CN106176707B - Application of terpenoid camphene composite ester - Google Patents

Abstract

An application of terpenoid camphene composite ester relates to terpenoid camphene composite ester. The terpenoid camphene composite ester is a compound Tschmgine with a molecular formula of C₁₇H₂₂O₃. The terpene camphene composite ester can be applied to the preparation of medicaments for preventing or treating diabetes, fatty liver, liver cirrhosis, obesity, hyperglycemia, hypertriglyceridemia, hypercholesterolemia, cardiovascular diseases, atherosclerosis, liver injury, inflammation, kidney diseases and the like; can be used for preparing health products for preventing or treating diabetes, fatty liver, liver cirrhosis, obesity, hyperglycemia, hypertriglyceridemia, hypercholesterolemia, cardiovascular diseases, atherosclerosis, liver injury, inflammation, kidney diseases, etc. The camphene composite ester can be used as an active ingredient to be mixed with other medicines, and then the active ingredient is mixed with pharmaceutically acceptable excipient/or administration carrier to prepare a pharmaceutical composition according to the requirements of conventional pharmaceutical method and process, wherein the pharmaceutical composition can be prepared by oral administrationA medicine taking preparation or an injection preparation.

Description

Application of terpenoid camphene composite ester

Technical Field

The invention relates to a terpenoid camphene composite ester, in particular to an application of the terpenoid camphene composite ester.

Background

The compound Tschmgine is a camphene compound ester which is terpene and can be extracted from the root of Ferula ovina of Ferula genus of Umbelliferae, and the molecular formula is C₁₇H₂₂O₃The structural formula is as follows:

tschimgine has been reported to have antibacterial activity against Staphylococcus aureus (Staphylococcus aureus 209), Escherichia coli (Escherichia coli WF +) and Candida albicans (Candida albicans 562).

MTT experiments show that Tschigmine can also inhibit the growth of melanoma cells (SK-MEL-28), ovarian cancer cells (CH1), lung cancer cells (A549) and breast cancer cells (MCF-7).

Diabetes, fatty liver, liver cirrhosis, obesity, hyperglycemia, hypertriglyceridemia, hypercholesterolemia, cardiovascular diseases and atherosclerosis, liver injury, inflammation, kidney diseases and other diseases seriously harm the health and the life of human beings, and the discovery of new drugs for preventing or treating the diseases has important social value and huge economic value.

Disclosure of Invention

The invention aims to provide application of terpenoid camphene composite ester.

The terpenoid camphene composite ester is a compound Tschmgine with a molecular formula of C₁₇H₂₂O₃The structural formula is as follows:

the terpene camphene composite ester can be applied to the preparation of medicines for preventing or treating diabetes, fatty liver, obesity, liver cirrhosis, hyperglycemia, hypertriglyceridemia, hypercholesterolemia, cardiovascular diseases, atherosclerosis, liver injury, inflammation, kidney diseases and the like.

The terpene camphene composite ester can be applied to the preparation of health care products for preventing or treating diabetes, fatty liver, obesity, liver cirrhosis, hyperglycemia, hypertriglyceridemia, hypercholesterolemia, cardiovascular diseases, atherosclerosis, liver injury, inflammation, kidney diseases and the like.

The diabetes may include, but is not limited to, diabetes with symptoms of hyperglycemia.

The fatty liver may include, but is not limited to, non-alcoholic fatty liver and/or fatty liver with hyperlipidemia symptoms.

The cirrhosis may include, but is not limited to, cirrhosis with excessive deposition of hepatic collagen and manifestation of liver fibrosis.

The obesity may include, but is not limited to, obesity with symptoms of hyperlipidemia, hyperglycemia.

The cardiovascular disease may include, but is not limited to, cardiovascular disease with symptoms of hyperglycemia, hypertriglyceridemia, and hypercholesterolemia.

The atherosclerosis may include, but is not limited to, atherosclerosis with symptoms of hypercholesterolemia and/or hyperlipidemia.

The kidney disease may include, but is not limited to, kidney diseases with abnormal manifestations of blood urea nitrogen levels, renal parenchymal lesions, uremia, and the like; the renal parenchymal lesions may include, but are not limited to, glomerulonephritis, interstitial nephritis, acute and chronic renal failure, intrarenal space occupying and destructive lesions, and the like.

The liver injury may include, but is not limited to, liver injury due to administration of a drug having a side effect of liver injury, acute liver injury due to alcohol, liver injury due to alcoholic fatty liver, liver injury due to immunological or chemical factors, liver injury due to viral infection; the drug having the side effect of liver injury includes, but is not limited to, a drug having acetaminophen as a main active ingredient.

The inflammation may include, but is not limited to, inflammation due to administration of a drug having an inflammatory reaction side effect including, but not limited to, a drug having acetaminophen as a main active ingredient, inflammation due to bacteria, viral hepatitis, acute hepatitis due to alcohol, hepatitis due to alcoholic fatty liver, hepatitis due to non-alcoholic fatty liver, hepatitis or nephritis due to diabetes or obesity complications.

The terpene camphene composite ester can be used as an active ingredient to be mixed with other medicines, and can be prepared into a pharmaceutical composition according to the conventional pharmaceutical method and process requirements after being mixed with an acceptable excipient or a drug delivery carrier in the pharmacy, wherein the pharmaceutical composition can be an oral preparation or an injection preparation and the like, and the oral preparation can be tablets, pills, capsules, granules, syrup and the like; the injection preparation can be injection or freeze-dried powder injection and the like.

Experiments prove that the Tschimgine has the effects of treating diseases such as diabetes, fatty liver, obesity, hyperglycemia, hypertriglyceridemia, hypercholesterolemia, cardiovascular diseases, atherosclerosis and the like. After 11 days of intraperitoneal injection of Tschimmine, the relevant metabolic indexes are detected by using db/db of a diabetes and obesity model mouse. The results show that the serum levels of glucose, cholesterol, triglycerides and free fatty acids are significantly reduced in the Tschimgine-treated model mice compared to the blank control. Results of HE staining and oil red staining of a paraffin section of a mouse liver show that the amount of fat in the liver tissue of the mouse in the Tschimgine treatment group is obviously lower than that of the mouse in the control group. These results indicate that Tschimgine has good therapeutic effects in treating diabetes, fatty liver, obesity, hyperglycemia, hypertriglyceridemia, hypercholesterolemia, cardiovascular diseases, atherosclerosis, and the like.

Experiments prove that the Tschimgine can reduce the expression of a marker gene of hepatic fibrosis in the liver, slow down the inflammatory reaction of the liver and effectively eliminate the accumulation of collagen in the liver, and the Tschimgine has a treatment function on cirrhosis.

Experiments prove that Tschimgine has the function of kidney function. Blood urea nitrogen is one of main indexes of renal function, and blood urea nitrogen is too high, which indicates various kidney parenchymal diseases, such as glomerulonephritis, interstitial nephritis, acute and chronic renal failure, intrarenal space occupying and destructive diseases, uremia and the like. The experimental result of a mouse shows that the Tschmgine can effectively reduce the level of blood urea nitrogen, and the Tschmgine has the functions of treating kidney diseases and protecting the kidney function.

Experiments prove that the liver protection and repair function of Tschimgine is proved by using a liver injury model mouse. The invention provides a detection result after 24 hours, wherein Tschimgine is injected into an abdominal cavity of a C57B6/J mouse, and then excessive acetaminophen (N-acetyl-para-aminophenol, abbreviated as APAP) is injected to cause liver injury. The results showed that the liver morphology and liver function of the mice injected with Tschimgine were essentially normal in the case of the control group exhibiting severe liver injury. The Tschimgine has excellent functions of preventing and treating liver injury.

Experiments prove that the Tschimgine has the treatment effect on inflammation on a mouse model. Excessive use of APAP causes inflammation of the liver in mice. The invention provides a method for detecting the level of inflammatory factors in liver tissues of a C57B6/J mouse by injecting Tschimmine into the abdominal cavity of the mouse for 5 days, then injecting excessive APAP to cause liver inflammation and detecting the level of the inflammatory factors in the liver tissues of the mouse. The results show that compared with mice treated by blank control, the gene expression level of various inflammatory factors in the liver tissues of the mice injected with Tschigmine in the abdominal cavity is obviously reduced. The results of the examples at the cellular level show that tschimbine can down-regulate the expression of inflammatory factors due to LPS induction. The Tschimgine has a good function in the aspect of inflammation treatment.

Tschimgine is a natural product extracted from plants, and has no report on the pharmacy for treating diabetes, fatty liver, obesity, hyperglycemia, hypertriglyceridemia, cardiovascular diseases, hypercholesterolemia, atherosclerosis, liver injury, inflammation, kidney diseases and other diseases. Therefore, the Tschmgine provided by the invention has a therapeutic function on the diseases, and is related to the new function of the Tschmgine.

Tschimgine is a natural product extracted from a plant of genus Ferula of family Umbelliferae, has been commercialized, and is easily available as a raw material for pharmaceuticals. Diabetes, fatty liver, obesity, hyperglycemia, hypertriglyceridemia, hypercholesterolemia, cardiovascular diseases, atherosclerosis, liver injury, inflammation, kidney diseases and other diseases are important diseases affecting human health and life, so the commercialized compound Tschmgine provided by the invention has a practical function in preparing medicinal preparations for treating the important diseases, and has important social value and great economic value.

In some embodiments, the compounds of the methods of the invention may be formulated as pharmaceutical compositions for use in some dosing regimens. The pharmaceutical compositions of the invention may comprise the compound itself and a pharmaceutically acceptable salt or carrier therefor. Such compositions may also optionally comprise other therapeutic agents.

Useful delivery vehicles in the above-described pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer media such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose preparations, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, lanolin and self-emulsifying drug delivery systems such as α tocopherol, polyethylene glycol 1000 succinate, or other similar polymeric delivery modes.

As used herein, "metabolic disease" refers to any disease or disorder associated with metabolism. For example, metabolic diseases include, but are not limited to: hyperglycemia, insulin resistance, hyperlipidemia, hypercholesterolemia, diabetes, obesity, metabolic syndrome, metabolic disorders and related diseases, diseases of the liver (liver disease), fatty liver diseases (hepatic lipidosis), nonalcoholic fatty liver disease, hepatitis, nonalcoholic steatohepatitis, cirrhosis, liver fibrosis, chronic and acute liver failure, biliary cirrhosis, primary sclerosing cholangitis, cholestasis, cholelithiasis atherosclerosis, inflammation, cancer, and co-morbidities of the above diseases.

In certain pharmaceutical compositions having only the compounds described herein as their active ingredient, the method of administration may further comprise treating the subject with additional agents or therapies. Such agent therapies include, but are not limited to: anemia treatment, diabetes treatment, hypertension treatment, cholesterol treatment, neurological drugs, drugs that modulate cardiovascular function, drugs that modulate inflammation, immune function, hematopoiesis; hormones and antagonists, chemotherapeutic agents affecting gastrointestinal function, microbial disease, and/or chemotherapy of neoplastic disease.

Certain agents or therapies may be administered in combination with the compounds of the present invention, such as matrix metalloproteinase inhibitors, lipoxygenase inhibitors, cytokine antagonists, immunosuppressive agents, cytokines, growth factors, immunomodulators, prostaglandins or anti-vascular hyperproliferative compounds.

The term "combination" and its related terms as used herein correspond, in turn, to the administration of a therapeutic agent concurrently or sequentially with a compound of the invention. For example, the compound may be administered in a single unit dosage form with another therapeutic agent, either simultaneously or sequentially. Accordingly, the present invention provides a single unit dosage form comprising the compound, an additional therapeutic agent. In many dosing regimens, a patient or individual is generally considered to act in a "combination" if the patient or individual exhibits the relevant therapeutic effect of the agents used simultaneously in a particular target tissue or sample (e.g., in the brain, in serum, etc.) when exposed to two or more agents at the same time.

If the compositions are prepared as pharmaceutically acceptable salts of the compounds of the present invention, the salts are preferably derived from inorganic or organic acids and bases. The acid salt includes: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentane, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanes, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate. Base salts include ammonium salts, alkali metal salts (e.g., sodium and potassium salts), alkaline earth metal salts (e.g., calcium and magnesium salts), and salts with organic bases (e.g., dicyclohexylamine salts, N-methyl-D-glucamine salts, or salts with amino acids such as arginine, lysine, etc.).

The compound compositions of the present invention may also include, depending on the desired formulation, a pharmaceutically acceptable non-toxic carrier or diluent. It is generally defined as a vehicle for formulating pharmaceutical compositions for animal or human use. The appropriate diluent should be selected so as not to affect the biological activity of the composition. Such diluents are: distilled water, physiological phosphate buffered saline, ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic non-therapeutic non-immunogenic stabilizers, and the like.

The compound compositions of the present invention may also include wetting agents, emulsifying agents, and lubricating agents, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants.

The pharmaceutical composition of the present invention may be formulated in solid or liquid form. Suitable administration forms include the following: (1) oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets, buccal, sublingual and systemic absorbents, boluses, powders, granules, pastes for sublingual use; (2) parenteral administration, prepared as sterile solutions or suspensions or sustained release formulations by subcutaneous, intramuscular, intravenous or epidural injection; (3) topical applications, such as creams, ointments, controlled release patches or sprays for the skin, lungs or oral cavity; (4) intrarectal administration, e.g., as a cream or foam; (5) and others: sublingual, ophthalmic, transdermal or nasal, pulmonary and other mucosal uptake.

The process for preparing the pharmaceutical compositions of the compounds comprises combining a compound of the invention with a carrier or a plurality of accessory ingredients using any of the procedures. In general, the compounds of the invention can be prepared in intimate, uniform combination with a carrier, which is a liquid carrier, a finely divided solid carrier, or both, and the product can be shaped as desired.

In some cases, the efficacy of the drug can be prolonged by slowing the rate of slow absorption of the drug from the subcutaneous or intramuscular injection. This can be achieved by preparing a liquid suspension of crystalline or amorphous material which is poorly water soluble. The rate of absorption of the drug depends on its rate of dissolution, which in turn may depend on crystal size and crystalline form. Alternatively, absorption by parenteral administration is delayed by dissolving or suspending the drug in an oily vehicle.

Injectable drug depot formulations can be prepared by combining the compound with a biodegradable polymer (e.g., polylactide-polyglycolide) in a microencapsulation matrix. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Other biodegradable polymers include polyorthoesters and polyanhydrides. Injectable drug depot formulations compatible with body tissues can also be prepared by entrapping the drug in liposomes or microemulsions.

The pharmaceutical compositions of the present invention may be administered orally in any orally acceptable dosage form, including, but not limited to: capsules, tablets, and aqueous suspensions and solutions. For oral tablets, lactose and corn starch are commonly used as coatings, and a lubricant, such as magnesium stearate, is usually added. For oral capsule formulations, useful diluents include lactose and dried corn starch. When administered orally in aqueous suspensions and solutions, as well as propylene glycol formulations, the pharmaceutically active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.

Dosage forms suitable for oral administration as described herein may be in the form of capsules, cachets, pills, tablets, lozenges (usually sucrose and acacia or tragacanth as flavoring substances), powders, granules, or dissolved in an aqueous or non-aqueous liquid to make a solution or suspension, or as a water-in-oil or oil-in-water liquid emulsion, or as an elixir or syrup, or as pastilles (using inert bases such as gelatin and glycerin or sucrose and acacia) and/or by mouth rinsing. Various dosage forms contain a predetermined amount of a compound of the present invention as one of its active ingredients. The compounds of the invention may also be administered in the form of a bolus, electuary or paste.

Liquid dosage forms for oral administration of the compounds of the present invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters sorbitol, and mixtures thereof.

Such administration may be selected when the pharmaceutical composition of the present invention is more effective by topical administration to the targeted treatment area or organ. For topical application to the skin, the active ingredient may be suspended or dissolved in a delivery vehicle and the pharmaceutical composition formulated as an ointment. Topical carriers for the compounds of the present invention include, but are not limited to: mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, the active compound may be suspended or dissolved in a carrier, and the pharmaceutical composition formulated in a suitable lotion or cream, such carriers including, but not limited to: mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of the present invention may also be applied topically to the lower intestinal tract by making into rectal suppositories or suitable enema preparations. The invention also includes the preparation of the compounds into transdermal patches for topical administration.

The pharmaceutical composition of the present invention can be administered by making it into an aerosol or an inhalant. Such agents may be prepared according to techniques widely used in the art of pharmaceutical formulation, and also as saline solutions. Such prior art often uses benzyl alcohol or other suitable preservatives, fluorocarbons and/or other solubilizing or dispersing agents, absorption promoters to enhance their bioavailability.

An additional advantage of transdermal patches is the controlled delivery of the compounds of the present invention to the body. Such dosage forms may be made by dissolving or dispersing the compound in a suitable medium. Absorption enhancers may be used to increase the absorption of the compound by the skin. Control of the rate of passage of the compound through the skin can also be achieved by employing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

Drawings

FIG. 1 shows that Tschimmine treatment can reduce blood glucose in db/db mice.

FIG. 2 is a graph showing that Tschimmine treatment decreased serum triglyceride levels in db/db mice.

FIG. 3 is a graph showing that Tschimmine treatment decreased serum free fatty acid levels in db/db mice.

FIG. 4 is a graph showing that Tschimmine treatment decreased serum total cholesterol levels in db/db mice.

FIG. 5 is a graph showing that Tschimmine treatment decreased blood urea nitrogen levels in db/db mice.

FIG. 6 shows HE staining of paraffin sections of liver showing that Tschigmine treatment reduced adipocyte formation in liver tissue of db/db mice.

FIG. 7 is an oil red stain of cryo-sections of liver tissue demonstrating that Tschimmine treatment reduces fat in liver tissue of db/db mice.

FIG. 8 is a HE staining of paraffin sections of mouse liver showing that Tschimmine can repair APAP-induced liver damage.

FIG. 9 shows that Tschimmine treatment can reduce AST and ALT activities in serum of APAP-induced liver injury mice.

FIG. 10 shows that Tschimgine treatment can reduce LDH activity in serum of APAP-induced liver injury mice.

FIG. 11 is a graph showing that Tschimmine treatment can modulate the expression levels of genes involved in liver repair in mice with APAP-induced liver damage.

FIG. 12 shows that Tschimmine inhibits inflammatory responses due to APAP.

FIG. 13 shows that Tschimmine can inhibit the expression level of the inflammatory factor MIP-1 α in inflammatory responses induced by LPS.

FIG. 14 shows that Tschimmine can suppress the expression level of the inflammatory factor iNOS in inflammatory responses induced by LPS.

FIG. 15 shows that Tschimmine can inhibit the expression level of inflammatory factor TNF α in inflammatory responses induced by LPS.

FIG. 16 shows that Tschimmine can inhibit the expression level of the inflammatory factor IFN gamma in the inflammatory response induced by LPS.

FIG. 17 shows that Tschimmine can inhibit the expression level of the inflammatory factor IL-1 β in inflammatory responses induced by LPS.

FIG. 18 shows that Tschimmine can inhibit the expression level of IL-6, an inflammatory factor in inflammatory responses induced by LPS.

FIG. 19 shows that Tschimmine can inhibit the expression level of the inflammatory factor CD36 in inflammatory responses induced by LPS.

FIG. 20 shows Masson staining showing that Tschigmine reduces collagen deposition in mouse liver.

FIG. 21 is a photograph of sirius red stain showing that Tschmgine can reduce mouse liver collagen fibers.

FIG. 22 shows that Tschimmine can down-regulate the expression of liver fibrosis marker gene in liver.

In fig. 1 to 5,9 to 12, and 22, p <0.05 and p < 0.01.

Detailed Description

The first embodiment is as follows: tschimgine has a role in the treatment of metabolic diseases.

Test method: db/db mouse (strain name BKS. Cg-Dock 7^m+/+Lepr^dbJnju) belongs to a type II diabetes model, and animals start to eat and become fat at one month, and then symptoms such as hyperglycemia, hyperinsulinemia and the like are generated.

The serum samples were collected after 9 p.m. by feeding db/db mice, which were about 10 weeks old, in SPF-grade animal rooms of the university of Xiamen laboratories, which were fed with conventional feed and were freely drunk, Tschimgine was dissolved in DMSO at a high concentration, and then 40% HBC (2-hydroxypypropyl- β -cyclodextin) was prepared at a working concentration such that the DMSO concentration was 10%, and 100. mu.l of Tschimgine solution was intraperitoneally injected at a final working concentration of 10mg/kg (drug/mouse body weight), respectively, a blank control group was directly diluted with an equal amount of DMSO in 40% HBC solution, and was injected at 9 p.m. once daily, and was fasted for 16h after 6 days of Tschigma injection, and after 6h of German was cut off, the mice were tested for glucose metabolism-related indicators including serum triglycerides, free fatty acids and glycerol levels, and serum fraction of Beilange Beijing Lam (BRA) and blood glucose-measuring test strips were collected for routine examination of liver tissue by the ELISA method, and by conventional methods, and by taking a kit for obtaining serum fraction (liver tissue staining).

The oil red dyeing method comprises the following steps: liver tissue was snap frozen in isopentane in liquid nitrogen, embedded at optimal cutting temperature, and cut on a cryostat.

Preparing oil red fuel: 0.5g of oil red O and 100ml of isopropanol (the content is more than 98 percent) are fully dissolved in an oven at 60 ℃ for 24h to form a storage solution. When the dyeing solution is used, 6ml of dyeing stock solution is taken, 4ml of distilled water is added, the dyeing stock solution is diluted, the obtained solution is kept for 5-10 min and then filtered, and the solution cannot be stored for more than 1-2 h.

The dyeing method comprises the following steps:

(1) freezing and slicing the slices to be 10 mu m thick, fixing 10% paraformaldehyde for 10-15 min, and then washing with water;

(2) placing in an oil red dye solution in a sealed container for 5 h;

(3) separating color with 60% ethanol, soaking and slicing until cytoplasm is red and bottom color is white;

(4) washing with water at 37 deg.C for 5 min;

(5) performing hematoxylin light nuclear staining for 10-20 s; washing with water or 1% dimethyl hydrogen phosphate to turn blue;

(7) glycerin water-mixing sealing sheet. And (5) judging a result: fat is red and nuclei are blue.

The differences between the control and drug-treated groups in all examples were analyzed using student's t-test.

And (3) test results: tschimgine treated db/db mice had significantly lower blood glucose after the sixth dose compared to the blank control (FIG. 1). The serum triglyceride (fig. 2), free fatty acid (fig. 3) and total cholesterol (fig. 4) levels of the mice treated with Tschimgine were significantly lower than those of the control group. The Tschimgine can effectively treat the diseases of the diabetic mice.

Non-alcoholic fatty liver disease is a clinical pathological syndrome characterized mainly by fat accumulation in liver cells caused by various reasons without history of excessive drinking, and diffuse liver cell bullous steatosis and lipid accumulation, and is considered as a pathological manifestation of metabolic syndrome in liver. The HE staining results of liver tissue sections showed that significant vacuoles were present in liver cells in the control group, whereas no significant vacuoles were formed in liver cells of Tschimgine-treated mice (fig. 6). The oil red O is fat-soluble dye, can be highly dissolved in fat, and can specifically color neutral fat such as triglyceride in tissue. In routine pathological diagnosis and scientific research work, fat in tissues is often stained with oil red O. To further prove that vacuoles in the HE-stained pictures of liver sections are aggregates of neutral fats such as triglycerides, we analyzed the liver sections by oil red staining. The results show that a plurality of large-drop oil particles are displayed in the liver section of the control group, while the oil particles in the liver section of the mouse treated by the Tschmgine are obviously smaller than those in the control group, the fat content is obviously lower than that in the control group (figure 7), and the results are consistent with the results of HE staining, which shows that the Tschmgine has good curative effect on the fatty liver.

And (4) conclusion: tschimgine has good curative effect on metabolic diseases such as diabetes, non-alcoholic fatty liver, obesity, hyperglycemia, hypertriglyceridemia, hypercholesterolemia and the like.

Excessive blood glucose levels and blood triglyceride levels in the blood are a warning indicator of cardiovascular disease, and excessive levels of these indicators reflect an extremely high risk of cardiovascular disease in the body. Therefore, the Tschmgine treatment in the embodiment can obviously reduce the blood sugar and the blood triglyceride level, and the Tschmgine has a therapeutic effect on cardiovascular diseases.

Research shows that high total cholesterol is in positive correlation with the lesion degree of the carotid atherosclerotic plaque. Therefore, in the embodiment, the Tschigmpine can effectively reduce the serum total cholesterol level when being used for treating the mice, and the Tschigmpine has a curative effect on atherosclerosis.

Example two: application of Tschimgine in treating kidney diseases

The test method comprises the following steps: the kidney is the major organ for urea excretion, and urea is reabsorbed in all tubules after being filtered from the glomerulus, and the higher the flow rate of urine in the tubules, the less reabsorption. When renal function is impaired, glomerular filtration rate decreases, and blood urea nitrogen concentration increases rapidly when filtration rate falls below 50% of normal. Various kinds of renal parenchymal diseases, such as glomerulonephritis, interstitial nephritis, acute and chronic renal failure, intrarenal space occupation and destructive diseases, can increase blood urea nitrogen. Therefore, blood urea nitrogen is one of the main indicators of renal function. Elevated blood urea nitrogen may also be used as a diagnostic indicator of uremia.

Chronic kidney disease is one of the complications of diabetes. This example uses db/db mouse serum treated in example one and uses the urea nitrogen (BUN) test kit (urease method) built into technology Co., Ltd. from Nanjing as a protocol for measuring the level of urea nitrogen in serum.

And (3) test results: the level of blood urea nitrogen was significantly down-regulated in Tschimgine-treated mice compared to the blank control group (fig. 5), indicating the maintenance of renal function and therapeutic effect of Tschimgine on renal disease.

And (4) conclusion: tschimgine has therapeutic effect on various renal parenchymal diseases with blood urea nitrogen level rise, such as glomerulonephritis, interstitial nephritis, acute and chronic renal failure, intrarenal space occupying and destructive diseases, and kidney diseases such as uremia.

Example three: tschimgine tests for liver protection and repair function.

The test method comprises the following steps: the mouse acute drug-induced liver injury model caused by excessive APAP is one of the models commonly used for researching liver-protecting drugs, and APAP is a typical representative of drug-induced liver injury caused by active oxygen or active nitrogen. Excessive use of APAP causes increase of active oxygen in liver, excessive consumption of reduced substances such as GSH in vivo causes decrease of GSH level in vivo, and activity of aspartate aminotransferase AST, alanine aminotransferase ALT, lactate dehydrogenase LDH and the like is increased, so that inflammation or necrosis of liver tissue is caused. In this embodiment, the model is used to detect the protection and repair functions of Tschimgine on liver injury induced by APAP.

Tschimgine was dissolved in DMSO at a high concentration in 8-week-old C57B6/J wild-type mice in SPF-grade animal rooms at the center of the laboratory animals of Xiamen university, and free drinking water was added, Tschimgine was dissolved in DMSO at a high concentration, 40% HBC (2-hydroxypypropyl- β -cyclodextin) was used as a working concentration, DMSO was adjusted to a concentration of 10%, 100. mu.l of Tschimgine solution was intraperitoneally injected at a final working concentration of 10mg/kg and 40mg/kg (drug/mouse body weight), respectively, blank control group was directly diluted in 40% HBC solution with DMSO at a final working concentration of 10mg/kg and 40mg/kg (drug/mouse body weight), mice were sacrificed after 500mg/kg of APAP.24h prepared by fresh dissolution in PBS at 3 pm on the fifth day, a part of liver tissue was fixed with paraformaldehyde, paraffin, sliced, stained HE, frozen in a part of liver tissue, RNA was extracted with Trizol reagent, and then cDNA was subjected to RT-PCR for real-time detection of fluorescence-PCR using Masterx PCR for quantitative detection of serum-PCR.

And (3) test results: the results of the HE staining of the liver sections are shown in FIG. 8, and pathological sections of the blank control group show that the infiltration of cells in liver lobules is obvious, the liver lobules are vacuolated and necrotic, a large amount of inflammatory cells infiltrate in the lobules and the manifold area, the cells are turbid, the nuclei are condensed or dissolved and broken, and the liver cell cords are fuzzy. The liver morphology of mice injected with Tschimgine appeared essentially normal compared to the significantly severe liver injury of the blank control group. AST and ALT activities (as in FIG. 9) and LDH activities (as in FIG. 10) in serum of mice using Tschimmine were significantly lower than the high activities caused by APAP in the blank control group. The results of the expression levels of the genes related to liver injury in liver tissues also show that Tschmgine can up-regulate the expression levels of functional genes GCLM, GSTa3, GPX1 and UGT1a1 which are closely related to the metabolism of exogenous toxic substances (as shown in figure 11), and the Tschmgine can regulate, protect and repair the liver function through the expression regulation of the genes related to the metabolism of exogenous drugs.

And (4) conclusion: tschimgine has good protection and repair functions in the aspect of liver injury.

Example four: tschimgine has a therapeutic effect on inflammation.

The test method comprises the following steps: inflammatory factors are substances produced and secreted by cells during inflammation and involved in inflammatory reactions, and are markers of reactive inflammation. In this example, the expression level of inflammatory factors in liver tissue was detected by real-time fluorescent quantitative PCR using realmastermix (sybr Green i) kit from tiangen biochem ltd after extracting RNA from mouse liver tissue using Trizol reagent and reverse transcribing the RNA into cDNA using wild type C57B6/J mice treated in example three, which induced liver inflammation by APAP.

Compared with a blank control group, the gene expression levels of various inflammatory factors such as iNOS, TGF β, TNF α, COX2, IL-1 β, IFN gamma, MIP-1a and the like in the liver tissue of a mouse injected with Tschmgine in the abdominal cavity are obviously lower than those of the blank control group (figure 12), and the Tschmgine can inhibit inflammatory reaction.

And (4) conclusion: tschimgine has therapeutic effects on inflammation, including inflammation caused by chemical factors.

Example five: tschimgine inhibits inflammatory responses induced by LPS

The test method comprises the following steps: inflammation is a common and important basic pathological process in animals, and any factor capable of causing tissue damage can cause inflammation. Bacterial Lipopolysaccharides (LPS) are a major pathogenic factor of gram-negative bacteria, which can cause inflammatory reactions in the body. The cell or organism inflammatory reaction induced by LPS is often used for the research of the action and mechanism of gene or medicine in inflammation.

This example uses primary hepatocytes extracted from C57B6/J wild-type mice, the cells being arranged at 5X 10⁵One cell per well, which is divided into 6-well plates, after the cells in two groups are cultured overnight in high-sugar DMEM (containing 10% fetal calf serum) culture medium, the fresh culture medium (drug group) containing 20 mu M Tschimidine (dissolved in DMSO) is replaced, the fresh culture medium containing DMSO in the same volume of the drug treatment group is used as a control group (control group), after 18 hours of treatment, LPS (drug-induced group) in 20 mu g/ml is added in one group of the drug group, and DMSO in the same volume is added in the other group of the drug group as a control group (drug-non-induced group); one group of the control groups was supplemented with LPS at 20. mu.g/ml (control-induced group), and the other group was supplemented with an equal volume of DMSO as a control (control-non-induced group). LPS is added for treatment for 6h, cells are harvested, total RNA extraction kit of Omega company is used for extracting cell RNA, reverse transcription kit of TAKARA company is used for reverse transcription to form cDNA, real-time fluorescence quantitative PCR reaction is carried out by RealMasterMix (SYBR GreenI) kit of Tiangen Biochemical technology limited company, and the expression level of inflammatory factors in the cells is detected.

The test results show that compared with a non-induced group, the expression of each inflammatory factor in cells induced by LPS is obviously increased, which indicates that the inflammatory reaction of the cells induced by LPS is generated, and the expression levels of a plurality of inflammatory factors including MIP-1a, iNOS, TNF α, IFN gamma, IL-1 β, IL-6, CD36 and the like are obviously reduced in the cells of the induced group treated by Tschimgine, which indicates that the Tschimgine can effectively reduce the level of the inflammatory factors in the cells and relieve inflammatory symptoms.

And (4) conclusion: tschimgine has therapeutic effects on inflammation, including bacterial inflammation.

Example six efficacy of Tschimgine on liver cirrhosis.

The test method comprises the following steps: fatty liver is a liver disease caused by excessive accumulation of fat in the liver. Excessive fat is accumulated in the liver for a long time, which affects the supply of blood and oxygen of the liver and the metabolism of the liver, gradually causes a large amount of liver cell swelling, inflammatory infiltration and degenerative necrosis, and once fibroplasia and false lobule are formed, cirrhosis can be caused, and the risk of liver cancer is greatly increased. The liver tissue of the patients with liver cirrhosis has increased content of various collagens, and the type I collagen fiber is increased and participates in the formation of connective tissue. As the degree of cirrhosis progresses and worsens, the deposition of type I collagen increases and the ratio of type I collagen to other types of collagen increases. Therefore, this example identifies whether the Tschimgine treatment can alleviate the symptoms of collagen deposition in the liver tissue of mice with excessive fat accumulation in the liver by the Masson's staining method and the sirius red staining method, and detects the expression level of collagen by the fluorescent quantitative PCR method.

The method comprises the following steps:

one of the staining methods used by Masson's stainer to visualize fibers in tissue is the authoritative and classical technique for staining collagen fibers. The Masson's staining method is based on the difference of the gap and size of different tissues and cells, so the permeability of the tissues is different, wherein the collagen fiber has a relatively loose structure and high permeability, so that the dye of macromolecule such as aniline blue (blue) can enter into the staining, and the red blood cell and muscle fiber part in the tissues have smaller gap, so that the aniline blue can not enter into the staining. The method can distinguish the presence and accumulation degree of collagen fibers in the tissue. Widely used for research of connective tissues, muscle tissues and collagen, etc. The dyeing method has the advantages of simple operation, stable performance and clear color development.

In this example, Masson staining was performed on the liver of the mouse treated in the first example according to the kit specification using Masson rapid staining kit (product number D026) from south beijing technologies ltd, and the staining results were: the collagen fibers are blue, the cytoplasm, muscle, cellulose and glia are red, and the nucleus is bluish purple.

The second method comprises the following steps:

the four types (I, II, III and IV) of collagen are known to distinguish and quantify the four types of collagen fibers, and have certain significance for researching the occurrence mechanism and the evolution process of pathological changes. By utilizing the characteristics of different collagen polymerization and winding spiral arrangement, the four types of collagen fibers can be respectively displayed according to different birefringence and coloring under a polarized light microscope by using a Sirius Red bitter (Sirius Red) acid dyeing method.

The specific method comprises the following steps:

preparing saturated picric acid: adding purified water into the picric acid powder, and dissolving for two days to obtain saturated solution with precipitate. Sirius red saturated bitter acid: 0.1g of Sirius Red, dissolved in 100ml of saturated picric acid solution.

And (3) dyeing: neutral formalin was used to fix the tissue, paraffin sections were cut and dewaxed to water by routine. Hematoxylin staining for 1min, and washing with distilled water for 3 times. The saturated bitter acid of the sirius red is heavily infected for 15-30min, and the absolute ethyl alcohol is directly differentiated and dehydrated. Xylene transparent, neutral gum blocking.

And (3) dyeing results: the sections after being dyed and sealed need to be observed and photographed by a polarizing microscope in time to keep bright colors. Four types of collagen fibers were observed under a polarization microscope. Type i collagen fibers: closely arranged, exhibiting strong birefringence, and yellow or red fibers. Type ll collagen fibers: shows weak double refraction and is in loose net distribution with various colors. Dish-type collagen fiber: shows weak double-refraction and is a green fine fiber. Type iv collagen fibers: the basement membrane, which showed weak birefringence, was pale yellow.

In this example, the collagen fiber in the liver tissue of the mouse treated in the first example was stained by sirius red staining method.

The third method comprises the following steps:

real-time fluorescent quantitative PCR: in the first example, a part of liver tissue of a mouse was frozen with liquid nitrogen, RNA was extracted from the liver tissue using Trizol reagent, and after reverse transcription into cDNA using a reverse transcription kit from Takara, real-time fluorescent quantitative PCR reaction was carried out using RealMasterMix (SYBR Green I) kit from Tiangen Biochemical technologies, Inc., and reaction was carried out using CFX96 fluorescent quantitative PCR instrument from Bio-rad. Detecting the expression of the collagen related gene in the liver tissue.

As shown in FIG. 20, in the results of the Masson staining, it can be seen that the control group liver slices have obvious blue-stained collagen deposition, and the Tschimgine-treated mouse liver slices have no obvious blue collagen deposition, the Tianlangasin red staining result is consistent with the Masson staining result (as shown in FIG. 21), the control group liver slices have obvious red collagen fibers, while the Tschimgine-treated mouse liver slices have no obvious red collagen deposition, the fluorescent quantitative PCR result shows (as shown in FIG. 22), the expression level of the hepatic fibrosis marker genes α 1(I) collagen, α 2(I) collagen, α -SMA and MMP2 genes in the Tschimgine-treated mouse liver is obviously lower than that of the control group.

And (4) conclusion: in combination with the results of the above examples, Tschimgine can slow down inflammatory reaction of liver cells and tissues, reduce fat accumulation of liver tissues, and effectively eliminate collagen accumulation in liver, so that Tschimgine can prevent and treat liver cirrhosis diseases.

Claims (1)

1. The use of camphene composite ester Tschmgine as terpenoid in preparing medicine for treating nonalcoholic fatty liver disease, hypertriglyceridemia and hypercholesterolemia; the terpenoid camphene composite ester is a compound Tschmgine with a molecular formula of C₁₇H₂₂O₃The structural formula is as follows:

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