TW201416075A - Use of jenstadin-analogs for improving feed-efficiency and reducing feeding-rate and adipose-tissue weight of obesity animal - Google Patents

Abstract

The synthesized piperazium salt of disclosed in the present invention is characterized by presented pharmaceutics having functions to improve feed-efficiency and to reduce feeding-rate and adipose tissue weight of obesity animal.

Description

Jianzhiling compound increases meat exchange rate, reduces feeding rate and obesity in obese animals Organizational benefits

The invention relates to a Jianzhiling compound or a Jianzhiling compound compound, and the invention is applied to an animal, and the invention has recently found that the blood fat content and the obesity state can be improved, in particular, the reduction of the fat tissue excess, the increase of the meat exchange rate and the reduction of the feed intake. Reduce the weight of obese tissue and mix the potency of the above state-related patterns.

KMUP-1, which is modified on the seventh nitrogen base of the xanthine derivative with theophylline as the skeleton, activates the endothelial nitric oxide synthase (eNOS) of the epithelium and endothelium, and partially activates the smooth muscle soluble guanylate ring. Inhibition of soluble guanylyl cyclase (sGC) and phosphodiesterase (PDE). KMUP-1 has been shown to affect cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) and Cyclic guanosine monophosphate (cGMP)/protein kinase G ( PKG) and other pathways, and will increase the production of nitric oxide in tracheal epithelial cells, thereby activating sGC, KMUP-1 or directly activating sGC in tracheal smooth muscle cells, and increasing the amount of cGMP to activate PKG.

The inventor is equal to the 2004 British journal of pharmacology reported that KMUP-1 also activates adenylate cyclase (AC) to increase the amount of cAMP and activate PKA. Both PKA and PKG cause smoothing of potassium channel in smooth muscle cells. Finally, the tracheal smooth muscle is relaxed. cAMP and cGMP are intracellular secondary messengers, and regulate a variety of physiological responses, including cell growth and differentiation, apoptosis, glycolysis and esterification, immune and inflammatory reactions. The study reports that KMUP-1 not only induces endogenous nitric oxide release, but also has similar supply of nitric oxide (NO). Pharmacological action of donor). Therefore, in the past, an anti-hypertensive, 095112923 treatment of prostate hypertrophy, 094129421 anti-pulmonary hypertension, and a US 12/878451 composite salt application of the invention application No. 096121950 were proposed.

The piperazine group of the KMUP compound can be prepared by chemical synthesis, using mineral acids, organic acids, and statin derivatives containing carboxylic acid groups, anti-inflammatory drugs, prostacyclin, and anti-asthmatic drugs. Become a KMUP compound.

The inventors have conceived a KMUP composite compound prepared by chemical synthesis using a KMUP-like compound or piperazine. The synthesis reaction of the KMUP composite compound can be carried out by mixing a KMUP compound with a mixed solution of a C1-C4 low alcohol and water, and a sufficient amount of mineral acid to form a quaternary ammonium salt. In addition, it is a mineral acid or organic acid of a KMUP compound mixed with a mixed solution of a C1-C4 low alcohol and water, and the amount of the KMUP compound is considered to be sufficient for the reaction of the "RX" group such as Statin in the mixed solution. a carboxylic acid derivative, an ester derivative of Statin, a derivative of a protective group statin, an anti-inflammatory drug, prostacyclin, and a carboxylic acid group-containing reactant such as anti-asthmatic drugs Montelukast, Cromolyn sodium, Nedocromil, etc. According to the nature of the water and the reaction temperature, select the C1-C4 low alcohol and adjust the amount of the mixed solution. The preferred choice is ethanol, isopropanol with 5%-30% moisture, 10% moisture with 90% ethanol or Isopropyl alcohol. In the reaction of the alkaline catalyst to hydrolyze the ester derivative of Statin, the amount of the ester derivative of Statin is added to the mixed solution, and is about 10 mmol to 1 mol per liter. The mixed solution is refluxed to accelerate the reaction. The temperature of the mixed solution should be raised to about 40 ° C to 70 ° C to form KMUP-1 KMUP composite compound. After filtration, it should be dissolved again in the mixed solution, preferably at room temperature. Recrystallization is carried out. The above mineral acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid (H ₃ PO ₄ ), sodium dihydrogen phosphate (NaH ₂ PO ₄ ), disodium hydrogen phosphate (Na ₂ HPO ₄ ), and the like. . The organic acid is selected to include citric acid, glycyrrhizic acid, fumaric acid, maleic acid, nicotinic acid, isonicotinic acid, tartaric acid, succinic acid, adipic acid, fatty acid, methanesulfonic acid, benzene. Oxyvaleric acid and the like. All of them are disclosed in the national patent application filed on January 29, 2010, with the number 099102735.

In addition, the tea contains caffeine of the formula (A) and theophylline of the formula (B). For example, Patent Publication No. 201201794 uses a composition formed of caffeine, theophylline and other compounds. Lipolysis.

Chen N. et al. reported lipolysis of caffeine and activation of Hormone sensitive lipase (HSL), peroxisome proliferator-priming receptor-gamma (PPAR-γ), and thermogenic effects ( Thermogenesis) is associated with lipid metabolism (Nutr Res. 2009 Nov; 29(11): 784-93).

In view of the incompleteness of the prior art, the inventors have carefully experimented and researched, and have a perseverance spirit to finally conceive the case that "Jianzhiling compounds can increase the meat exchange rate and reduce the benefits of obesity". The shortcomings of the prior art, the following is a brief description of the case.

According to its concept, in a suitable embodiment, the effective amount of the main component is selected from the group consisting of the Jenstadin-analogs compound of the formula (I) or the Jenstadin-analogs complex compound of the formula (II). The above-mentioned improved obesity state can be exhibited by adding an appropriate amount of excipients, treating the prepared composition by a preparation method, and administering the various dosage forms in the animal through a suitable manner, in particular, the blood fat content, the fat tissue content, and the meat change. The rate increases or decreases the amount of feed intake, obesity tissue, and the potency of the relevant patterns that mix the above states. The animal, including humans, and birds, animals, and fish for meat. Such as chickens, ducks, geese, turkey poultry, such as pigs, cattle, deer, horses, sheep livestock or mountain pigs, dogs, rabbits, such as crickets, pigeons, ostrich birds or squid, Shiban fish, Wu Guoyu fish and so on. In order to reduce fat tissue and increase lean meat for veterinary animals, it will be beneficial to provide animal meat with less fat to consumers. Adding the Jianzhiling compound as an additive to food and feed, resulting in the feed consumption of the farm's streamlined feeding animal, is beneficial to the economical effect of the farm's operating expenses.

According to its concept, the disubstituted piperazine analogs are represented by the formula of the formula (I), wherein R ₂ and R ₄ are each selected from the group consisting of hydrogen groups and halogens. a substituent of an amine group or a nitro group; a substituent of a C 1-5 alkyl group; a substituent of a C 1-5 alkoxy group.

In a suitable embodiment, the Jianzhiling compound is a Jianzhiling compound such as Jianzhiling-1, Jianzhiling-2, Jianzhiling-3 and Jianzhiling-4. The description of the present invention is directed to the description of the present invention, and the nutrient-free compound or the Jenstadin-analogs complex compound is not specifically described.

The inventors further conceived that the lipid-lowering compound can be chemically synthesized with a compound having a carboxylic acid group structure such as a Statin drug, a sodium carboxymethylcellulose, a high molecular polymer or a polyglutamic acid group derivative. Prepared as a Jenstadin-analogs complex compound of formula (II).

According to the concept, a Jianzhiling compound has a structure represented by the formula (II), wherein R ₂ and R ₄ are each independently selected from the group consisting of a hydrogen group, a halogen, an amine group, and a nitro group. a substituent; a substituent having a carbon number of 1-5 alkyl; a substituent having a carbon number of 1 to 5 alkoxy; and RX is selected from the group consisting of carboxylic acid groups: Statin, carboxymethyl A sodium CMC, a high molecular polymer or a polyglutamic acid derivative derivative drug; and RX ^- may be a negatively charged anion of the above group.

In a suitable embodiment, the Jianzhiling compound is a statin drug, carboxylate of Jianzhiling-1, Jianzhiling-2, Jianzhiling-3 and Jianzhiling-4 and a carboxylic acid group-containing derivative. A compound of the compound which is synthesized by a compound such as sodium carboxylate (sodium CMC), a high molecular polymer, a polyglutamic acid derivative derivative drug, and a mixed form thereof.

The compound of the formula (I) or the Jianzhiling compound of the formula (II), wherein the Jianzhiling compound can be represented by Jianzhiling-1, Jianzhiling-2, Jianzhiling-3, and Jianjian Zhiling-4 and Jianzhiling-5, etc., such as (7-2-4-(2-chlorophenyl) piperazinyl]ethyl]-1,3-dimethylxanthine (7-[2-[ 4-(2-chloro-phenyl)piperazinyl]-ethyl]-1,3-dimethylxanthine, Jianzhiling-1), Jianzhiling-2 is 7-2-4-(2-methoxyphenyl)piperazine Ethyl]-1,3-dimethylxanthine (7-[2-[4-(2-methoxybenzene)-piperazinyl]ethyl]-1,3-dimethylxanthine), Jianzhiling-3 is 7- [2-[4-(4-Nitrophenyl)piperazinyl]ethyl]-1,3-dimethylxanthine (7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1 , 3-dimethyl-xanthine); Jianzhiling-4 is 7-[2-[4-(phenyl)piperazinyl]ethyl]-1,3-dimethylxanthine (7-[2-[4 -benzene piperazinyl]-ethyl]-1,3-dimethylxanthine); and Jianzhiling-5 is 7-[2-[4-(2-fluorophenyl)piperazinyl]ethyl]-1,3-dimethyl 7-[2-[4-(2-fluorobenzene)piperazinyl]-ethyl]-1,3-dimethylxanthine).

The RX group of the above formula (II) may be selected from the group consisting of Statins, sodium carboxylate (sodium CMC), high molecular weight polymer (Co-polymers), and polyglutamic acid (γ-). A PGA) group derivative and a mixed type thereof, such as a carboxylic acid group-containing drug, ^- RX may be a negatively charged anion of the above group, and the halogen may be a group such as fluorine, chlorine, bromine or iodine. In a suitable embodiment, the Statin-containing drug containing a carboxylic acid group, if necessary, is a commercially available statin-containing drug having a carboxylic acid group, including atorvastatin (Atorvastatin), cerivastatin. (Cerivastatin), Fluvastatin, Lovastatin, Mevastatin, Pravastatin, Rosuvastatin, Simvastatin and their Mixed type. Co-polymers, if necessary, high molecular weight polymeric molecules containing carboxylic acid groups, including hyaluronic acid, polyacrylic acid, polymethacrylates, and Eudragit, dextran sulfate, heparan sulfate, polylactic acid or polylactide (PLA), polyglycolic acid (PGA), sodium polylactic acid ( Polylactic acid sodium (PLA sodium), polyglycolic acid sodium (PGA sodium) and a mixed version thereof. Hyaluronic acid, also known as uronic acid, is a high molecular polymer composed of D-glucuronic acid and N-Acetyl-D-Glucosamine (NAG) units. Polymethacrylates (PMMA) are high molecular weight polymers of methacrylic acid, and Eudragit is a product of polymethacrylates. Glucan sulfate and heparin sulfate are polysaccharide molecules polymerized by sulfate groups and mixed forms thereof.

In a suitable embodiment, a carboxylic acid group-containing poly-γ-polyglutamic acid (γ-PGA) group derivative, if necessary, is a carboxylic acid group-containing sodium alginate (alginate sodium). ), poly-γ-polyglutamic acid (γ-PGA), poly-γ-polyglutamic acid sodium (γ-PGA sodium) or polylysine and sea Alginate-poly-lysine-alginate (APA), polylactic acid sodium (PLA sodium), polyglycolic acid sodium (PGA sodium) and mixed forms thereof.

According to its concept, the Jianzhiling compound is selected from a drug containing a carboxylic acid group such as a Statin drug, sodium carboxymethylcellulose (sodium CMC), a polymeric drug, and a polyglutamic acid derivative drug. Mixing, or by synthesizing the Jianzhiling compound compound of formula (II), can increase the meat exchange rate of animals, reduce the feeding rate of animal feed and obesity tissues of animals.

According to the above concept, the Jianzhiling compound of the formula (II), wherein the reacted RX group is added in a unit molar amount, synthesizes a composite compound synthesized from the above formula (II) and a monovalent carboxylic acid group. On the basis of the amount of the reaction acid and the steric bond, if the amount of RX participating in the reaction is sufficient, it may be two or more moles, and the dimer carboxylic acid group represented by the formula (III) may be present. a composite compound.

The medicinal composition of the saponin compound can be used as a pharmaceutical composition by adding a proper amount of the excipient, and can be processed into various dosage forms suitable for administration to mammals by the preparation method, and the animal meat exchange rate is increased. Reduce the feeding rate of animal feed and animal obesity and other functions.

2-chloroethyl theophylline, 2-chlorobenzene 2-chlorophenyl piperazine was dissolved in an alkaline solution of hydrous ethanol as a percentage of molecular weight and heated for 3 hours. After standing overnight, the supernatant was decanted, concentrated under reduced pressure to remove the solvent, and then dissolved in 1 volume of ethanol and 3 times by volume of 2N hydrochloric acid (HCl), placed in a water bath at 50 to 60 ° C to form a pH 1.2 saturation. Solution. It was decolorized by activated carbon, filtered, allowed to stand overnight, and filtered to obtain white crystals of Jianzhiling-1 HCl.

2-Chloroethyl theophylline and 4-nitrophenylpiperazine were dissolved in an aqueous ethanol solution according to the molecular weight and heated under reflux for 3 hours. After cooling overnight, the supernatant was decanted, concentrated and dried under reduced pressure, and then a 1 volume of ethanol and 3 times by volume of 2N hydrochloric acid were added to dissolve in a water bath at 50 to 60 ° C to a saturated solution of pH 1.2. The yellow crystal of Jianzhiling-3 HCl can be obtained by decolorizing activated carbon, filtering, placing overnight, and filtering.

2-chloroethyl theophylline, 2-methoxybenzene piperazine is dissolved in a solution of alkaline solution of hydrous ethanol according to the molecular weight and heated Reflux for 3 hours. After standing overnight, the supernatant was decanted, concentrated under reduced pressure to remove the solvent, and then dissolved in 1 volume of ethanol and 3 times by volume of 2N hydrochloric acid (HCl), placed in a water bath at 50 to 60 ° C to form a pH 1.2 saturation. Solution. It was decolorized by activated carbon, filtered, allowed to stand overnight, and filtered to obtain white crystals of Jianzhiling-2 HCl.

2-Chloroethyl theophylline and phenyl piperazine were dissolved in an alkaline solution of hydrous ethanol as a percentage of molecular weight and heated and refluxed for 3 hours. After standing overnight, the supernatant was decanted, concentrated under reduced pressure to remove the solvent, and dissolved in 1 volume of ethanol and 3 times by volume of 2N hydrochloric acid (HCl), and placed in water at 50 to 60 ° C. The bath formed a saturated solution of pH 1.2. It was decolorized by activated carbon, filtered, allowed to stand overnight, and filtered to obtain white crystals of Jianzhiling-1 HCl.

2-Chloroethyl theophylline and 2-fluorophenyl piperazine were dissolved in an alkaline solution of hydrous ethanol in a percentage of molecular weight and heated for 3 hours. After standing overnight, the supernatant was decanted, concentrated under reduced pressure to remove the solvent, and then dissolved in 1 volume of ethanol and 3 times by volume of 2N hydrochloric acid (HCl), placed in a water bath at 50 to 60 ° C to form a pH 1.2 saturation. Solution. It was decolorized by activated carbon, filtered, allowed to stand overnight, and filtered to obtain white crystals of Jianzhiling-1 HCl.

Sodium carboxylate (sodium CMC) or high molecular polymer, polyglutamic acid salt is dissolved in an alkaline solution, and added with Jianzhiling or its hydrochloride is placed at 50 to After 70 ° C water bath reaction, add ethanol at room temperature overnight to carry out crystallization, and filter to obtain Jianzhiling-carboxymethyl cellulose Jianzhiling compound compound, Jianzhiling-polymer or Jianzhiling-poly A glutamic acid group complex compound. The above alkaline solution is selected from the group consisting of sodium hydroxide (NaOH) or sodium hydrogencarbonate (NaHCO ₃ ).

According to the above concept, the Jianzhiling compound of the formula (II) of the present invention is a carboxylic acid group-containing structure selected from the group consisting of a Statin drug, a sodium carboxymethylcellulose, a high molecular polymer or a polyglutamic acid group derivative. The drug is suitable for improving the blood fat content and the obesity state and the weight of the body weight imbalance. Specifically, it is a compound of Jianzhiling-1-atorvastatin, Jianzhiling-2-atorvastatin compound, Jianzhiling-3-atorvastatin compound, Jianzhiling-4-A Atorvastatin compound, Jianzhiling-5-atorvastatin compound compound; Jianzhiling-1-sivaratin compound, Jianzhiling-2-sivaratin compound, Jianzhiling-3-sivaratin compound, Jianzhiling-4-sivarstatin compound Compound, Jianzhiling-5-cevastatin compound; Jianzhiling-1-fluvastatin compound, Jianzhiling-2-fluvastatin compound, Jianzhiling-3-fluvastatin compound , Jianzhiling-4-fluvastatin compound, Jianzhiling-5-fluvastatin compound; Jianzhiling-1-Rovastatin compound, Jianzhiling-2-Rovastatin compound , Jianzhiling-3-Rovastatin compound compound, Jianzhiling-4-Rovastatin compound compound, Jianzhiling-5-Rovastatin compound compound; Jianzhiling-1-Mervastatin compound Compound, Jianzhiling-2-Mevastatin compound compound, Jianzhiling-3-Mevastatin compound compound, Jianzhiling-4-Mevastatin compound compound, Jianzhiling-5-Mevastatin compound compound; Jianzhiling-1-Pravastatin Compound, Jianzhiling-2-Pravastatin Compound, Jianzhiling-3-Pravastatin Compound, Health Ling-4-Pravastatin Compound Compound, Jianzhiling-5-Pravastatin Compound Compound; Jianzhiling-1-Shushustatin Compound Compound, Jianzhiling-2-Shushustatin Compound Compound, Jianzhiling-3-jianshurustatin compound, Jianzhiling-4-jianshurustatin compound; Jianzhiling-1-simvastatin compound, Jianzhiling-2-simvastatin compound Compound, Jianzhiling-3-simvastatin compound, Jianzhiling-4-simvastatin compound, Jianzhiling-5-simvastatin compound; Jianzhiling-1-carboxymethyl cellulose composite Compound, Jianzhiling-2-carboxymethylcellulose composite compound, Jianzhiling-3-carboxymethylcellulose composite compound, Jianzhiling-4-carboxymethylcellulose composite compound, Jianzhiling-5- Carboxymethyl cellulose composite compound; Jianzhiling-1-hyaluronic acid compound compound, Jianzhiling-2-hyaluronic acid compound compound, Jianzhiling-3-hyaluronic acid compound compound, Jianzhiling-4-hyaluronic acid compound compound, fat Ling-5-hyaluronic acid composite compound; Jianzhiling-1-polyacrylic acid compound compound, Jianzhiling-2-polyacrylic acid compound compound, Jianzhiling-3-polyacrylic acid compound compound, Jianzhiling-4-polyacrylic acid compound Compound, Jianzhiling-5-polyacrylic acid composite compound; Jianzhiling-1-polymethacrylate compound, Jianzhiling-2-polymethacrylate compound, Jianzhiling-3-polymethyl Acrylate compound, Jianzhiling-4-polymethacrylate compound, Jianzhiling-5-polymethacrylate compound; Jianzhiling-1-Youtech compound, Jianzhiling-2 - Utech compound, Jianzhiling-3-Youtech compound, Jianzhiling-4-Youtech compound, Jianzhiling-5-Youteqi compound; Jianzhiling-1-polymer Lactic acid complex compound, Jianzhiling-2-polylactic acid composite compound, Jianzhiling-3-polylactic acid composite , Jianzhiling-4-polylactic acid composite compound, Jianzhiling-5-polylactic acid composite compound; Jianzhiling-1-polyglycolic acid compound compound, Jianzhiling-2-polyglycolic acid compound compound, fat Ling-3-polyglycolic acid composite compound, Jianzhiling-4-polyglycolic acid composite compound, Jianzhiling-5-polyglycolic acid composite compound; Jianzhiling-1-sulfate dextran compound compound, Jianzhiling -2-sulfate dextran compound, Jianzhiling-3-sulfate dextran compound, Jianzhiling-4-sulfate dextran compound, Jianzhiling-5-sulfate dextran compound; Lipid-1-sulfate heparin heparin compound, Jianzhiling-2-acetic acid heparin heparin compound, Jianzhiling-3-sulfate heparin heparin compound, health Lipid-4-sulfate heparin heparin compound, Jianzhiling-5-acetate heparin compound; Jianzhiling-1-alginate compound, Jianzhiling-2-alginate compound, health Zhiling-3-alginate compound compound, Jianzhiling-4-alginate compound compound, Jianzhiling-5-alginate compound compound; Jianzhiling-1-polyglutamic acid compound compound, fat Ling-2-polyglutamic acid composite compound, Jianzhiling-3-polyglutamic acid composite compound, Jianzhiling-4-polyglutamic acid composite compound, Jianzhiling-5-polyglutamic acid composite compound; Jianzhiling-1-polyglutamate complex compound, Jianzhiling-2-polyglutamate complex compound, Jianzhiling-3-polyglutamate complex compound, Jianzhiling-4-polygluten Sodium amide complex compound, Jianzhiling-5-poly glutamate compound compound; Jianzhiling-1-polyalginate compound compound, Jianzhiling-2-polyalginate compound compound, Jianzhiling- 3-polyalginate compound compound, Jianzhiling-4-polyalginate compound compound, Jianzhiling-5-polyalginate compound compound and the like.

The alkyl group is a monovalent saturated hydrocarbon radical which is bonded to a carbon atom in a single chain, and the hydrocarbon may form a straight-chain, branched or cyclic. The "carbon number C1-C5 alkyl group" is an alkyl group having 1 to 5 carbon atoms, preferably the C1-C5 alkyl group is a methyl group, an ethyl group or an n-propane group ( N-propyl), isopropanyl (isopropyl), n-butyl, iso-butyl, sec-butyl, tert-butyl ), n-pentyl, iso-pentyl, tert-pentyl, neo-pentyl.

Alkoxy is a hydrocarbon group substituted with a single oxygen atom One of the carbon atoms of the alkyl group. The C1-C5 alkoxy group has a carbon number, preferably a methoxyl group, an ethoxyl group, an n-propoxyl group, or an isopropoxyl group. N-butoxyl, iso-butoxyl, sec-butoxyl, tert-butoxyl, n-pentaneoxy ( N-pentoxyl), iso-pentoxyl, tert-pentoxyl.

The Jianzhiling compound is a xanthine derivative based on theophylline, which synthesizes the theophylline containing the substituent and the piperazine containing the substituent to form caffeine. A modified compound of the theophylline bulk structure. The lipid-lowering compound activates endothelial nitric oxide synthase (eNOS) in the epithelium and endothelium, partially activates soluble guanylyl cyclase (sGC) and phosphodiesterase (PDE) inhibition. effect. It has been confirmed that the lipid-lowering compounds can affect cyclic adenosine monophosphate (cAMP)/protein kinase (PKA) and Cyclic guanosine monophosphate (cGMP)/protein kinase. G (PKG) and other pathways, and will increase the production of nitric oxide in tracheal epithelial cells, thereby activating sGC in tracheal smooth muscle cells, and directly activate sGC in tracheal smooth muscle cells, so that the amount of cGMP is increased to activate PKG. It can also activate adenylate cyclase (AC) to increase the amount of cAMP and activate PKA. Both PKA and PKG will cause the potassium channel of smooth muscle cells to open, and finally relax the smooth muscle of the trachea. cAMP and cGMP are intracellular secondary messengers, and regulate a variety of physiological responses, including cell growth and differentiation, apoptosis, glycolysis and esterification, immune and inflammatory reactions.

RhoA/ROCK and subsequent Peroxisome Proliferative Activated Receptor-γ (PPAR-γ) are involved in the increase of high-density lipoprotein (HDL). Both Statins and statins can rely on the NO/cGMP pathway to inhibit the major mechanisms by which RhoA/ROCK exhibits Pleiotropic effects. Rho-associated protein kinase (ROCK) has become another new focus of its pathogenesis.

The Jianzhiling compound not only induces endogenous nitric oxide release, but also has a pharmacological effect similar to the supply of nitric oxide. eNOS is the main source of endothelium-derived nitric oxide (NO), which involves RhoA/ROCK, which can increase the animal meat exchange rate, reduce the feed rate of animal feed and the obesity of animal body. Recent evidence suggests that statins induce eNOS expression in vascular endothelial cells to improve endothelial function in blood vessels, and inhibition of HMGR leads to an increase in eNOS mRNA genes. Reducing the Rho GTPase protein response increases the production and bioavailability of endothelial-derived NO, and regulates eNOS via Rho GTPases protein as an important mechanism for protecting cardiovascular function. The chemical structure of the Jianzhiling compound based on the Astragalus structure is different from that of the statins. We infer that the increase of eNOS/cGMP and the reduction of RhoA/ROCK are beneficial to the pleiotropic effects of the Jianzhiling compound.

Anti-hyperlipidemia agent can increase high-density lipoprotein (HDL), and low-density lipoprotein cholesterol content constitutes another risk factor for cardiovascular disease; increasing high-density lipoprotein can reverse cholesterol transport pathway To prevent atherosclerosis (Brewer HB; 2004).

Visceral adipose tissues (VAT) can be mainly divided into epididymal fat, retroperitoneal fat and dorsal fat. The lipid-lowering compound can regulate Enos and reduce RhoA/ROCK activity through the recovery of PPARγ, thereby increasing the decomposition of adipose tissue and reducing the weight of adipose tissue and inhibiting obesity.

The animal obesity state and the fatty liver disease are caused by the secretion of free fatty acids after being decomposed by the obese state of the visceral fat tissue (VAT), resulting in an abnormal state. However, due to the obesity of animals fed with high-fat diet (HFD), the stimulation of beta-agonist stimulates the excessive decomposition of obese tissue, which will cause hydrolysis of intracellular triglycerides. And increasing the amount of accumulated fatty acids via phosphorylated hormone-sensitive lipase (p-HSL), forcing the fatty acid secretions to move out of the cell, thereby reducing the volume of adipose tissue.

The Jianzhiling Compound (I) or the Jianzhiling Compound of the formula (II) was tested to exhibit the activity shown below.

1. Jianzhiling-1 and Simvastatin affect the weight and feed intake of mice.

The body weight and feed intake of the rats fed for 8 weeks were recorded every 3 days as shown in Table 1. The increase in body weight of the mice fed the high-fat diet was approximately 2.3 times higher than that of the standard feed (STD) control group. Administration of Jianzhiling-1 (2.5-5 mg/kg/day) or simvastatin (5 mg/kg/day) can effectively reduce the increase in body weight. In terms of feed intake, mice fed a high-fat diet, regardless of whether they were administered with Jianzhiling-1 or simvastatin, showed a reduced intake compared to the standard fed control group. And dose of 1-5 with Jianzhiling-1 There was no difference between the mg/kg/day or simvastatin (5 mg/kg/day) group and the high-fat diet (HFD) group, indicating that Jianzhiling-1 or simvastatin did not cause the appetite to decrease and decrease in mice. The phenomenon of feed intake. It can be inferred that the effects of Jianzhiling-1 and simvastatin on improving the abnormal effect of hyperlipemia and preventing weight gain are not caused by the decrease in the amount of feed intake by mice.

(Note) 1. Each group of experimental mice, 6 high-fat feed (HFD) 2. Jianzhiling-1-b Jianzhiling-1 dosage 2.5 mg/kg/day, Jianzhiling-1-c The dose of Zhiling-1 was 5 mg/kg/day, the dose of simvastatin was 5 mg/kg/day 3. #P <0.05 compared with standard feed; ^* P <0.05 compared with high fat diet

Effects of Jianzhiling-1 and simvastatin on serum biochemical values of mice with dyslipidemia induced by high fat diet

A. serum total cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL), low density lipoprotein (LDL) for the evaluation of dyslipidemia No biochemical indicators.

Serum biochemical values of mouse heart blood after 8 weeks of feeding as shown in Table 2. Serum total cholesterol (TC), triglyceride (TG), and low-density lipoprotein (LDL) values in the high-fat diet group were significantly higher than those in the control group, while high-density lipoprotein (HDL) was also higher than the standard-feed control group. Slightly higher, it shows that rats fed high-fat diets can indeed induce abnormalities in bleeding fat content.

(Note) 1. Jianzhiling-1-b Jianzhiling-1 dosage 2.5 mg/kg/day, Jianzhiling-1-c Jianzhiling-1 dosage 5 mg/kg/day, 2. Jian Lipid-1-Simvastatinic Acid dosage 5 mg/kg/day, high fat diet (HFD)

High fat diet group induced dyslipidemia in mice, and administration of Jianzhiling-1 can significantly improve hyperlipidemia, and oral administration of Jianzhiling-1 (1-5 mg/kg/day) group and simple Compared with the high-fat diet group, serum total cholesterol (TC), triglyceride (TG), and low-density lipoprotein (LDL) were significantly reduced.

(Note) 1. High Density Lipoprotein Cholesterol (HDL-C) 2. Low density lipoprotein cholesterol (LDL-C), which is significantly different from high fat diet (HFD).

High-density lipoprotein (HDL) serum values, regardless of 1-5 mg/kg/day, were also increased in the Jianzhiling-1 group compared with the simple high-fat diet group. Compared with the simple high-fat diet group, Simvastatin also reduced the serum values of TC, TG, LDL and increased the effect of high-density lipoprotein. The results in Table 3 show that the Jianzhiling compound or Jianzhiling compound and Simvastatin have the effect of improving hyperlipemia.

(Note) Data were presented as mean ± SEM, number of animals (n = 9). HFD = fauna induced by high fat diet; C57BL/6J mice sacrificed at week 23; group A = high fat diet Induced fauna, 1.0 mg/kg/day of Jianzhiling, A-1 (Jianzhiling-1), A-2 (Jianzhiling-2), A-4 (A-4) were administered at the 10th to 23rd week. Jianzhiling-4), A-5 (Jianzhiling-5); Group B = high fat diet-induced fauna, 2.5 mg/kg/day of Zhizhiling, 5 weeks after the 19th week, B -1 (Jianzhiling-1), B-2 (Jianzhiling-2), B-4 (Jianzhiling-4), B-5 (Jianzhiling-5); Group C = induced by high fat diet The fauna was administered with 5.0 mg/kg/day of Jianzhiling, C-1 (Jianzhiling-1), C-2 (Jianzhiling-2) and C-4 (5 weeks after the 19th week). Jianzhiling-4), C-5 (Jianzhiling-5); compared with high fat diet ^* P <0.05, compared with week 10 and week 23 #P<0.05.

Influence weight

As shown in Table 5, the body weight of mice fed a high fat diet showed a significant increase at weeks 19 and 23. The weight of mice fed a high-fat diet showed a significant decrease in the administration of the Jianzhiling compound. With the doses of 1, 2.5 and 5 mg/kg/day, the weight gain at week 17 was significantly different from that at week 10 (P<0.05). Group A (1 mg/kg/day) treatment with Jianzhiling compound, compared with the weight gain at week 23 and week 18, no significant difference. However, group B (2.5 mg/kg/day) and group C (5 mg/kg/day) of the lipid-lowering compound showed significant differences in body weight gain at week 23 and week 18.

(Note) Data were presented as mean ± SEM, number of animals (n = 9). Comparison with high fat diet (HFD) ^* P < 0.05, HFD = fauna induced by high fat diet; C57BL/6J small Rats sacrificed at week 23; group A = high fat diet-induced fauna, and 1.0 mg/kg/day of Zhizhiling, A-1 (Jianzhiling-1), A- 2 (Jianzhiling-2), A-4 (Jianzhiling-4), A-5 (Jianzhiling-5); Group B = high fat diet-induced fauna, 5 weeks after the 19th week 2.5 mg/kg/day of Jianzhiling, B-1 (Jianzhiling-1), B-2 (Jianzhiling-2), B-4 (Jianzhiling-4), B-5 (Body Fat) Ling-5); Group C = fauna induced by high fat diet, administered with 5.0 mg/kg/day of Jianzhiling, C-1 (Jianzhiling-1), C-2, 5 weeks after the 19th week. (Jianzhiling-2), C-4 (Jianzhiling-4), C-5 (Jianzhiling-5).

Feeding rate (Feeding-Rate)

As shown in Table 6, the feeding rate (Feeding-Rate) is the percentage of whole-week mouse feed, and the obese mice were given high-fat diet at the 18th week, and the mice with similar body weight were weighed. There was a significant decrease in the feeding rate at week 22 or week 23. Among them, with the different dosages of the Jianzhiling compound, it showed a significant saving in feed addition, which led to a decrease in the feeding rate of high fat diet.

(Note) Data were presented as mean ± SEM, number of animals (n = 9). Comparison with high fat diet (HFD) ^* P < 0.05, compared with week 18 # P <0.05; data for week 18 For the control group data before administration. HFD = fauna of high fat diet; C57BL/6J mice sacrificed at week 23; group A = high fat diet-inducing fauna, administered with 1.0 mg/kg/day of Jianzhiling from week 19, A -1 (Jianzhiling-1), A-2 (Jianzhiling-2), A-4 (Jianzhiling-4), A-5 (Jianzhiling-5); Group B = induced by high fat diet The fauna, from the 19th week, was administered 2.5 mg/kg/day of Jianzhiling, B-1 (Jianzhiling-1), B-2 (Jianzhiling-2), B-4 (fatty fat). Ling-4), B-5 (Jianzhiling-5); Group C = animal group induced by high fat diet, from the 19th week, respectively, 5.0 mg/kg/day of Jianzhiling, C-1 (Health Zhiling-1), C-2 (Jianzhiling-2), C-4 (Jianzhiling-4), C-5 (Jianzhiling-5).

(Note) Data are presented as mean ± SEM, number of animals (n = 9). Comparison with high fat diet # P < 0.05 HFD = fauna with high fat diet; C57BL/6J mice at week 23 Sacrifice; Group A = high fat diet-induced fauna, from the 19th week on the 1.0 mg / kg / day of Jianzhiling, A-1 (Jianzhiling-1), A-2 (Jianzhiling-2 ), A-5 (Jianzhiling-5); Group B = high fat diet-induced fauna, from the 19th week on the 2.5 mg / kg / day of Jianzhiling, B-1 (Jianzhiling - 1), B-2 (Jianzhiling-2), B-5 (Jianzhiling-5); Group C = fauna induced by high fat diet, which was administered at 5.0 mg/kg/day from the 19th week. Jianzhiling, C-1 (Jianzhiling-1), C-2 (Jianzhiling-2), C-5 (Jianzhiling-5); Jianzhiling and high fat diet ^** P < 0.01.

Two Epididymal Fat Pads-Weight/Body-Weight Ratio ratio

As shown in Table 7, the ratio of the two epididymal Fat Pads-Weight/Body-Weight Ratio decreased during the 5 weeks indicated by Jianzhiling, indicating that Jianzhiling can inhibit the proliferation of body fat.

Feed-efficiency

The meat change rate is the ratio of the weight gain (g) of the experimental week to the food intake of the high fat feed. As shown in Table 8, the 14th week of the 14th week, the 23rd week, the injection of Jianzhiling, showing that Jianzhiling can increase the meat exchange rate.

Table 8 Comparison of the meat exchange rate of the high fat feed at the 23rd week with the treatment group and the 14th week

(Note) The data were presented as mean ± SEM, the number of animals (n=8). The comparison between the meat exchange rate of the high fat feed at the 23rd week and the meat change rate at the 14th week, #P <0.05; Comparison of treatment groups, ^* P <0.05 HFD = fauna of high fat diet; C57BL/6J mice weighed weekly increase in body weight of mice per week at week 14, 18, 23 / amount of feed consumed per mouse per week (Feed-efficiency) A-1 (Jianzhiling-1) group = high fat diet-induced fauna, 1.0 mg/kg/day of Jianzhiling from week 14 to week 23; A-2 (lipid) Ling-1) group = fauna induced by high fat diet, administered with 2.5 mg/kg/day of Jianzhiling from the 19th week to the 23rd week; B-1 (Jianzhiling-2) group = induced by high fat diet Group of animals, 1.0 mg/kg/day of Jianzhiling from week 14 to week 23; B-2 (Jianzhiling-2) group = high fat diet-induced fauna, from week 19 to week 23 2.5 mg/kg/day of Jianzhiling; C-1 (Jianzhiling-3) group = high fat diet-induced fauna, and 1.0 mg/kg/day of Jianzhiling from week 14 to week 23; C-2 (Jianzhiling-3) group = fauna induced by high fat diet, administered with 2.5 mg/kg/day of Jianzhiling from the 19th week to the 23rd week; D-1 (lipid Ling-3) group = high fat diet-induced fauna, 1.0 mg/kg of Jianzhiling from week 14 to week 23; D-2 (jianzhiling-4) group = high fat diet-induced fauna , from the 19th week to the 23rd week, 2.5 mg/kg/day of Jianzhiling; E-1 (Jianzhiling-3) group = high fat diet-induced fauna, and administered from the 14th week to the 23rd week, 1.0 mg /kg/day of Jianzhiling; E2 (Jianzhiling-5) group = high fat diet-induced fauna, from the 19th week to the 23rd week, 2.5 mg/kg/day of Jianzhiling.

Average daily feed weight (Food Intake) from week 19 to week 23

As shown in Table IX, the daily feed (g) of the high-fat diet at week 23 was higher than that of the treatment group, and the daily feed at the 23rd week of treatment was lower than the daily feed amount at the 19th week.

(Note) The data were presented as mean ± SEM, number of animals (n=8). Comparison of the daily feed volume of the high fat feed at the 23rd week with the daily feed amount of the treatment group at the 23rd week, #P <0.05 HFD = fauna of high fat diet; C57BL/6J mice weighed in the 19th to 23rd week (g); A-1 (Jianzhiling-1) group = high fat diet induced fauna, From the 19th week to the 23rd week, 1.0 mg/kg/day of Jianzhiling; A-2 (Jianzhiling-1) group = high fat diet-induced fauna, from the 19th week to the 23rd week 2.5 mg/kg/day of Jianzhiling; B-2 (Jianzhiling-2) group = high fat diet-induced fauna, 2.5 mg/kg/day of fat from week 19 to week 23 Ling; C2 (Jianzhiling-3) group = high fat diet-induced fauna, from the 19th week to the 23rd week, 2.5 mg/kg/day of Jianzhiling; D2 (Jianzhiling-4) group = faeces induced by high fat diet, 2.5 mg/kg/day of Jianzhiling from the 19th week to the 23rd week; E2 (Jianzhiling-5) group = high fat diet-induced fauna, 19th From the beginning of the week to the 23rd week, 2.5 mg/kg/day of Jianzhiling was administered.

(Note) Data were presented as mean ± SEM, number of animals (n=6); compared with high fat diet ^* P <0.05; control group, standard feed comparison ^# P <0.05; HFD = high fat feed Group of animals; C57BL/6J mice measured vascular thickness at 19th to 23rd week; A-1 (Jianzhiling-1) group = high fat diet-induced fauna, from the 10th week to the 23rd week, 1.0 mg /kg/day of Jianzhiling; A-2 (Jianzhiling-1) group = high fat diet-induced fauna, from the 10th week to the 23rd week, 2.5 mg/kg/day of Jianzhiling; B-2 (Jianzhiling-2) group = high fat diet-induced fauna, 2.5 mg/kg/day of Jianzhiling from the 10th week to the 23rd week; C2 (Jianzhiling-3) group = high fat diet-induced fauna, 2.5 mg/kg/day of Zhizhiling from the 10th week to the 23rd week; D2 (Jianzhiling-4) group = high fat diet-induced fauna, 10th From the beginning of the week to the 23rd week, 2.5 mg/kg/day of Jianzhiling; E2 (Jianzhiling-5) group = high fat diet-induced fauna, from the 10th week to the 23rd week, 2.5 mg / Kg/day's Jianzhiling.

Anti-atherosclerosis and morphology

High fat diet induces changes in arterial tissue structure, including a reduction in the thickness ratio of the vessel wall. As shown in Table 10, the difference in the dose of the Jianzhiling was observed to prevent the decrease in the thickness of the blood vessel wall, which was a dose-dependently.

Decomposition activity and protein expression of 3-T-3 adipocytes

Assess cAMP/cGMP activity as the second information to activate hormone-sensitive lipolytic enzyme (HSL). HSL, a rate-limiting enzyme that regulates the breakdown of fat cells, then catalyzes the triglyceride and promotes the breakdown of fat, releasing free fatty acids (FFA) and glycerol. Phosphodiesterases (PDE) hydrolyze cyclic adenosine monophosphate (cAMP) to become an enzyme of 5'-adenosine (5'-AMP), which reduces the decomposition of fat. PDE inhibitors increase cAMP levels. A non-specific PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX), which acts as a PDE inhibitor. As shown in Table 11, the decomposition of PPAR/eNOS in fat is regulated by Jianzhiling-1, resulting in an increase in HSL and p-HSL.

Simvastatin inhibited the formation of [14C] mevalonate by 86.6 ± 4.2% compared to the control group. Jianzhiling-1 (10 μM) does not inhibit HMGR, while reducing the formation of [14C] mevalonate. The 10 μM dose of the Jianzhiling-1-Simvastatinic Acid complex inhibited the formation of [14C] mevalonate of 23.0±2.3%. The effect of the serotonin compound simvastatin on the production of [14C] mevalonate was shown.

The above excipients, or "pharmaceutically acceptable carriers or excipients", "bioavailable carriers or excipients", include solvents, dispersing agents, coatings, antibacterial or antifungal agents, or Any suitable compound suitable for the preparation of a dosage form such as an absorbent is delayed. Usually such carriers or excipients do not themselves have the activity of treating diseases, and the derivatives disclosed in the present invention, together with pharmaceutically acceptable carriers or excipients, are prepared for administration to animals or humans. Causes adverse reactions, allergies or other inappropriate reactions. Thus, the derivatives disclosed herein, in combination with pharmaceutically acceptable carriers or excipients, are suitable for clinical and human use. The therapeutic effect can be achieved by administering the dosage form of the compound of the present invention intravenously, orally, by inhalation or by local or sublingual administration such as nasal, rectal, vaginal or the like. For patients with different conditions, about 0.1 mg to 100 mg of active ingredient is administered daily.

The carrier will vary with each dosage form, and the sterile injectable compositions may be solution or suspended in a non-toxic intravenous diluent or solvent such as 1,3-butanediol. A carrier acceptable therebetween may be Mannitol or water. In addition, the oil is fixed or a synthetic single or bis-glycoside oil suspension medium is a commonly used solvent. fat Acids, such as oleic acid, olive oil or castor oil, and its oleic acid oil ester derivatives, especially in the form of polyoxyethylation, can be used as an injection preparation and are natural pharmaceutically acceptable oils. These oil solutions or suspensions may contain long-chain alcohol diluents or dispersants, carboxymethylcellulose or similar dispersing agents. Other commonly used surfactants such as Tween, Spans or other similar emulsifiers or bioavailable enhancers for use in general pharmaceutical manufacturing for use in pharmaceutically acceptable solid, liquid or other formulation developments.

The composition for oral administration is in any of the orally acceptable dosage forms, and the forms thereof include capsules, troches, tablets, emulsifiers, liquid suspensions, dispersants, solvents. Oral dosage forms are generally used as carriers, and in the case of tablets, lactose, corn starch, and a lubricant such as magnesium stearate are basic additives. The diluent used in the capsules includes lactose and dried corn starch. The liquid suspension or emulsifier dosage form is prepared by suspending or dissolving the active substance in an oily interface combined with an emulsifier or a suspending agent, and adding a moderate sweetener, a flavoring agent or a pigment as needed.

Nasal gasifying sprays or inhalant compositions can be prepared according to known formulation techniques. For example, the composition is dissolved in physiological saline, benzyl alcohol or other suitable preservative, or an absorbent is added to enhance bioavailability. The compositions of the compounds of the invention may also be formulated as a suppository for rectal or vaginal administration.

The compounds of the present invention may also be administered "intravenous administration", including subcutaneous, intraperitoneal, intravenous, intramuscular, or intra-articular, intracranial, intra-articular, intraspinal injection, aortic injection, intrathoracic injection, intralesional injection. , or other suitable drug delivery technology.

In the feed composition of the present invention, at least one feed of the "feed material" defined below is mixed by a subdividing method, a softening method, a fermentation method, a drying method or the like, within a range not affecting the effects of the present invention. The term "feed ingredients" means corn, Buckwheat, wheat, rye, sorghum, soybean, soybean powder and other cereals; bran, rice bran, wheat bran, millet and other mites; soybean residue, bean curd residue, starch residue, coconut pulp, wine residue, soy sauce residue , the residue of fruit and vegetable juice after juice extraction or its crushed residue; even the use of peanut meal, rapeseed meal, sesame meal, sunflower seeds and other oil residue; yeast powder, casein, skim milk powder, dried whey, Meat and bone meal, meat powder, feather meal, and other animal feeds; plant extracts such as Kudzu, Saidou, Hazelnut, Clover, Valerian, Pennisetum, and Pangu.

Excipients, extenders, nutritional supplements, feed supplements and the like may be mixed in the above-mentioned feed composition within the range not impairing the effects of the present invention. The excipient may be selected from cellulose derivatives such as hydroxymethylcellulose, the extender may be selected, for example, cyclodextrin or starch, the nutritional supplement may be selected, for example, from vitamins or minerals, and the feed supplement may be, for example, an enzyme. Agent or fungicide.

The feed composition of the present invention can be administered to livestock alone, or added to a general feed, and mixed and then administered to livestock. For example, in the breeding of pigs, only breast milk is given at the time of the first birth, and the breast milk plus the pre-primary feed (pre-early feed) is administered in parallel from 1 to 2 weeks.

The invention converts the weaned piglets into the initial feed (early feed), and then the middle pig or the big pig uses the finishing feed for the fattening period to feed the feed composition at the same time.

The feed composition of the present invention can be administered to all livestock and has a superior effect on non-ruminants. For example, pigs, chickens, horses, rabbits, domestic ducks, turkeys, quails, ostriches and other non-ruminants can achieve an excellent increase in meat exchange rate and a reduction in feed intake, especially for pigs. .

The "Jianzhiling Compounds" proposed in this case increase the meat exchange rate and reduce the obesity group. The benefits of the present invention will be fully understood from the following description of the embodiments, so that those skilled in the art can do so. However, the implementation of the present invention is not limited by the following embodiments, and those skilled in the art are familiar with the art. Still other embodiments may be devised in accordance with the spirit of the disclosed embodiments, and such embodiments are within the scope of the invention.

Experimental materials and methods: Activity experiment:

The 5-week-old male C57BL/6J was purchased from the National Animal Experimental Center and fed at the Animal Experimental Center of Kaohsiung Medical University.

Configuration of experimental drugs:

(1). Jianzhiling, the KMUP or its complex ammonium salt used in the experiment is a compound synthesized in the laboratory. Dissolve in deionized water to a concentration of 10 ^-2 M and dilute with deionized water at the desired concentration.

(2). Y27632((R)-(+)-trans-4-(1-Aminoethyl)-N-(4-Pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate is dissolved in deionized water to a concentration of 10 ^-2 M, Dilute with deionized water at the desired concentration.

(3). Simvastatin (2,2-dimethylbutanoic acid (1S,3R,7S,8S,8aR)-1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-[2 -[(2R,4R)-tetra-hydro-4-hydroxy-6-oxo-2H-pyran-2-yl]ethyl-1-maphthalenyl ester,Simvastatin) is dissolved in methyl hydrazine (DMSO) to make it concentration It is 10 ^-2 M and is diluted with deionized water at the desired concentration.

Experimental drugs: B. West German Merck:

Methanol (CH ₃ OH), sodium chloride (NaCl), sodium hydroxide (NaOH)

C. Sigma-Aldrich, USA:

Bovine Serum Albumin (BSA)

Glycerol

Heparin Sulfate

Tris (hydroxymethyl) amino-methane HCl, Tris-HCl

Tween-20

Phosphate-Buffered Saline (PBS) buffer (10 times)

D, US Bio-Rad:

Ammonium Persulfate (APS)

30% Acrylamide/Bis-acrylamide (Acrylamide/Bis) (37.5:1) Reagent

2-Mercapethanol

Tetramethylethylenediamine ( N,N,N', N'-Tetramethyl-Ethylene Diamine, TEMED)

Protein Assay Dye

Sodium Dodecyl Sulfate (SDS)

Tris Base

Glycine

E. US Roche:

Mixed tablets (Complete Mini Cocktail table t)

Protease-inhibiting mixed tablets (Complete, Mini Protease Inhibitor Cocktail Table ts)

F. US Millipore Corporation:

Polyvinylidene Difluoride (PVDF) film

Enhanced Chemiluminescence (ECL)

G. Japan OSAKA Corporation:

Sodium carboxymethyl cellulose (sodium CMC)

H. Antibody (Antibody): (1) Primary antibody

Anti-eNOS: Upstate Laboratories, U.S.A.

Anti-ROCK: Upstate, U.S.A.

Anti-RhoA: Santa Cruze, U.S.A.

Anti-5-HT _2B : Upstate,USA

Anti-AKT: Santa Cruz Biotechnology, U.S.A.

Anti-phosphor-AKT: Santa Cruz Biotechnology, U.S.A.

Anti-5-HTT: Chemicon Biotechnology, U.S.A.

Anti-ERK1/2: Santa Cruz Biotechnology, U.S.A.

Anti-phosphor-ERK1/2: Santa Cruz Biotechnology, U.S.A.

Anti-β-actin: Sigma-Aldrich, U.S.A.

(2) Second antibody (Second antibody):

Horseradish peroxidase-labeled goat anti-mouse antibody (Goat anti-mouse IgG Horseradish Peroxidase Conjugate: Santa Cruz Biotechnology, U.S.A)

Horseradish peroxidase-labeled goat anti-rabbit IgG Horseradish Peroxidase Conjugate: Santa Cruz Biotechnology, U.S.A

Horseradish peroxidase-labeled goat anti-goat antibody (Goat anti-goat IgG Horseradish Peroxidase Conjugate: Santa Cruz Biotechnology, U.S.A)

I. Preparation of experimental buffer solution:

(1) 1.5 M Tris-HCl pH 8.8: 27.23 g of Tris base was dissolved in 80 mL of deionized water to adjust the pH with 1 N sodium chloride. Value to 8.8, finally add deionized water to a final volume of 150 mL, stored at 4 °C.

(2) 0.5 M Tris-HCl, pH 6.8: Dissolve 6.0 g of Tris base in 60 mL of deionized water, adjust the pH to 6.8 with 1N hydrochloric acid, and finally add deionized water to a final volume of 100 mL and store at 4 °C. .

(3) 10% sodium dodecyl sulfate (SDS): 10 g of SDS was mixed in 90 mL of deionized water, and finally deionized water was added to make a final volume of 100 mL, which was stored at room temperature.

(4) Tissue or Cell lysis buffer: Pre-cooled with 10 ml of Mammalian Protein Extraction Reagent (T-PER ^TM ), followed by addition of a protease inhibitor mixture (Complete mini Protease inhibitor cocktail). Store at -80 ° C.

(5) Cell culture: 10% fetal bovine serum (Fetal Bovine Serum, FBS), 20 ml glutamine, 20 ml antibiotic, 3 g sodium bicarbonate (NaHCO ₃ ) and lowest The essential medium (Minumum Essential Medium, MEM) powder was mixed and added with deionized water to 2 liters, and the pH was adjusted to 7.2.

(6) Cell Cryopreservation medium: 10% fetal bovine serum (FBS), 7% Dimethyl sulfoxide (DMSO) and 1 x minimum essential medium (MEM medium)

(7) Trypsin cell digest: 0.25% trypsin mixed with 0.02% ethylenediaminetetraacetates (EDTA)

(8) Hank's balanced salt solution (HBSS): 8 g of sodium chloride (NaCl), 0.4 g of potassium chloride (KCl), 0.1 g of magnesium sulfate (MgSO ₄ .7H ₂ O), magnesium chloride (MgCl. 6H ₂ O) 0.1 g, anhydrous calcium chloride (CaCl ₂ ) 0.14 g, glucose 1.0 mg, disodium hydrogen phosphate (NaHPO ₄ ) 0.154 g, potassium dihydrogen phosphate (KH ₂ PO ₄ ) 0.06 g, added 0.4 after mixing 5 ml of phenol red liquid, deionized water to 1 liter, and no phenol red.

(9) Extraction buffer: Tris hydroxymethylaminomethane (Tris) 10 mM, sodium chloride 140 mM, phenylmethyl-sufonyl-fluoride (PMSF) 2 mM Dithiothreitol (DTT) 5 mM, ethyl phenyl polyethylene glycol (Nonidet, NP-40) 0.5%, 0.05 mM peptidin (pepstatin) and 0.2 mM leupeptin.

(10) RIPA extraction buffer: 50 mmol/L Tris (hydroxymethyl) aminomethane (Tris/HCl), pH 8.0, 150 mmol/L sodium chloride , 1% ethyl phenyl polyethylene glycol (Nonidet, NP-40) 1% sodium deoxycholate (sodium deoxycholate), 0.1% sodium dodecyl sulfate (SDS), 0.1 mmol / L dithiothreose Alcohol (dithiothreitol, DTT), 0.05 mmol/L phenylmethyl-sufonyl-fluoride (PMSF), 0.002 Mg/mL aprotinin (aprotinin), 0.002 mg/mL leupeptin.

(11) MDI medium: Dulbecco'Modified Eagle' Medium (DMEM) 10% bovine serum, 1 times antibiotic-antimycotic, 10 μg/mL insulin, 39 μg/mL dicortisol ( Dexamethasone), 115 μg/mL 3-isobutyl-1-methylxanthine.

12. Sodium alkyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) solution preparation

(1) 5X Separation Colloidal Buffer ( Running buffer) : Trimethylolamine 15g Tris Base, 72 g Glycine, 5 g sodium dodecyl sulfate (SDS) Add deionized water to 1 L and dilute to 1 running buffer with deionized water before use.

(2) Transfer buffer : 12.1 g of tris, 57.652 g of glycine, 800 mL of methanol, 3200 ml of deionized water.

(3) Washing buffer (t-TBS) : 9.68 g of tris, 32 g of sodium chloride, 4 mL of Tween-20, deionized water to 1 L, 6 N of hydrochloric acid to adjust the pH pH = 7.6.

(4) 5X Sample Solution: 3.8 mL of deionized water, 1.0 mL of Tris (0.5 M, pH 6.8), 0.8 mL of Glycerol, 1.6 mL (10%) Sodium lauryl sulfate, 0.4 mL of 2-Mercaptoethanol, and 0.4 mL of bromophenol blue (1%) were foamed to make a 5-fold concentrate and stored at room temperature.

(5) Blocking buffer: 100 ml of washing buffer, 5.0 g of skimmed milk powder

Preparation of SDS-PAGE film (Laemmli buffer system)

(1) Preparation of 7.5% Separating gel solution: 4.85 mL of deionized water, 2.5 mL of trishydroxymethylaminomethane hydrochloride (1.5 M, pH 8.8), 100 μL (10% w/v) Sodium dialkyl sulfate, 2.5 mL of Acrylamide Bis (30% stock), 50 μL of ammonium persulfate (APS, 10%), 5 μL of tetramethylethylenediamine (TEMED) ), the total volume of uniform mixing is 10 mL.

(2) Preparation of 8.5% Separating gel solution: 4.52 mL of deionized water, 2.5 mL of trishydroxymethylaminomethane hydrochloride (1.5 M, pH 8.8), 100 μL (10% w/v) Sodium dialkyl sulfate, 2.83 mL of acrylamide/Bisamine (30% stock), 50 μL (10%) ammonium persulfate, 5 μL of tetramethylethylenediamine, uniformly mixed in total volume 10mL.

(3) Preparation of 12% Separating gel solution: 3.35 mL of deionized water, 2.5 mL of trishydroxymethylaminomethane hydrochloride (1.5 M, pH 8.8), 100 μL (10% w/v) Sodium dialkyl sulfate, 4 mL of acrylamide/bis acrylamide (30% stock), 50 μL (10%) ammonium persulfate, 5 μL of tetramethylethylenediamine, uniformly mixed to a total volume of 10 mL.

(4) Preparation of 14% Separating gel solution: 2.68 mL of deionized water, 2.5 mL of trishydroxymethylaminomethane hydrochloride (1.5 M, pH 8.8), 100 μL (10% w/v) Sodium dialkyl sulfate, 4.67 mL acrylamide/bis acrylamide (30% stock), 50 μL (10%) ammonium persulfate, 5 μL tetramethylethylenediamine, uniformly mixed to a total volume of 10 mL. After mixing evenly, pour into the MINI-PTOTEAM II unit and pressurize with deionized water to allow for about 40 ~50 minutes to be formed. Select the film according to the molecular weight of the protein to be explored: the protein with low molecular weight is suitable for high concentration film, and vice versa.

(5) Preparation of 4% stacking gel solution: 3.05 mL of deionized water, 1.25 mL of trishydroxymethylaminomethane hydrochloride (1.5 M, pH 8.8), 50 μL (10% w/v) Sodium dialkyl sulfate, 0.65 mL of acrylamide/bis acrylamide (30% stock), 25 μL (10%) of ammonium persulfate, and 5 μL of tetramethylethylenediamine were uniformly mixed in a total volume of 5 mL. Pour off the water layer on the upper layer of the separation gel, add the uniform mixture solution, and then put the sample tooth mold (Comb) and let it stand at room temperature for coagulation. If the liquid level drops, the colloidal solution must be replenished at any time.

laboratory apparatus:

(1) pH meter (SP-701): SUNTEX, Taiwan

(2) Enzyme-Linked Immunosorbant Assay reader (MRX): DYNEX Technologies, Germany

(3) Mini-PROTEAM ^® 3 Cell: Bio-Rad Laboratories Inc., USA

(4) Mini Trans-Blot ^® Electrophoretic Transfer Cell: Bio-Rad Laboratories Inc., USA

(5) Power supply/POWER PAC HC: Bio-Rad Laboratories Inc., U.S.A.

(6) Refrigerated centrifuge: Kubota 8800, Japan

(7) Microcentrifuge: Eppendorf 5415 C, Taiwan

(8) Automatic filming machine and darkroom project: M43 716-7957, Kodak, U.S.A

(9) Fluorescence photometer (spectroflurophotometer: Shimadzu, RF-5301PC, Japan)

(10) computer interface microscope (computer-interfaced light microscope (Eclipse TE2000-S microscope, Nikon, Tokyo, Japan)

experimental method

Analysis of proteins in 3T3-L1 fat cultured cells by dot blot analysis (Western blotting analyses of proteins in 3T-3 cell culture)

3T3-L1 adipocytes were placed in a 100 mm culture dish and stimulated with MDI medium and DPA-1 according to the experiment. After stimulation with a phosphatase inhibitor 1 mmol/L sodium vanadate (NaVO ₃ ), the cells were extracted by RPPA extraction buffer. The protein content was determined by a protein assay concentrate (Bio-Rad stain), and the experiment was carried out in accordance with the protein content assay method.

Western blotting method for protein expression in mouse liver and HepG2 cell line

The isolated rat liver diced and HepG2 cell strain were loaded into an extraction buffer to extract proteins, which were then centrifuged at 12,500 g for 30 minutes. The Western blotting method was used to measure the performance of the protein.

C57BL/6J mouse experiment

Six-week-old male C57BL/6J mice were randomly divided into 5 groups, two control groups and three administration groups. During the 8 weeks, the control group received standard diet (STD) or high-fat diet (HFD). The rats in the treatment group received high-fat diet and orally administered simvastatin (5 mg/kg) or Jianzhiling compound (1, 2.5, 5 mg/kg).

The two control groups compared the body-weight gain and fat content of the high fat diet to the mice. The liver eNOS and ROCKII performance of the administration group were evaluated 1 to 24 hours after the sacrifice of the mice.

A.) DPA-1 and Simvastatin affect serum biochemical values in mice with dyslipidemia a. Animal pattern of dyslipidemia:

1. Feed standard diet (STD) group for 8 weeks

2. Feeding high-fat diet (HFD) for 8 weeks to cause dyslipidemia: composition of 60 cal% fat, 21.3 cal% carbohydrates, and 18.7 cal% protein

3. Administration of high-fat diet for 8 weeks while oral administration (p.o.) 1 mg/kg DPA-1

4. Administration of high-fat diet for 8 weeks while oral administration of 2.5 mg/kg DPA-1

5. Administration of high-fat diet for 8 weeks while oral administration of 5 mg/kg DPA-1

6. Administer high-fat diet for 8 weeks while oral administration of 5 mg/kg Simvastatin

b.) Extract mouse tissue and heart blood:

After 8 weeks of feeding in each group of C57BL/6J mice, after anesthesia with urethane, the mice were gently pressed against the thigh of the index finger or middle finger to feel the heartbeat and the position was determined. Then, 1 cc syringe was used. Insert the needle into the heart, draw blood from the heart (the process should avoid hemolysis), immediately separate the serum from the plasma by centrifugation at 3000 rpm for 15 minutes, and store the serum at -80 °C to separate serum total cholesterol (TC) and triglyceride. Determination of biochemical values of (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) . The above biochemical value test was performed using Hitachi Clinical Analyzer 7070 (Hitachi High-Technologies Co. Tokyo, Japan) and commercial kit (Sigma-Aldrich, St Louis, MO, USA).

After the heart blood is drawn, the left lobe liver of the mouse is immediately taken out and stored in a -80 ° C refrigerator (to be tested by the Western blot method).

B.) Inhibition of obesity in mice

The 10-week male C57BL/6J mice were fully supplied with 50 g of feed, the room temperature was 25 ° C, the light-dark cycle was 12-h, and the high-fat feed (HFD) and water were consumed at random.

I. Western Blotting to investigate the effects of DPA-1 and Simvastatin on the expression of Rho kinase, PPARγ, HMGR and ABCA1 in liver tissues of mice with dyslipidemia 1. Organizational processing:

The left liver of the above-mentioned refrigerator refrigerated in -80 ° C was taken out, soaked in tissue lysis buffer, placed on ice to make it small and healthy, and the tissue was homogenized on ice by ultrasonic homogenizer. After centrifugation (15,000 rpm, 30 min, 4 ° C), the supernatant was taken.

2. Determination of protein content:

The protein content was determined by a protein assay concentrate (Bio-Rad stain). Bio-Rad dye is an acid solution containing Coomassie Brilliant Blue G-250. When the dye and protein are combined to form a complex, the maximum absorption wavelength of the spectrum is shifted from 456 nm to 595 nm. According to this characteristic, the standard curve of the standard solution can be directly measured by an enzyme immunoassay analyzer (ELISA reader), and the amount of protein contained in the test solution can be known by measuring the absorbance value of the sample at a wavelength of 595 nm.

Standard solution configuration - Bovine serum albumin (BSA) at a concentration of 0.1 mg/ml as a protein content standard and configured as a standard solution of various known protein contents (protein content 0, 2, 4, 8, 12) , 16, 20 and 30 μg/ml).

Determination of the concentration of protein extract - take 237 μl deionized water plus 3 μl of the cell extract was added, and 60 μl of the protein was added to measure the concentration of the concentrate. The absorbance at a wavelength of 595 nm was measured by an enzyme immunoassay analyzer, and the concentration of the protein extract was estimated by reference to a standard curve.

3. Protein electrophoresis separation:

After quantitatively dissolving and diluting the protein extract, a quarter of the sample buffer was added and heated in boiling water for 5 minutes. Then, the test samples were sequentially added to each well of 10% sodium dodecyl sulfate-polymerized acrylamide gel (SDS-PAGE), and at the same time, prestained protein molecular weight standards (prestained protein molecular weight standards) ) Align the molecular weight position. Adding a separation colloid buffer to the electrophoresis tank, electrophoresis was carried out for 30 minutes at a voltage of 80 volts, and then electrophoresis was continued at 180 volts until the dye solution of SDS-PAGE ran out of SDS-PAGE, then the power was stopped. .

4. Immunoblotting method (Immunoblotting):

After performing the electrophoresis analysis, carefully remove the film and place it in a transfer buffer. In addition, a 3 mm filter paper of the same size as the film was cut and allowed to immerse in the transfer buffer for equilibration. The polyvinylidene difluoride (PVDF) film of the same size as the film was preliminarily conditioned with methanol for 30 seconds, deionized water for 2 minutes, and finally infiltrated in the transfer buffer for 15 minutes or more. After the film, filter paper, and polyvinylidene fluoride film are balanced, place two filter papers on the saturated and absorbing graphite negative plate, cover the film, cover a PVDF film, and drop a little of the buffer to PVDF. The membrane, covered with two filter papers, and be careful not to leave air bubbles, and finally covered with a saturated and absorbent graphite positive plate to form a "sandwich" sandwich. The transfer process was carried out using a Wet blotter system, and the sandwich sandwich was placed in a transfer buffer to fix the current for 1.5 hours. The value of the fixed current is set according to the calculated value of the film area × 0.8 milliamperes (mA/cm ² ). After the film protein was completely transferred to the PVDF membrane, the PVDF membrane was removed and infiltrated into an appropriate amount of blocking buffer, placed on a horizontal rotary shaker, and shaken slowly for 2 hours at room temperature. Subsequently, the washing buffer ( T-TBS) was successively washed 6 times for 5 minutes each time, and then the diluted primary antibody solution was uniformly coated on the PVDF membrane overnight at 4 ° C to effect. After washing for 5 minutes every other day with washing buffer for 5 minutes, dilute the horseradish peroxidase (HRP)-labeled secondary antibody solution to a suitable concentration and evenly pour into the PVDF membrane. The upper shock reaction was 1 hour. After the reaction was completed, the washing buffer was further washed 7 times for 5 minutes each time. Finally, the detection buffer (ECL) was added for 2 minutes, and after being dried slightly, the film was tableted and washed. The results were analyzed and quantified by density and analytical software.

2. Analysis of the activity of hydroxymethyl pentane coenzyme A reductase (HMGR) 1. Principle:

Reaction formula of HMG-CoA reductase (HMGR): HMG-CoA+2NADPH+2H ⁺ →mevalonate+2NADP ⁺ +CoA-SH Determination of wavelength 340nm by enzyme immunoassay, reducing absorption The wavelength of 340 nm is the reaction amount of nicotinamide adenine dinucleotide phosphate (NADPH). The more the reaction amount of NADPH, the higher the activity of HMGR, and the drug which inhibits the activity of HMGR is administered. The lower the amount of NADPH involved in the reaction, the more effective the inhibition of HMGR activity between drugs.

2. Steps:

a. Set the enzyme immunoassay analyzer to 37 ° C and 340 nm, and add appropriate reagents to the 96-well reaction plate according to Table 5.

b. After adding appropriate reagents to each well, immediately put it into the enzyme immunoassay analyzer to read the absorbance. The first reading must be shaken for 10 seconds, then read every 30 seconds for 30 minutes.

c. The value read is taken into the kinetic formula: TV=Total volume of the reaction in ml, 0.2 ml for plates

V = volume of enzyme used in the assay (ml)

LP = Light path in cm, 0.55 for plates

Units/mg-protein=(△A340/min _sample -△A340/min _blank ) ^* TV/12.44 ^* V ^* 0.6 ^* LP

The unit is: μmol/min/mg-protein.

HMG-CoA reductase activity analysis test reagent added to each well

(Note) 1. Buffer is 1 × Assay buffer 2. Drug is selected for Simvastatin or DPA-1

Test for 3-hydroxy-3-methylpentadienyl Coenzyme A reductase (HMG Co-A reductase activity and [ ¹⁴ C] mevalonate formation)

Recombinant human 3-hydroxy-3-methylpentadienyl Coenzyme A reductase (HMG CoA reductase) was expressed by Escherichia coli (Sigma, H7039), pre-administered with Jianzhiling compound, simvastatin cultured at 37 ° C phosphate buffer Liquid (pH 7.5) for 15 minutes. Further, with 2.5 μM [ ¹⁴ C]HMG-CoA, the reaction was started after a 20-minute incubation period, and the reaction was terminated by additionally adding 1 N hydrochloric acid to calculate the amount of [14C] valproic acid formed.

Example 1 Preparation of 7-[2-[4-(2-chlorobenzene)piperazinyl]ethyl]-1,3-dimethyl xanthine HCl)

Take Jianzhiling-1 (8.3 g) dissolved in ethanol (10 mL) and 1 N hydrochloric acid (60 mL), react at 50 ° C for 20 minutes, add ethanol (20 mL) at room temperature overnight. Crystallization was carried out to obtain Jianzhiling-1 hydrochloride (6.4 g).

Example 2 Preparation of 7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethyl xanthine HCl)

Take Jianzhiling-1 (8.3 g) dissolved in ethanol (10 mL) and 1 N hydrochloric acid (60 mL), react at 50 ° C for 20 minutes, add at room temperature Ethanol (20 mL) was added to stand overnight to crystallize, and filtered to obtain Jianzhiling-3 hydrochloride (6.6 g).

Example 3 Preparation of Jianzhiling-2-polyacrylic acid composite compound

Take Jianzhiling-2 (8 g) dissolved in ethanol (10 mL) and polyacrylic acid (2.5 g), react at 50 ° C for 10 minutes, add ethanol (20 mL) at room temperature overnight to crystallize The gelatin-1 polyacrylic acid complex compound (7.4 g) was obtained by filtration.

Example 4 Preparation of Jianzhiling-3-polyalginate composite compound

Take 12.5 g of sodium alginate (APA) dissolved in 40 ml of sodium hydroxide (5%) in water, add 8.3 g of Jianzhiling-3 hydrochloride, and react at 50 ° C for 10 minutes. Add ethanol at room temperature. (20 mL) was allowed to stand overnight for crystallization, and filtered to obtain a Jianzhiling-3-polyalginate compound (31.4 g).

Example 5 Preparation of Jianzhiling-1-polyglutamic acid composite compound

Take Jianzhiling-1 (7.9 g) dissolved in ethanol (10 mL) and 3.8 g of polyglutamic acid solution, react at 50 ° C for 10 minutes, add ethanol (20 mL) at room temperature overnight to crystallize The Jianzhiling-1-polyglutamic acid complex compound (10.4 g) was obtained by filtration.

Example 6 Preparation of Jianzhiling-1-carboxymethylcellulose composite compound

20 g of sodium carboxymethylcellulose (sodium CMC) was dissolved in 40 ml of sodium hydroxide (5%) in an aqueous solution, and 16 g of Jianzhiling-1 hydrochloride was added and reacted at 50 ° C for 10 minutes. Add ethanol (20 mL) under temperature Crystallization was carried out overnight, and the Jianzhiling-1-carboxymethylcellulose composite compound (31.4 g) was obtained by filtration.

Example 7 Preparation of Jianzhiling-2-hyaluronic acid composite compound

Take Jianzhiling-2 (8 g) dissolved in ethanol (10 mL) and hyaluronic acid (2.5 g), react at 50 ° C for 10 minutes, add ethanol (20 mL) at room temperature overnight to crystallize, The Jianzhiling-2-hyaluronic acid complex compound (9.4 g) was obtained by filtration.

Example 8 Preparation of Jianzhiling-4-Ethyl sulfate heparin composite compound

Take Jianzhiling-4 (8.3 g) dissolved in ethanol (10 mL) and acesulfate heparin (8.5 g), react at 50 ° C for 10 minutes, add ethanol (20 mL) at room temperature overnight. Crystallization was carried out, and the Jianzhiling-4-acetic acid heparin heparin complex compound (9.2 g) was obtained by filtration.

Example 9 Preparation of Jianzhiling-1-sulfuric acid dextran composite compound

Take Jianzhiling-1 (8 g) dissolved in a solution of ethanol (10 mL) and dextran sulfate (3.5 g), react at 50 ° C for 10 minutes, and add ethanol (20 mL) at room temperature overnight. Crystallization was carried out, and a Jianzhiling-1-sulfuric acid dextran composite compound (10.6 g) was obtained by filtration.

Example 10 Preparation of Jianzhiling-4-polyglutamic acid composite compound

Take Jianzhiling-4 (8.3 g) in a solution of ethanol (10 mL) and polyglutamic acid (3.8 g), react at 50 ° C for 10 minutes, and add ethanol (20 mL) at room temperature overnight. Crystallization, filtration to obtain Jianzhiling-4-poly glutamine Acid complex compound (9.2 g).

Example 11 Preparation of Jianzhiling-1-polyglutamic acid composite compound

Take Jianzhiling-1 (8.3 g) dissolved in ethanol (10 mL) and polyglutamic acid (3.8 g), react at 50 ° C for 10 minutes, add ethanol (20 mL) at room temperature overnight. Crystallization was carried out, and the Jianzhiling-1-polyglutamic acid complex compound (9.2 g) was obtained by filtration.

Example 12 Preparation of Jianzhiling-1-polylactic acid composite compound

Take Jianzhiling-1 (8.3 g) dissolved in ethanol (10 mL) and polylactic acid (3.2 g), react at 50 ° C for 10 minutes, add ethanol (20 mL) at room temperature overnight to crystallize The Jianzhiling-1-polyglutamic acid complex compound (9.4 g) was obtained by filtration.

Example 13 Preparation of Jianzhiling-1-polyglycolic acid composite compound

Take Jianzhiling-1 (8.3 g) dissolved in a solution of ethanol (10 mL) and polyglycolic acid (3.5 g), react at 50 ° C for 10 minutes, and add ethanol (20 mL) at room temperature overnight. Crystallization, filtration obtained the Jianzhiling-1-polyglutamic acid complex compound (9.6 g).

Example 14 Composition for preparing a tablet

The following components are separately weighed according to the amount, mixed and filled in a tableting machine to prepare a tablet Corn flour qs

Example 15 Composition for preparing a tablet

The following components are separately weighed according to the amount, mixed and filled in a tableting machine to prepare a tablet

Example 16 Daily feed composition of piglets

Among them, crude protein accounts for 18.4%, crude fat accounts for 6.5%, and others include general oils such as oil esters 25kg and calcium powder, which can provide vitamins or minerals.

Example 17 Daily feed composition of pigs

Among them, crude protein accounts for 17.5%, crude fat accounts for 4.6%, and others include general esters such as oil esters and calcium carbonates, which can provide vitamins or minerals.

Example 18 Daily feed composition of large pigs

Among them, crude protein accounts for 17.5%, crude fat accounts for 4.6%, and others include general esters such as oil esters and calcium carbonates, which can provide vitamins or minerals.

Other embodiments

1. A physiochemical compound for improving hyperlipemia, such as quaternary ammonium of the structure represented by formula (II), wherein R ₂ and R ₄ may each be selected from the group consisting of a hydrogen group, a halogen, an amine group, a substituent of a nitro group; a substituent having a carbon number of 1 to 5 alkyl groups; and a carbon number of 1 to 5 alkoxy groups. a substituent; RX which is selected from the group consisting of Statin-like drugs, sodium carboxymethylcellulose (sodium CMC), high molecular weight polymers or polyglutamic acid group derivative drugs; And ^- RX can be a negatively charged anion of the above groups.

2. The Jianzhiling compound of Example 1, wherein the carboxylic acid group-containing polyglutamic acid group derivative is selected from the group consisting of alginate sodium, polyglutamic acid (γ-PGA), and polybrene. Sodium amide (γ-PGA sodium) or sodium alginate (APA).

3. The compound of claim 1, wherein the Statin-containing drug having a carboxylic acid group is selected from the group consisting of atorvastatin, cerivastatin, fluvastatin, and rosavastat. Lovastatin, mevastatin, Pravastatin, Rosuvastatin, and Simvastatin.

4. The Jianzhiling compound of Example 1, wherein the high molecular polymer is selected from the group consisting of hyaluronic acid, polyacrylic acid, Polymethacrylates, Eudragit, dextran sulfate, heparan sulfate, polylactic acid (PLA), polyglycolic acid (Polyglycolic acid, PGA), poly(lactic acid sodium), PLA sodium, poly(glycolic acid sodium, PGA sodium).

5. An improved high-lipid blood lipid complex compound selected from the following quaternary ammonium salts: a quaternary ammonium salt synthesized from a saponin compound and a Statin drug; a saponin compound and a carboxymethyl fiber a quaternary ammonium salt synthesized by sodium sulphate (sodium CMC); a quaternary ammonium salt synthesized from a saponin compound and a high molecular polymer; and a serotonin compound and a glutamic acid group containing a carboxylic acid group A quaternary ammonium salt synthesized by a group derivative.

6. The Jianzhiling compound of Example 5, wherein the Jianzhiling compound is selected from the group consisting of 7-2-4-(2-chlorophenyl) piperazinyl]ethyl]-1,3-dimethylxanthine ( 7-[2-[4-(2-chlorophenyl)piperazinyl]ethyl]-1,3-dimethyl-xanthine, Jianzhiling-1); 7-2-4-(2-methoxyphenyl) piperazinyl Ethyl]-1,3-dimethylxanthine (7-[2-[4-(2-methoxybenzene)-piperazinyl]ethyl]-1,3-dimethylxanthine, Jianzhiling-2); 7-2 4-(4-nitrophenyl)piperazinyl]ethyl]-1,3-dimethylxanthine (7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3- Dimethylxanthine, Jianzhiling-3); 7-2-4-(2-nitrophenyl)piperazinyl]ethyl]-1,3-dimethylxanthine (7-[2-[4-(2-nitrobenzene)piperazinyl]ethyl]-1 ,3-dimethylxanthine,Jianzhiling-4)7-2-4-benzirazinyl]ethyl]-1,3-dimethylxanthine (7-[2-[4-benzene piperazinyl]ethyl]- 1,3-dimethylxanthine, Jianzhiling-4); and 7-[2-[4-(2-fluorophenyl)piperazinyl]ethyl]-1,3-dimethylxanthine (7-[2 -[4-(2-fluorobenzene)piperazinyl]-ethyl]-1,3-dimethylxanthine, Jianzhiling-5) and other lipid-lowering compounds.

7. The Jianzhiling composite compound of Example 5, wherein the carboxylic acid group-containing polyglutamic acid group derivative is selected from the group consisting of alginate sodium, polyglutamic acid (γ-PGA), and poly bran. Sodium amide (γ-PGA sodium) or sodium alginate (APA).

8. The Jianzhiling compound of Example 5, wherein the Statin-containing drug having a carboxylic acid group is selected from the group consisting of atorvastatin, cerivastatin, fluvastatin, and rovas Lovastatin, mevastatin, Pravastatin, Rosuvastatin, and Simvastatin.

9. A pharmaceutical composition for improving hyperlipemia comprising: a pharmaceutically acceptable carrier; and an effective amount of a main component of a quaternary ammonium structure represented by formula (II), wherein R ₂ and R ₄ may each be selected from the group consisting of a hydrogen group, a halogen, an amine group, a substituent of a nitro group; a substituent having a carbon number of 1 to 5 alkyl groups; and a carbon number of 1 to 5 alkoxy groups. a substituent; RX which is selected from the group consisting of Statin, sodium carboxymethylcellulose (sodium CMC), polymeric drug or polyglutamic acid derivative derivative; And ^- RX can be a negatively charged anion of the above groups.

10. A pharmaceutical composition for improving hyperlipemia comprising: a pharmaceutically acceptable carrier; and a quaternary ammonium salt selected from the group consisting of a quaternary ammonium salt synthesized from a saponin compound and a Statin drug; a quaternary ammonium salt synthesized from a compound of sodium carboxymethylcellulose (sodium CMC); a quaternary ammonium salt synthesized from a saponin compound and a high molecular polymer; and a serotonin compound and a carboxylic acid group A quaternary ammonium salt synthesized from a polyglutamic acid group derivative of the group.

11. A Jianzhiling compound compound for improving body weight imbalance, such as a quaternary ammonium salt of the structure represented by formula (II), wherein R ₂ and R ₄ may each be selected from the group consisting of a hydrogen group, a halogen, an amine group, a substituent of a nitro group; a substituent having a carbon number of 1 to 5 alkyl groups; and a carbon number of 1 to 5 alkoxy groups. a substituent; RX which is selected from the group consisting of Statin, sodium carboxymethylcellulose (sodium CMC), polymeric drug or polyglutamic acid derivative derivative; And ^- RX can be a negatively charged anion of the above groups.

12. A compound for improving body weight imbalance, which is selected from the group consisting of the following quaternary ammonium salts: a quaternary ammonium salt synthesized from a saponin compound and a Statin drug; a saponin compound and carboxymethyl cellulose a quaternary ammonium salt synthesized by sodium (sodium CMC); a quaternary ammonium salt synthesized from a saponin compound and a high molecular polymer; and a serotonin compound and a glutamic acid group containing a carboxylic acid group a quaternary ammonium salt synthesized by a derivative.

13. A pharmaceutical composition for improving weight loss, comprising: a pharmaceutically acceptable carrier; and an effective amount of a quaternary ammonium structure as shown in formula (II), wherein R ₂ and R ₄ may each be selected from the group consisting of: a hydrogen group, a halogen, an amine group, a substituent of a nitro group; a substituent having a carbon number of 1 to 5 alkyl groups; and a carbon number of 1 to 5 alkoxy groups. a substituent; RX which is selected from the group consisting of sodium carboxymethyl cellulose (sodium CMC), polymeric drug or polyglutamic acid derivative derivative; and ^- RX An anion that is negatively charged for the above groups.

14. A pharmaceutical composition for improving weight loss comprising: a pharmaceutically acceptable carrier; and a quaternary ammonium salt selected from the group consisting of a quaternary ammonium salt synthesized from a saponin compound and a Statin drug; a quaternary ammonium salt synthesized by the compound and sodium carboxymethyl cellulose (sodium CMC); a quaternary ammonium salt synthesized from a saponin compound and a high molecular polymer; and a guaranolin compound and a carboxylic acid group A quaternary ammonium salt synthesized from a polyglutamic acid group derivative.

15 The Jianzhiling compound according to the above embodiment, wherein the Jianzhiling compound is selected from the group consisting of 7-2-4-(2-chlorophenyl) piperazinyl]ethyl]-1,3-dimethylxanthine (7) -[2-[4-(2-chlorophenyl)piperazinyl]ethyl]-1,3-dimethyl-xanthine, Jianzhiling-1); 7-2-4-(2-methoxyphenyl) piperazinyl] Ethyl]-1,3-dimethylxanthine (7-[2-[4-(2-methoxybenzene)-piperazinyl] ethyl]-1,3-dimethylxanthine, Jianzhiling-2); 7-2-4-(4-nitrophenyl)piperazinyl]ethyl]-1,3-dimethylxanthine (7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1 , 3-dimethylxanthine, Jianzhiling-3); 7-2-4-phenylpiperazinyl]ethyl]-1,3-dimethylxanthine (7-[2-[4-benzenepiperazinyl]ethyl]- 1,3-dimethylxanthine, Jianzhiling-4); and 7-[2-[4-(2-fluorophenyl)piperazinyl]ethyl]-1,3-dimethylxanthine (7-[2 -[4-(2-fluorobenzene)piperazinyl]-ethyl]-1,3-dimethylxanthine, Jianzhiling-5) and other lipid-lowering compounds.

16. The Jianzhiling composite compound according to the above embodiment, wherein the carboxylic acid group-containing polyglutamic acid group derivative is selected from the group consisting of alginate sodium, polyglutamic acid (γ-PGA), and poly bran. Sodium amide (γ-PGA sodium) or sodium alginate (APA).

17. The Jianzhiling compound according to the above embodiment, wherein the Statin-containing drug having a carboxylic acid group is selected from the group consisting of atorvastatin, cerivastatin, fluvastatin, and rovas Lovastatin, mevastatin, Pravastatin, Rosuvastatin, and Simvastatin.

18. The pharmaceutical composition according to the above embodiment, wherein the Jianzhiling compound is selected from the group consisting of 7-2-4-(2-chlorophenyl) piperazinyl]ethyl]-1,3-dimethylxanthine (7- 2-[4-(2-chlorophenyl)piperazinyl]ethyl]-1,3-dimethyl-xanthine, Jianzhiling-1); 7-2-4-(2-methoxyphenyl) piperazinyl]ethyl]-1,3-dimethylxanthine (7-[2-[4-(2-methoxybenzene)-piperazinyl]ethyl] -1,3-dimethylxanthine, Jianzhiling-2); 7-2-4-(4-nitrophenyl)piperazinyl]ethyl]-1,3-dimethylxanthine (7-[2- [4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine, Jianzhiling-3); 7-2-4-phenylpiperazinyl]ethyl]-1,3-dimethylxanthine (7-[2-[4-benzenepiperazinyl]ethyl]-1,3-dimethylxanthine, Jianzhiling-4); and 7-[2-[4-(2-fluorophenyl)piperazinyl]ethyl]- 1,3-Dimethylxanthine (7-[2-[4-(2-fluorobenzene)piperazinyl]-ethyl]-1,3-dimethylxanthine, Jianzhiling-5) and other lipid-lowering compounds.

19. The pharmaceutical composition according to the above embodiment, wherein the high molecular polymer is selected from the group consisting of hyaluronic acid, polyacrylic acid, polymethacrylates, Eudragit, and sulphuric acid. Dextran sulfate, heparan sulfate, polylactic acid or polylactide (PLA), polyglycolic acid (PGA), polylactic acid sodium (PLA sodium), poly Polyglycolic acid sodium (PGA sodium).

20. The pharmaceutical composition according to the above patent application, wherein the Statin-containing drug having a carboxylic acid group is selected from the group consisting of atorvastatin, cerivastatin, fluvastatin, and rovas Lovastatin, mevastatin, pravastatin (Pravastatin), Rosuvastatin, and Simvastatin.

21. The pharmaceutical composition according to above, wherein the high molecular polymer is selected from the group consisting of hyaluronic acid, polyacrylic acid, polymethacrylates, Eudragit, and dextran sulfate ( Dextran sulfate), heparan sulfate, polylactic acid (polylactic acid or polylactide, PLA), polyglycolic acid (PGA), polylactic acid sodium (PLA sodium), polyglycolic acid Polyglycolic acid sodium (PGA sodium).

A composition for improving obesity comprising: a main component selected from the group consisting of a Jianzhiling compound and a Jianzhiling compound.

23. The composition according to the embodiment, wherein the Jianzhiling compound is one of Jianzhiling-1, Jianzhiling-2, Jianzhiling-3, Jianzhiling-4 and Jianzhiling-5 compound.

24. The composition according to the embodiment, wherein the Jianzhiling compound is a Jianzhiling compound and a Jianzhiling compound synthesized from the following compound containing a carboxylic acid group structure: a Statin drug, a carboxymethyl group Sodium cellulose (sodium CMC), high molecular polymer, polyglutamic acid group derivatives and their mixed forms.

25. The composition according to the embodiment, wherein the high molecular polymer is selected from the group consisting of hyaluronic acid, polyacrylic acid, polymethacrylates, Eudragit, and sulphuric acid. Dextran sulfate, heparan sulfate, polylactic acid or polylactide (PLA), polyglycolic acid (PGA), polylactic acid Polylactic acid sodium (PLA sodium), polyglycolic acid sodium (PGA sodium) and mixed forms thereof.

26. The composition of any of the embodiments, wherein the polyglutamic acid group derivative is selected from the group consisting of alginate sodium, polyglutamic acid (γ-PGA), and sodium polyglutamate (γ-PGA sodium). Or polyalginate (APA) and its mixed form.

27. The composition of any of the embodiments, wherein the Statin drug is selected from the group consisting of atorvastatin, cerivastatin, fluvastatin, lovastatin, and melamine. Mevastatin, Pravastatin, Rosuvastatin, Simvastatin, and their mixed forms.

28. The composition of any of the preceding embodiments, wherein the obesity is related to a state of fat content in the blood, an increase in the rate of meat exchange or a decrease in feed intake, obesity tissue, and a correlation pattern in which the above state is mixed.

29. A method of improving obesity comprising: a composition selected from the group consisting of a lipophyllin compound and a main component of a Jianzhiling compound; and administering an effective amount of the composition to the animal.

30. The method of embodiment wherein the animal is a human, a bird, a livestock, or a fish.

31. The method according to the embodiment, wherein the livestock animal is a livestock of pigs, cattle, deer, horses, sheep or livestock of mountain pigs, dogs and rabbits.

32. The method of any of the preceding claims, wherein the avian bird is a bird of chicken, duck, geese, turkey or a bird of cockroach, pigeon or ostrich.

33. The method according to the embodiment, wherein the Jianzhiling compound is Jianzhiling-1, Jian One of the compounds of Zhiling-2, Jianzhiling-3, Jianzhiling-4 and Jianzhiling-5; the Jianzhiling compound is a compound of Jianzhiling and a compound selected from the following compounds containing a carboxylic acid group Jianzhiling compound: Statin drug, sodium carboxymethyl cellulose (sodium CMC), high molecular polymer, polyglutamic acid group derivatives and their mixed forms.

references

1. Robidoux J, Martin TL, Collins S. Beta-adrenergic receptors and regulation of energy expenditure: a family affair. Annu Rev Pharmacol Toxicol 2004;44:297-323.

2. Andrea Armani, Vincenzo Marzolla, Giuseppe M.C. Rosanol, Andrea Fabbri and Massimiliano Caprio. Phosphodiesterase type 5 (PDE5) in the adipocyte: a novel player in fat metabolism? Trends in Endocrinology and Metabolism. 2011; 22, 404-411.

3. Koh EH, Kim M, Ranjan KC, Kim HS, Park HS, Oh KS, Park IS, Lee WJ, Kim MS, Park JY, Youn JH, Lee KU. eNOS plays a major role in adiponectin synthesis in adipocytes. Am J Physiol Endocrinol Metab. 2010; 298(4): E846-53.

4. Chen N, et al., Green tea, black tea, and epigallocatechin modify body composition, improve glucose tolerance, and differentially alter metabolic gene expression in rats fed a high-fat diet. Nutr Res. 2009 Nov;29(11) :784-93.

Claims (10)

A composition for improving the meat change rate, comprising: a main component selected from the group consisting of a Jianling compound and a Jianzhiling compound. The composition of claim 1, wherein the Jianzhiling compound is Jianzhiling-1, Jianzhiling-2, Jianzhiling-3, Jianzhiling-4 and Jianzhiling-5 compound. One. The composition of claim 1, wherein the Jianzhiling compound is a Jianzhiling compound and a Jianzhiling compound synthesized from the following compound containing a carboxylic acid group structure: Statin, carboxymethyl Sodium cellulose (sodium CMC), high molecular polymer, polyglutamic acid group derivatives and mixed forms thereof. The composition according to claim 1, wherein the meat exchange rate is related to the following: blood fat content, adipose tissue content, meat change rate increase or decrease of feed intake, obesity tissue, and mixing of the above states. Related patterns. The composition of claim 1, which is for use in birds, livestock, fish. A composition for improving obesity comprising: a main component selected from the group consisting of a Jianling compound and a Jianzhiling compound. The composition of claim 6 is for use in humans, birds, livestock, fish. A method for improving obesity, comprising: providing a composition formed from a main component of a compound of a Zhilingling compound and a Jianzhiling compound; An effective amount of the composition is administered to the animal. The method of claim 8, wherein the animal is a human, a bird, an animal, or a fish. The method of claim 8, wherein the Jianzhiling compound is one of Jianzhiling-1, Jianzhiling-2, Jianzhiling-3, Jianzhiling-4 and Jianzhiling-5 compound. ; Jianzhiling compound is a Jianzhiling compound and a Jianling compound compound selected from the following compounds containing a carboxylic acid group structure: Statin drugs, sodium carboxymethyl cellulose (sodium CMC), high molecular weight polymer , polyglutamic acid group derivatives and their mixed forms.