performed 3 weeks after continuous administration. The following materials were drawn after four consecutive administrations, and the heart, liver, blood Total Cholesterol (TC), low density lipoprotein cholesterol (LDL-C), Triglyceride (TG), glycosylated hemoglobin (HbA1C), insulin and creatinine were measured to calculate the insulin resistance index.

1.2 blood glucose testing

The test is carried out by using an ACCU-CHEKPorma of a Roche excellence type blood glucose meter and a matched test strip.

1.3 IPGTT

Animals are fasted for 6h (administration is carried out when the animals are fasted for 5 h), blood sugar is measured for 0min, glucose solution (1g/kg) is administered to abdominal cavity, blood is taken from tail tip to measure blood sugar load, and blood sugar is measured for 15min, 30min, 60min, 90min and 120 min.

1.4 ITT

Animals are fasted for 6h (administration is carried out when fasted for 5 h), blood sugar is measured for 0min, insulin (0.4IU/kg) is injected subcutaneously, blood is taken from tail tips, and blood sugar is measured for 15min, 30min, 60min, 90min and 120min after the insulin is injected.

1.5 TC detection

A10-fold diluted blood sample was taken from the mouse and measured according to the instructions of the kit (Zhongsheng Bei Zhi Biotech Co., Ltd.).

1.6 LDL-C assay

1.7 TG detection

1.8 HbA1c detection

EDTA anticoagulated whole blood was taken and then measured according to the instructions of the kit (Beijing Haimai bioengineering Co., Ltd.).

1.9 insulin detection

Mouse plasma was diluted 2-fold and then assayed according to kit instructions (ALPCO).

1.10 Creatinine assay

EDTA anticoagulated whole blood was taken and then measured according to the instructions of a kit (Zhongsheng Bei Zhi Biotech Co., Ltd.).

1.11 insulin resistance index calculation

HOMA-IR ═ fasting blood glucose (mmol/l) × fasting insulin (uU/ml)/22.5.

2. Results of the experiment

2.1 As shown in figure 4, after 2 weeks of intraperitoneal administration, the positive drug rosiglitazone significantly improved the glucose tolerance of ob/ob mice, and compared with the control group, andrographolide (20mg/kg) also significantly reduced fasting blood glucose and glucose level after glucose loading of ob/ob mice, improved glucose tolerance, significantly reduced area under the blood glucose curve, and no significant effect on the body weight of mice after 2 weeks of administration.

2.2 As shown in figure 5, after 3 weeks of intraperitoneal administration, 20mg/kg of andrographolide significantly reduced fasting blood glucose in ob/ob mice, improved insulin tolerance in mice, and significantly reduced area under the blood glucose curve compared with the control group.

2.3 As shown in FIG. 6, andrographolide significantly reduced glycosylated hemoglobin levels, as well as hyperinsulinemia and insulin resistance index in ob/ob mice, after 4 weeks of administration. And these effects are comparable to the positive drug rosiglitazone.

2.4 As shown in FIG. 7, the positive drug rosiglitazone significantly increased the blood Total Cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) levels in ob/ob mice, while andrographolide did not increase LDL-C and also decreased TC. The positive drug rosiglitazone reduces Triglyceride (TG) but increases liver weight significantly, suggesting increased liver lipid synthesis; andrographolide improves triglyceride without increasing liver weight.

2.5 rosiglitazone increased cardiac index in ob/ob mice as shown in FIG. 8, suggesting that rosiglitazone increased edema and cardiac hypertrophy. While andrographolide did not increase the cardiac index of ob/ob mice. Compared with rosiglitazone, andrographolide can also obviously reduce the blood creatinine level of ob/ob mice, and indicates that andrographolide improves the renal function of ob/ob mice.

3. Conclusion of the experiment

(1) The andrographolide administration for 2-4 weeks significantly improved ob/ob mouse insulin resistance. Improving glucose tolerance improves insulin tolerance, and decreases the hyperinsulinemia and insulin resistance index in ob/ob mice.

(2) The andrographolide can be administered for 2-4 weeks to significantly reduce fasting blood glucose of diabetic mice.

(3) Andrographolide administration did not have a significant effect on body weight for 2-4 weeks.

(4) Unlike the positive medicine rosiglitazone, andrographolide can improve lipid disorders of ob/ob mice and reduce cholesterol and triglyceride.

(5) Unlike the positive medicine rosiglitazone, andrographolide does not increase ob/ob cardiac index, and can improve renal function of diabetic mice.

Claims

1. the application of andrographolide shown in the following formula and its pharmaceutically acceptable salt in the preparation of LTB4 receptor inhibitor;

2. the application of andrographolide shown in the following formula and its pharmaceutically acceptable salt in the preparation of medicines for preventing or treating obesity, insulin resistance, abnormal glucose tolerance, hyperlipidemia, diabetes and its complications;

3. The application according to claim 2, wherein the diabetic complications include diabetic nephropathy, diabetic cardiovascular complications, diabetic cerebrovascular diseases, diabetic eye complications or diabetic foot.

4. the application of a pharmaceutical composition in the preparation of LTB4 receptor inhibitor, it is characterized in that, described pharmaceutical composition comprises andrographolide of effective dose and pharmaceutically acceptable salt or pharmaceutically acceptable carrier thereof or excipients.

5. a preparation method of extracting, separating and purifying and obtaining the andrographolide of claim 1 from Andrographis paniculata medicinal material, the method comprises the following steps:

(1) After crushing the aerial part of Andrographis paniculata, extract with 65% ethanol for 3 times at 50°C for 2 hours each time, and recover the solvent under reduced pressure at 40-50°C to 1/10 of the extract to obtain a crude extract of Andrographis paniculata;

(2) andrographis paniculata crude extract is left standstill, separate out crystallization, filter; After further dissolving and decolorizing recrystallization, obtain andrographolide;

(3) MCI column chromatography was carried out on the crude extract of Andrographis paniculata that precipitated andrographolide, eluted with 30% and 50% ethanol, two column volumes for each gradient elution, and 1/10 of the column volume was regarded as a fraction , a total of 40 copies were received;

(4) merge the fractions eluted with 30% ethanol, leave standstill after concentrating, and separate out to obtain andrographiscoside;

(5) The fractions eluted with 50% ethanol were combined, concentrated and left to stand for precipitation to obtain andrographolide.

6. The method according to claim 5, wherein in step (1), the ratio of the weight of the medicinal material of Andrographis paniculata to the weight of the medicinal material of 65% ethanol used each time to the volume of the solvent is 1:10.