Application of n-butylphthalide

Abstract

The invention provides an application of n-butylphthalide. According to the application, n-butylphthalide is used for providing a combination for inducing stem cells to differentiate into fat cells; the invention relates to an application of n-butylphthalide in preparation of a composition. The composition is used for inhibiting white fat accumulation, promoting white fat to be converted into brown fat, inhibiting weight gain, inhibiting obesity, reducing the liver fat content of an individual, reducing the fat content of liver cells, improving the antioxidant activity of the liver and / or reducing the content of triglyceride, glucose and total cholesterol in blood. The present invention relates to a pharmaceutical composition for preventing or treating obesity, preventing metabolic syndrome associated with obesity, preventing or treating fatty liver, improving liver lesion caused by fatty liver, and / or reducing liver damage in an individual, and to a use of n-butylphthalide in the manufacture of a pharmaceutical composition for preventing or treating obesity, preventing metabolic syndrome associated with obesity, preventing or treating fatty liver, and to a use of n-butylphthalide in the manufacture of a pharmaceutical composition for preventing or treating liver lesion caused by fatty liver.

Description

[0001] This application is a divisional application. The application number of the original application is CN202110354354.6, the application date is April 1, 2021, and the name of the invention is Application of n-butylphthalide. Technical Field

[0002] The present invention relates to the use of n-butylphthalide, particularly the use of n-butylphthalide to induce stem cells to differentiate into brown adipocyte-like cells and related applications thereof, including the use of n-butylphthalide to inhibit white fat accumulation, promote the conversion of white fat into brown fat, inhibit weight gain, inhibit obesity, reduce liver fat content in an individual, reduce fat content in hepatocytes, enhance liver antioxidant activity, and / or reduce blood triglyceride, glucose, and total cholesterol levels; and the use of n-butylphthalide to prevent or treat obesity, prevent obesity-related metabolic syndrome, prevent or treat fatty liver, improve liver lesions caused by fatty liver, and / or reduce liver damage in an individual. Background Art

[0003] White fat is composed of white adipocytes, giving it a white appearance. Its morphology consists of single, large fat droplets, and its primary function is energy storage. Brown fat, on the other hand, is composed of brown adipocytes that contain numerous mitochondria, and its primary function is energy metabolism. Uncoupling protein 1 (UCP1) is also expressed at higher levels in brown adipocytes. It can undergo oxidation via the electron transport chain in the mitochondrial uncoupled respiratory chain, but cannot undergo phosphorylation, thus not producing ATP. This reaction promotes nutrient and oxygen consumption, but the free energy released in the process is dissipated as heat. Regular exercise and cold stimulation are known to promote browning of fat, stimulating energy expenditure and reducing body fat. Numerous studies have confirmed that browning of fat, in addition to its application in obesity treatment, also plays a crucial role in aging, pathogenic infection, inflammation, heart disease, cancer, and neurodegenerative diseases. Therefore, although research has shown that brown fat cells can be injected into animals through cell transfusion, the industry is still committed to developing methods that effectively promote fat browning in animals and methods that can effectively solve the problem of difficulty in obtaining brown fat cells.

[0004] Statistics show that almost everyone with a body mass index (BMI) of 30 or greater has fatty liver disease. However, even with a normal BMI, people with unhealthy lifestyles or excess abdominal fat may be at high risk for fatty liver disease. In the past, fatty liver disease was almost exclusively linked to alcoholism, but with modern eating disorders and a lack of exercise, it has become a 21st-century epidemic. Surveys show that a minimum weight loss of 9% is required to improve the histological structure of non-alcoholic fatty liver disease. While many medications marketed as effective for weight loss or management aim to achieve this, most of these work by increasing satiety, suppressing appetite, or inhibiting fat absorption. These medications often come with numerous side effects, such as headaches, hypoglycemia, constipation, insomnia, oily stools, and can even increase the risk of heart disease and stroke. Therefore, there is a need to continuously develop methods or medications with minimal side effects that are effective in preventing and treating obesity and its associated metabolic syndrome.

[0005] Approximately one-quarter of adults worldwide suffer from non-alcoholic fatty liver disease (NAFLD), which can develop into chronic liver disease. Severely fatty livers develop NAFLD, which increases the risk of developing fibrosis, cirrhosis, and liver cancer, ultimately leading to terminal liver disease and the need for a liver transplant. In Japan, fatty liver disease is the leading cause of liver diseases such as cirrhosis and liver cancer. According to statistics, the number of people diagnosed with NAFLD in the United States, Japan, and five European countries (the United Kingdom, France, Germany, Italy, and Spain) is expected to reach 18 million by 2027. However, standard lifestyle interventions (such as dietary restrictions or increased physical activity) are difficult to implement effectively, and if these interventions are not maintained, they cannot effectively control and improve NAFLD. Besides dietary restrictions or increased physical activity, bariatric surgery is another treatment option for NAFLD. However, studies have found that the prevalence of NAFLD is higher in obese patients who undergo bariatric surgery, and 12% of patients with NAFLD after surgery develop fibrosis in the later stages.

[0006] Since the US Food and Drug Administration has yet to approve any medications for the treatment of non-alcoholic fatty liver disease (NAFLD), the following 10 drug categories are used to treat various stages of the disease: antioxidants (such as vitamin E, glutathione, and ursodeoxycholic acid), peroxisome proliferator-activated receptor agonists (such as pioglitazone, bezafibrate, fenofibrate, and saroglitazar), diabetes medications, lipid-lowering drugs (such as ezetimibe, pemafibrate, and aramchol), hypertension medications (such as angiotensin II receptor antagonists), FXR (Farnesoid X-receptor) ligands (such as obeticholic acid), anti-inflammatory drugs (such as pentoxifylline), anti-apoptotic drugs (such as selonsertib), enteric bacteria, and anti-fibrotic drugs (such as cenicriviroc). However, all of these drugs in clinical trials for NAFLD have side effects to varying degrees. For example, while the anti-inflammatory effects of vitamins can improve non-alcoholic steatohepatitis (NAH), they can also increase the risk of prostate cancer and bleeding. While pioglitazone can improve fatty liver damage, insulin resistance, and increase liver enzymes by regulating glucose and lipid metabolism, clinical trials have shown significant side effects, including an increase of 3 to 5 kg in weight, heart damage, bone loss, and an increased risk of bladder cancer. The FXR ligand agonist obeticholic acid can improve liver fibrosis, but it is also associated with itching and dyslipidemia. Consequently, the industry remains committed to developing therapeutics and treatments for fatty liver disease and related liver diseases (hepatitis, fibrosis, cirrhosis, and liver cancer). Summary of the Invention

[0007] n-Butylphthalide is a small molecule compound. The inventors of this case have found that administering n-butylphthalide to mice induced to obesity by a high-fat diet can provide the following effects: (1) inhibiting weight gain; (2) reducing the accumulation of subcutaneous fat and visceral fat; (3) promoting the conversion of white fat into brown fat; (4) reducing the levels of triglycerides, glucose and total cholesterol in the blood of mice; (5) reducing the level of low-density lipoprotein cholesterol, atherosclerosis index, cardiovascular risk index and triglyceride-glucose index (TyG); (6) reducing liver damage index (alanine aminotransferase, aspartate aminotransferase); (7) improving antioxidant activity indicators (SOD, CAT and GPx); (8) improving oxygen consumption rate and energy consumption rate; and (9) reducing the accumulation of lipid droplets in liver cells. In addition, cell experiments found that n-butylphthalide treatment can provide the following effects: (1) inhibit the accumulation of oil droplets in adipocytes; (2) reduce the triglyceride content of adipocytes; (3) reduce the fat content of hepatocytes; and (4) reduce the accumulation of lipid oil droplets in hepatocytes.

[0008] Therefore, one object of the present invention is to provide a use of n-butylphthalide in the manufacture of a composition, wherein the composition is used to inhibit white fat accumulation, promote the conversion of white fat to brown fat, inhibit weight gain, inhibit obesity, reduce liver fat content, reduce liver cell fat content, enhance liver antioxidant activity, and / or reduce blood triglyceride, glucose, and total cholesterol levels. Preferably, the composition is a pharmaceutical composition, a food composition, or a feed composition. More preferably, the food composition is a health food, a health supplement, a functional food, a nutritional supplement, or a special nutritional food.

[0009] Another object of the present invention is to provide a use of n-butylphthalide in the manufacture of a pharmaceutical composition, wherein the pharmaceutical composition is used to prevent or treat obesity, prevent obesity-related metabolic syndrome, prevent or treat fatty liver disease, ameliorate liver disease caused by fatty liver disease, and / or reduce liver damage in a subject. Preferably, the obesity-related metabolic syndrome is at least one of diabetes, cerebrovascular disease, cardiovascular disease, hypertension, and nephropathy; the fatty liver disease is non-alcoholic fatty liver disease; the liver disease is liver fibrosis, cirrhosis, hepatitis, or liver cancer; and the liver damage is at least one of liver tissue damage and liver function damage.

[0010] Another object of the present invention is to provide a combination comprising: (1) a component that induces stem cells to differentiate into adipocytes; and (2) n-butylphthalide. The component that induces stem cells to differentiate into adipocytes is selected from the group consisting of dexamethasone, insulin, 3-isobutyl-1-methylxanthine (IBMX), rosiglitazone, doxycycline, triiodothyronine (T3), indomethacin, transferrin, selenium, and combinations thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The graph shows the changes in body weight of mice in the "standard diet" group, the "high-fat diet" group, the "NBP (80 mg / kg)" group, and the "NBP (200 mg / kg)" group during the feeding period (weeks 1 to 24). The mice in the "standard diet" group and the "high-fat diet" group were fed a standard diet and a high-fat diet, respectively; the mice in the "NBP (80 mg / kg)" group and the "NBP (200 mg / kg)" group were fed a high-fat diet and additionally administered 80 mg / kg and 200 mg / kg of n-butylated phenyl peptide, respectively, by tube feeding.

[0012] Figure 2 Shown is a graph showing the changes in food intake of the above groups of mice during the feeding period;

[0013] Figure 3 Shown are photographs of the body appearance of the mice in each group on the last day of the feeding period;

[0014] Figure 4 Shown is a bar graph of the food conversion efficiency of the above groups of mice;

[0015] Figure 5 The figure shows the oxygen consumption (VO2) of mice in the "high-fat diet" group and the "NBP (200 mg / kg)" group over a 24-hour period.

[0016] Figure 6 Shown are the exhaled carbon dioxide (VCO2) of mice in the "high-fat diet" group and the "NBP (200 mg / kg)" group over a 24-hour period;

[0017] Figure 7 Shown is the energy expenditure of mice in the "high-fat diet" group and the "NBP (200 mg / kg)" group over a 24-hour period;

[0018] Figure 8 The figure shows the respiratory exchange ratio of mice in the "high-fat diet" group and the "NBP (200 mg / kg)" group over a 24-hour period;

[0019] Figure 9 Shown are photographs of the appearance of subcutaneous adipose tissue (two images on the upper left), epididymal adipose tissue (two images on the lower left), heart (upper right), liver (middle right), and kidney (lower right) of the above-mentioned groups of mice;

[0020] Figure 10 Shown is a bar graph of the subcutaneous fat weight of the above-mentioned groups of mice;

[0021] Figure 11 Shown is a bar graph of epididymal fat weights of the above groups of mice;

[0022] Figure 12 Shown is a bar graph of the liver weights of the above groups of mice;

[0023] Figure 13 Shown is a bar graph of the blood glucose levels of the above groups of mice;

[0024] Figure 14 Shown is a bar graph of the triglyceride content in the blood of the above-mentioned groups of mice;

[0025] Figure 15 Shown is a bar graph of the total cholesterol content in the blood of the above-mentioned groups of mice;

[0026] Figure 16 Shown is a bar graph of the low-density lipoprotein cholesterol levels in the blood of the above-mentioned groups of mice;

[0027] Figure 17 Shown is a bar graph of the ratio of low-density lipoprotein cholesterol to high-density lipoprotein cholesterol in the blood of the above-mentioned groups of mice;

[0028] Figure 18 Shown are bar graphs of the atherosclerosis index of the above groups of mice;

[0029] Figure 19 Shown is a bar graph of the cardiovascular risk index of the above-mentioned groups of mice;

[0030] Figure 20 Shown are bar graphs of the triglyceride-glucose index of the above-mentioned groups of mice;

[0031] Figure 21 Shown is a bar graph of alanine transaminase activity in the above-mentioned groups of mice;

[0032] Figure 22 Shown is a bar graph of aspartate transaminase activity in the above-mentioned groups of mice;

[0033] Figure 23 Shown is a bar graph of superoxide dismutase activity in the above groups of mice;

[0034] Figure 24 Shown is a bar graph of catalase activity in the above groups of mice;

[0035] Figure 25 Shown is a bar graph of glutathione peroxidase activity in the above groups of mice;

[0036] Figure 26 Shown are photographs of the staining results of liver tissue sections of the above-mentioned groups of mice;

[0037] Figure 27 Shown are photographs of the staining results of subcutaneous adipose tissue sections of the above-mentioned groups of mice;

[0038] Figure 28 Shown are photographs of the staining results of epididymal adipose tissue sections of the above-mentioned groups of mice;

[0039] Figure 29Shown are photographs of the results of red oil staining of adipose-derived stem cells (i.e., the "undifferentiated" group), as well as the "differentiated" group, the "differentiated + NBP (2 μg / ml) group," the "differentiated + NBP (10 μg / ml) group," and the "differentiated + NBP (50 μg / ml) group." The "differentiated" group cells were obtained by culturing adipose-derived stem cells in an adipogenic differentiation medium for 14 days. The cells in the "differentiated + NBP (2 μg / ml) group," the "differentiated + NBP (10 μg / ml) group," and the "differentiated + NBP (50 μg / ml) group" differed from the cells in the "differentiated" group only in that different concentrations of n-butylated phenyl peptide were added to the culture medium on day 8 of culture.

[0040] Figure 30 Shown are bar graphs of the relative absorbance values at 490 nanometers (nm) of the cells in the "undifferentiated" group, "differentiated" group, "differentiated + NBP (2 μg / ml) group," "differentiated + NBP (10 μg / ml) group," and "differentiated + NBP (50 μg / ml) group" after red oil staining;

[0041] Figure 31 Shown are the percentage increases in oil droplets in the above-mentioned groups of cells;

[0042] Figure 32 Shown are the triglyceride contents in the cells of the above groups;

[0043] Figure 33 Shown are photographs of the cell morphology of hepatocytes after different treatments. DETAILED DESCRIPTION

[0044] The detailed technical contents and some specific implementation plans of the present invention will be described in the following content so that ordinary technicians in the field to which the present invention belongs can understand the characteristics of the present invention; however, without departing from the spirit of the present invention, the present invention can also be practiced in a variety of different forms of solutions, and the scope of protection of the present invention should not be interpreted as limited to the contents specifically stated in the description.

[0045] Unless otherwise specified herein, the terms "a", "an", "the" and similar terms used in this specification (especially in the claims) should be understood to include both singular and plural forms; the so-called "individual" refers to a human or non-human mammal (e.g., a dog or cat).

[0046] n-Butylphthalide has a neuroprotective effect and has been approved by the China Food and Drug Administration as a clinical drug for ischemic stroke. The inventors of this case have found that administering n-butylphthalide to obese mice induced by a high-fat diet can provide the following effects: (1) inhibit weight gain; (2) reduce the accumulation of subcutaneous fat and visceral fat; (3) promote the conversion of white fat into brown fat; (4) reduce the levels of triglycerides, glucose, and total cholesterol in the blood of mice; (5) reduce the level of low-density lipoprotein cholesterol, atherosclerosis index, cardiovascular risk index, and triglyceride-glucose index (TyG); (6) reduce liver damage index (alanine aminotransferase, aspartate aminotransferase); (7) enhance antioxidant activity indicators (SOD, CAT, and GPx); (8) enhance oxygen consumption rate and energy consumption rate; and (9) reduce the accumulation of lipid droplets in liver cells. In addition, cell experiments found that n-butylphthalide treatment can provide the following effects: (1) inhibit the accumulation of oil droplets in adipocytes; (2) reduce the triglyceride content of adipocytes; (3) reduce the fat content of hepatocytes; and (4) reduce the accumulation of lipid oil droplets in hepatocytes.

[0047] Therefore, the present invention relates to the use of n-butylphthalide, and in particular, to the use of n-butylphthalide to induce stem cells to differentiate into brown adipocyte-like cells and related applications thereof, including: using n-butylphthalide to provide a composition for inducing stem cell differentiation into adipocytes; using n-butylphthalide to manufacture a composition for inhibiting white fat accumulation, promoting the conversion of white fat into brown fat, inhibiting weight gain, inhibiting obesity, reducing liver fat content in a subject, reducing fat content in hepatocytes, increasing liver antioxidant activity, and / or reducing blood triglyceride, glucose, and total cholesterol levels; and using n-butylphthalide to manufacture a pharmaceutical composition for preventing or treating obesity, preventing obesity-related metabolic syndrome, preventing or treating fatty liver, ameliorating liver lesions caused by fatty liver, and / or reducing liver damage in a subject.

[0048] The n-butylphthalide of the present invention is a small molecule compound that can be extracted and purified from celery seeds, or purchased commercially or prepared by known synthetic methods. The composition provided by the present invention can be a pharmaceutical composition, a food composition, or a feed composition.

[0049] In the application according to the present invention, examples of metabolic syndrome related to obesity include: diabetes, cerebrovascular disease, cardiovascular disease, hypertension and kidney disease; examples of fatty liver include: non-alcoholic fatty liver disease; examples of liver disease include: liver fibrosis, cirrhosis, hepatitis and liver cancer; and examples of liver damage include: liver tissue damage and liver function damage.

[0050] The pharmaceutical composition provided by the present invention can be used for systemic or local administration and can be delivered through various drug delivery systems (DDS), including oral drug delivery systems, transdermal drug delivery systems, injectable drug delivery systems, inhalation drug delivery systems, and transmucosal drug delivery systems. For example, but not limited to, the pharmaceutical composition provided by the present invention can be delivered by systems such as liposomes, microcapsules, nanoparticles, and microneedles to achieve the effects of improving bioavailability, controlling drug release rate, accurately delivering the drug to the lesion, and reducing drug side effects.

[0051] The pharmaceutical composition provided by the present invention can be in any suitable form without particular limitation, and can be in a suitable dosage form depending on the intended use. For example, but not limited to, the pharmaceutical composition can be administered orally, transdermally (e.g., patch, ointment, etc.), intravenously (including intravenous infusion and rapid injection), intramuscularly, subcutaneously, intraarterially, intraperitoneally, subcutaneously, interstitially, through the respiratory tract (e.g., spray, nasal drops, etc.), through the mucosa (e.g., orally disintegrating tablets, etc.) to an individual in need thereof. Depending on the form of use and the intended use, a pharmaceutically acceptable carrier can be selected to provide the pharmaceutical composition, wherein the carrier is known in the art and includes excipients, diluents, adjuvants, stabilizers, absorption promoters, disintegrating agents, solubilizers, emulsifiers, antioxidants, adhesives, binders, viscosity-increasing agents, dispersants, suspending agents, lubricants, hygroscopic agents, etc.

[0052] Taking oral dosage forms as an example, the pharmaceutical composition can be provided in a dosage form suitable for oral administration by any appropriate method. Liquid dosage forms suitable for oral administration include syrups, oral solutions, suspensions, elixirs, and the like. Solid dosage forms suitable for oral administration include powders, granules, buccal tablets, sugar-coated tablets, enteric-coated tablets, chewable tablets, blister tablets, film-coated tablets, capsules, and long-acting sustained-release tablets. The pharmaceutical composition provided according to the present invention may contain any pharmaceutically acceptable carrier that does not adversely affect the desired benefits of n-butylphthalide. For example, but not limited to, pharmaceutically acceptable carriers for the aforementioned liquid dosage forms include: water, saline, dextrose, glycerol, ethanol or its analogs, oils (such as olive oil, castor oil, cottonseed oil, peanut oil, corn oil, and germ oil), glycerol, polyethylene glycol, and combinations thereof; examples of pharmaceutically acceptable carriers for the aforementioned solid dosage forms include: cellulose, starch, kaolinite, bentonite, sodium citrate, gelatin, agar, carboxymethyl cellulose, gum arabic, algin, glyceryl monostearate, calcium stearate, and combinations thereof.

[0053] The above-mentioned dosage form suitable for transdermal administration may contain any pharmaceutically acceptable carrier that does not adversely affect the desired effect of n-butylphthalide contained in the pharmaceutical composition of the present invention, such as water, mineral oil, propylene glycol, polyethylene oxide, liquid paraffin, sorbitan monostearate, and polysorbate 60. The pharmaceutical composition may be provided in a dosage form suitable for transdermal administration by any suitable method, such as, but not limited to, an emulsion, cream, oil, gel (e.g., hydrogel), paste (e.g., dispersible paste, ointment), lotion, spray, and patch (e.g., microneedle patch).

[0054] In the aforementioned injection-compatible injection or drip, the pharmaceutical composition provided by the present invention may contain one or more components such as an isotonic solution, a salt buffer (e.g., a phosphate buffer or a citrate buffer), a solubilizer, an emulsifier, a 5% sugar solution, and other carriers, and the pharmaceutical composition may be provided in the form of an intravenous infusion solution, an emulsion intravenous infusion solution, a dry powder injection, a suspension injection, or a dry powder suspension injection. Alternatively, the pharmaceutical composition may be prepared as a pre-injection solid and, prior to administration to a subject in need thereof, the pre-injection solid may be dissolved in another solution or suspension or emulsified to provide the desired injection.

[0055] For dosage forms suitable for subcutaneous or interstitial implantation, the pharmaceutical composition provided herein may contain one or more ingredients such as excipients, stabilizers, buffers, and other carriers, and be provided in dosage forms such as wafers, tablets, pills, and capsules. This allows the pharmaceutical composition to be implanted into a subject, slowly and continuously releasing the n-butylphthalide contained therein to the tissue surrounding the administration site, achieving a localized, stable, high-dose effect. For example, but not limited to, the pharmaceutical composition provided herein may contain a biocompatible polymer, so that the pharmaceutical composition is in the form of a wafer for subcutaneous or interstitial implantation. The aforementioned biocompatible polymer can be purchased commercially or prepared by known synthetic methods. For example, a polyanhydride (e.g., "p(CPP-SA) copolymer") formed from bis(p-carboxyphenoxy)propane and sebacic acid can be used as the biocompatible polymer.

[0056] For pharmaceutical compositions administered through the respiratory tract, any suitable method may be used to aerosolize the pharmaceutical composition, if desired, to facilitate its delivery into the respiratory tract. For example, but not limited to, the pharmaceutical composition may be administered via a nebulizer or pressurized container (e.g., a nasal spray). Alternatively, the pharmaceutical composition may be formulated as nasal drops.

[0057] As for pharmaceutical compositions for transmucosal administration, the pharmaceutical compositions provided by the present invention may contain one or more penetrants, surfactants, viscosity modifiers, pH modifiers, preservatives, stabilizers, osmotic pressure regulators, and other carriers, and the pharmaceutical compositions may be provided in dosage forms such as orally disintegrating tablets, suppositories, nasal sprays, and nasal drops.

[0058] Optionally, the pharmaceutical composition provided by the present invention may further contain appropriate amounts of additives, such as toners and colorants that can enhance the feel of the composition during use, as well as buffers, preservatives, preservatives, antibacterial agents, antifungal agents, etc. that can improve the stability and storage properties of the composition.

[0059] The food composition provided according to the present invention can be a beverage, solid food, or semi-solid food, and can be provided in the form of a health food, a health food, a functional food, a nutritional supplement, or a special nutritional food. For example, but not limited to, the food composition can be a dairy product, processed meat product, bread, noodles, biscuits, ice cream, oral tablets, capsules, juices, teas, sparkling water, alcoholic beverages, sports drinks, nutritional drinks, and the like. Preferably, the food composition is provided in the form of a health food or a health food.

[0060] Furthermore, depending on the intended use and needs, the food composition provided by the present invention may contain any suitable food additives. For example, such food additives include, but are not limited to, preservatives, antiseptics, bactericides, antioxidants, bleaching agents, colorants, bulking agents, nutritional additives, colorants, flavorings (e.g., sweeteners), thickeners, binders, food industry chemicals, emulsifiers, and agents for quality improvement, brewing, and food manufacturing.

[0061] The outer packaging of the health food, supplemental food, functional food, nutritional supplement food, or special nutritional food provided by the present invention may be labeled with recommended dosage, usage standards and conditions, or recommendations for co-use with other foods or medicines, so that users can take the food without the guidance of a physician, pharmacist, or other relevant personnel without any safety concerns.

[0062] Depending on the intended use and needs, in addition to vegetable protein (e.g., crushed or cracked wheat, barley, corn, etc.), animal protein (e.g., blood meal, meat meal, fish meal, etc.), vegetable fat, and animal fat, the feed composition provided by the present invention may also contain various auxiliary components such as organic acids, phosphates, minerals, vitamins, amino acids, as well as at least one of a preservative, antiseptic, anti-influenza agent, antioxidant, antifungal agent, antimicrobial agent, nutritional supplement, growth promoter, and digestion-absorption enhancer. The feed composition provided by the present invention may also be used as a feed additive and used in conjunction with other components.

[0063] In addition, the feed composition provided by the present invention may contain any suitable dissolution promoter, buffer, diluent, dispersant, surfactant, viscosity modifier, binder, stabilizer, and lubricant to formulate the feed composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, capsules, granules, or tablets. The feed composition provided by the present invention may also be combined with a non-toxic carrier to prepare the feed composition as an immediate-release or sustained-release formulation. Depending on the form of use and application, any suitable edible carrier may be used to provide the feed composition. For example, but not limited to, examples of such edible carriers include: sugars and their derivatives (e.g., lactose, sucrose, mannitol, sorbitol), starches (e.g., corn starch, wheat starch, rice starch, potato starch), cellulose and its derivatives (e.g., methylcellulose, carboxymethylcellulose, ethylhydroxycellulose), protein derivatives (e.g., zein, gelatin), and synthetic polymers (e.g., polyvinyl alcohol, polyvinylpyrrolidone).

[0064] The feed compositions provided according to the present invention can be used in animal diets, i.e., as feed for a wide variety of animals, including mammals and poultry. These feed compositions can be used for commercially important mammals such as pigs, cattle, and sheep, zoo animals such as elephants and camels, or livestock such as dogs and cats. Commercially important poultry can include chickens, ducks, and geese. Ideally, the feed compositions provided according to the present invention will have a flavor, texture, and / or aroma that is favorable to the intended animal, thereby facilitating oral administration of the feed compositions.

[0065] The pharmaceutical compositions, food compositions, and feed compositions provided by the present invention may optionally contain one or more other active ingredients to further enhance the efficacy of the composition or increase the flexibility and adjustability of the formulation, as long as the other active ingredients do not adversely affect the benefits of n-butylphthalide contained in the composition of the present invention.

[0066] In the pharmaceutical composition, food composition or feed composition provided by the present invention, the content of n-butylphthalide is at least about 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.001, 0.0015, 0.002, 0.0025, 0.003, 0.0035, 0.004, 0.0045, 0.005, 0.0055, 0.006, 0.0065, 0.007, 0.0075, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, %, about 0.01 wt % to about 10 wt %, about 0.01 wt % to about 5 wt %, about 0.05 wt % to about 1 wt %, and about 0.05 wt % to about 0.5 wt %.

[0067] The pharmaceutical compositions, food compositions, and feed compositions provided by the present invention can be administered at various frequencies, such as once daily, multiple times daily, or once every few days, depending on the needs, age, weight, and health status of the individual being administered, as well as the intended purpose of administration. The content of n-butylphthalide in the pharmaceutical compositions, food compositions, and feed compositions provided by the present invention can also be adjusted based on actual application needs, for example, to a daily dosage for oral or topical use.

[0068] The kit provided by the present invention can effectively solve the problem of difficulty in obtaining brown adipocytes. In the kit, (1) the first component, which is a component for inducing stem cells to differentiate into adipocytes, and (2) the second component, n-butylphthalide, are typically packaged separately and stored in different storage spaces (e.g., plastic bags, plastic bottles, glass bottles, or ampoules). The two components can be shipped or sold separately, or they can be combined and distributed and sold together as a set. In addition, the kit can include an instruction manual to facilitate the user to mix the components on-site for processing and application according to the procedures and processes set out therein.

[0069] In the kit provided by the present invention, examples of ingredients suitable as the first component for inducing stem cell differentiation into adipocytes include: dexamethasone, insulin, 3-isobutyl-1-methylxanthine (IBMX), rosiglitazone, doxycycline, triiodothyronine (T3), indomethacin, transferrin, selenium, and combinations thereof. In some embodiments of the present invention, the ingredient for inducing stem cell differentiation into adipocytes is one or more of dexamethasone, insulin, 3-isobutyl-1-methylxanthine, and rosiglitazone.

[0070] In the kit according to the present invention, the source and type of n-butylphthalide are as described above.

[0071] The present invention is further illustrated by the following examples. These examples are provided for illustration only and are not intended to limit the scope of the present invention. The scope of the present invention is as set forth in the claims.

[0072] Example

[0073] In the following examples, the materials and equipment used are as follows:

[0074] 1. C57BL / 6J mice (average weight: 24 to 29 g): purchased from NLAC (https: / / www.nlac.narl.org.tw / ).

[0075] 2. Standard diet: purchased from LabDiet (Missouri, USA); product number: #5001; calories: 3.02 kcal / g, of which 13.496% of calories came from fat.

[0076] 3. High-fat diet: purchased from Research Diets (New Jersey, USA); product number: D12451; calorie content: 4.7 kcal / g, of which 45% of the calories came from fat.

[0077] 4. 3-N-butylphthalide (NBP): purchased from Toronto Research Chemicals (TRC); product number: B693850.

[0078] 5. Superoxide dismutase (SOD) assay kit: purchased from Cayman; product number: 706002.

[0079] 6. Catalase assay kit: purchased from Cayman; product number: 707002.

[0080] 7. Glutathione assay kit (purchased from Cayman; product number: 703002).

[0081] 8. Adipose-derived stem cells: purchased from ATCC; number: PCS-500-011 TM .

[0082] 9. Basic medium: KSFM (Keratinocyte serum free medium) (Gibco; Product No. 17005042) supplemented per 500 ml with 25 mg of bovine pituitary extract (BPE), 2.5 μg of human epidermal growth factor recombinant protein (EGF), 10% (v / v) fetal bovine serum (Gibco, Grand Island, USA), and 1% (v / v) penicillin / streptomycin solution.

[0083] 10. Adipogenic differentiation medium: Dulbecco's modified Eagle's medium (DMEM) (purchased from Gibco; product number: 11965092) supplemented with 10% (v / v) fetal bovine serum (Gibco, Grand Island, USA), 1% (v / v) penicillin / streptomycin solution, 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 170 nM insulin, 700 ng / mL doxycycline, and 0.5 μM rosiglitazone.

[0084] 11. Oil red stain: purchased from Sigma aldrich; product number: O1391-250ML.

[0085] 12. Mouse hepatocytes: purchased from ATCC; number: CRL-2254 TM .

[0086] 13.DMEM / F12 culture medium: purchased from Gibco; product number: 11320033.

[0087] Example 1: Animal Experiment

[0088] 1-1. Establishment of animal model

[0089] C57BL / 6J mice were randomly divided into four groups (10 mice in each group) and housed under the following conditions (maintaining a room temperature of 25°C and a 12-h day-night cycle; food and drinking water were provided ad libitum to the experimental animals during the experiment):

[0090] (1) “Standard feed” group (also called “control group”): fed with normal feed for 24 weeks.

[0091] (2) “High-fat diet + low-dose n-butylphthalide” group (also called “NBP (80 mg / kg) group”): The rats were fed a high-fat diet for 24 weeks, and in the last 8 weeks (i.e., weeks 17 to 24), they were additionally fed with olive oil containing n-butylphthalide (n-butylphthalide dose: 80 mg / kg body weight) once a day through a tube.

[0092] (3) “High-fat diet + high-dose n-butylphthalide” group (also called “NBP (200 mg / kg) group”): fed with a high-fat diet for 24 weeks, and in the last 8 weeks (i.e., weeks 17 to 24), additionally fed with olive oil containing n-butylphthalide (n-butylphthalide dose: 200 mg / kg body weight) once a day through a tube.

[0093] (4) “High-fat diet” group: fed with a high-fat diet for 24 weeks, and in the last 8 weeks (i.e., weeks 17 to 24), fed with olive oil once a day (the volume of olive oil administered was the same as that of the group fed with n-butylphthalide).

[0094] 1-2. Observation of body weight, food intake, and body shape

[0095] During the feeding process of Example 1-1, the weight and food intake of the mice were measured and recorded once a week, and the body shape of the mice was recorded by taking photos on the last day. The above observation results of weight, food intake and body shape are shown in Figures 1 to 3 The average bodyweight gain of each group of mice during the experimental process was then calculated using Formula A, where the starting weight was the average weight of the first week and the final weight was the average weight of the 24th week. The food efficiency ratio (also known as food conversion efficiency) of each group of mice was then calculated using Formula B. The results are shown in Figure 4 .

[0096] Formula A: Average weight gain (g) = final weight (g) - initial weight (g).

[0097] Formula B:

[0098] Depend on Figure 1 and Figure 3 It can be seen that compared with the "standard feed" group, the weight of the mice in the "high-fat feed" group was significantly higher and their body size was significantly larger; however, compared with the "high-fat feed", the weight of the mice in the "NBP (80 mg / kg)" group and the "NBP (200 mg / kg)" group was significantly lower and their body size was significantly smaller. Figure 4 Compared to the high-fat diet group, the food conversion efficiency of mice in the NBP (80 mg / kg) and NBP (200 mg / kg) groups was significantly lower. These results demonstrate that n-butylphthalide can effectively inhibit weight gain and obesity.

[0099] 1-3. Recording of oxygen consumption and exhaled carbon dioxide

[0100] During the feeding process of Example 1-1, the experimental animals were first placed in a feeding cage to acclimate for 24 hours. The room temperature was maintained at 25°C and the day-night cycle was 12 hours. Food and drinking water were provided to the experimental animals ad libitum during the experiment. The basic metabolic parameters of the experimental animals, such as oxygen consumption (VO2), carbon dioxide exhalation (VCO2), energy expenditure, heat production, and respiratory exchange rate (or "gas exchange rate"), were then recorded using CLAMS (Columbus Instruments) for 24 hours. The results are shown in Figures 5 to 8 .

[0101] Depend on Figures 5 to 7 It can be seen that compared with the "high-fat diet" group, the mice in the "NBP (200 mg / kg)" group had a higher oxygen consumption ( Figure 5 ), exhaled carbon dioxide ( Figure 6 ) and energy consumption ( Figure 7 ). In addition, Figure 8 This suggests that the increased gas exchange and energy expenditure caused by n-butylphthalide was not due to excessive respiratory rate in mice, indicating that the "NBP (200 mg / kg)" group had higher metabolic energy production. These results indicate that n-butylphthalide can increase oxygen consumption and energy expenditure. Compared to white fat, brown adipocytes / brown adipocytes have greater oxygen consumption and energy expenditure rates. Therefore, these experimental results suggest that n-butylphthalide administration should increase the proportion of brown fat in animals.

[0102] 1-4. Sample collection and observation

[0103] After completing the photographic records of Examples 1-2, the mice were fasted for 12 hours, then anesthetized. Blood was collected from the mice and placed in serum tubes for 30 minutes before centrifugation (4°C, 3000 rpm, 15 minutes). The supernatant (this is the blood sample) was then transferred to a microcentrifuge tube and frozen at -80°C for subsequent experimental analysis.

[0104] After completing the above blood sample collection, the mice were sacrificed and their subcutaneous fat tissue, epididymal fat tissue, liver, kidney and heart were removed and photographed. The results are shown in Figure 9 The average weights of subcutaneous fat tissue, epididymal fat tissue and liver of each group of mice were measured and recorded. The results are shown in Figures 10 to 12 ; and the collected organs were stored at -80°C for subsequent experimental analysis.

[0105] Depend on Figures 9 to 12Compared to the standard diet group, mice in the high-fat diet group had significant accumulation of white fat under the skin and in their internal organs (e.g., epididymis, liver, and kidneys). The livers were significantly whiter and heavier. However, compared to the high-fat diet, mice in the NBP (80 mg / kg) and NBP (200 mg / kg) groups showed significant improvement in subcutaneous and visceral fat accumulation, with liver color and weight returning to levels comparable to those in the standard diet group. These results demonstrate that n-butylphthalide effectively reduces subcutaneous and visceral fat accumulation, particularly liver fat content, potentially preventing or improving fatty liver disease.

[0106] 1-5. Blood biochemical value analysis

[0107] Blood samples from each group of mice provided in Examples 1-4 were taken and tested using SYSMEX K-1000 and TOSHIBATBA200FA fully automatic blood analyzers to analyze the levels of glucose, triglycerides (TG), total cholesterol (TC) and high-density cholesterol (HDL-C) in the mouse blood, as well as the activity of aspartate aminotransferase (AST) and aspartate aminotransferase (ALT). Thereafter, the low-density cholesterol (LDL-C) content of each group of mice was calculated using formula C, and the ratio of low-density to high-density cholesterol was further calculated. In addition, the atherogenic index of each group of mice was calculated using formula D, the cardiovascular risk index of each group of mice was calculated using formula E, and the triglyceride-glucose index (TyG) of each group of mice was calculated using formula F. The results of the aforementioned glucose, triglyceride, total cholesterol and low-density cholesterol levels are shown in Figures 13 to 16 The results of the ratio of low-density lipoprotein cholesterol to high-density lipoprotein cholesterol, atherosclerosis index, cardiovascular risk index, and triglyceride-glucose index (TyG) are shown in Figures 17 to 20 AST and ALT activities are shown in Figure 21 and Figure 22 .

[0108] Formula C: Low-density lipoprotein cholesterol = total cholesterol - high-density lipoprotein cholesterol - (triglycerides / 5).

[0109] Formula D:

[0110] Formula E:

[0111] Formula F:

[0112] Depend on Figures 13 to 20 Compared to the "standard diet" group, the "high-fat diet" group showed significantly higher blood glucose, triglyceride, total cholesterol, and low-density lipoprotein cholesterol (LDL-LDL-LLC) levels, the LDL-to-HDL cholesterol ratio, the atherosclerosis index, the cardiovascular risk index, and the triglyceride-glucose index (TyG). However, compared to the "high-fat diet," the "NBP (200 mg / kg)" group showed significantly lower values, even comparable to or lower than those of the "standard diet" group. These results suggest that n-butyl phenyl peptide can reduce blood glucose, triglyceride, total cholesterol, and low-density lipoprotein cholesterol levels, the atherosclerosis index, the cardiovascular risk index, and the triglyceride-glucose index (TyG), and may be used to prevent obesity-related metabolic syndrome.

[0113] It is known that alanine transaminase (AST) and aspartate transaminase (ALT) are amino acid metabolic enzymes that are widely present in the heart, liver, skeletal muscle, kidney and brain. When cells in organs are damaged, AST and ALT will be released into the blood. Therefore, the activity of AST and ALT can be used as damage indicators of these organs to monitor the damage to internal organs. The higher the activity, the more serious the damage to the internal organs. Among them, ALT is more abundant in the liver, and the higher the activity, the more serious the liver damage. Figure 21 and Figure 22 It can be seen that compared with the "standard diet" group, the "high-fat diet" group had significantly higher blood alanine transaminase (AST) and aspartate transaminase (ALT) activities. However, compared with the "high-fat diet", the "NBP (80 mg / kg)" and "NBP (200 mg / kg)" groups had significantly lower alanine transaminase (AST) and aspartate transaminase (ALT) activities, even comparable to those of the "standard diet" group. These results indicate that n-butyl phenyl peptide can improve liver lesions caused by fatty liver disease and reduce liver damage in individuals.

[0114] 1-6. Liver Sample Analysis

[0115] Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) are all known to be very important antioxidant enzymes. Superoxide dismutase (SOD) can convert reactive oxygen species (ROS) into less reactive H2O2, which is then converted into oxygen and water by catalase (CAT) and glutathione peroxidase (GPx).

[0116] To investigate the effects of n-butylphenyl peptide on lipid peroxidation, oxidative stress, and antioxidant capacity in mouse livers, liver samples from each group of mice provided in Examples 1-4 were taken and homogenized in 0.5 ml of PBS. The homogenized samples were centrifuged (4°C, 3500 rpm, 15 minutes). The supernatant samples were transferred to microcentrifuge tubes and analyzed using commercially available superoxide dismutase reagent kit, catalase reagent kit, and glutathione reagent kit, respectively, according to the user's manual. The results are shown in Figures 23 to 25 .

[0117] Depend on Figures 23 to 25 Compared to the standard diet group, the activities of superoxide dismutase, catalase, and glutathione peroxidase in mice fed a high-fat diet were significantly decreased. However, compared to the high-fat diet, the activities of superoxide dismutase, catalase, and glutathione peroxidase in mice fed the NBP (80 mg / kg) and NBP (200 mg / kg) groups were significantly increased. These results demonstrate that n-butylated phenyl peptide can effectively enhance liver antioxidant activity.

[0118] 1-7. Staining and Analysis of Subcutaneous and Visceral Adipose Tissue Sections

[0119] Liver samples, subcutaneous tissue samples, and epididymal fat tissue samples from each group of mice provided in Examples 1-4 were fixed in formalin, embedded in paraffin, and sectioned. The sections were stained with hematoxylin-eosin (H&E) to observe the cell morphology of the liver tissue of each group of mice. The results are shown in Figure 2. Figure 26 (liver tissue), Figure 27 (subcutaneous fat) and Figure 28 (epididymal fat).

[0120] Depend on Figures 26 to 28 The liver tissue, subcutaneous tissue, and epididymal adipose tissue of mice fed a high-fat diet were primarily composed of single, large white adipocytes. The liver tissue, subcutaneous tissue, and epididymal adipose tissue of mice fed the NBP (80 mg / kg) and NBP (200 mg / kg) diets displayed brown adipocyte morphology similar to that of mice fed a standard diet, with numerous small fat droplets. These results demonstrate that n-butylphthalide effectively converts subcutaneous and visceral white fat in mice induced by a high-fat diet into brown adipocytes, promoting fat browning.

[0121] Example 2: Fat cell experiment

[0122] 2-1. Cell pre-culture

[0123] Adipose-derived stem cells were cultured in basal medium at 37°C and 5% carbon dioxide. The medium was changed every 2 to 3 days. When the cells grew to 90% confluence, the morphology of the cells cultured before menstruation was observed under a microscope and photographed. The results are shown in Figure 29 (undifferentiated).

[0124] 2-2. Cell differentiation

[0125] The premenstrual adipose-derived stem cells provided in Example 2-1 were cultured in an adipogenic differentiation medium at 37°C and 5% carbon dioxide for 7 days. The cells were then divided into four groups and cultured in the following medium for 7 days in each group to differentiate the adipose-derived stem cells into adipocytes:

[0126] (1) “Differentiation” group: adipogenic differentiation medium.

[0127] (2) “Differentiation + NBP (2 μg / ml) group”: adipogenic differentiation medium supplemented with n-butyl phenyl peptide, wherein the concentration of n-butyl phenyl peptide in the medium is 2 μg / ml.

[0128] (3) “Differentiation + NBP (10 μg / ml) group”: adipogenic differentiation medium supplemented with n-butyl phenyl peptide, wherein the concentration of n-butyl phenyl peptide in the medium is 10 μg / ml.

[0129] (4) “Differentiation + NBP (50 μg / ml) group”: adipogenic differentiation medium supplemented with n-butyl phenyl peptide, wherein the concentration of n-butyl phenyl peptide in the medium is 50 μg / ml.

[0130] 2-3. Red oil dyeing

[0131] The adipose-derived stem cells (undifferentiated) provided in Example 2-1, as well as the cells in the "differentiation" group, "differentiation + NBP (2 μg / ml) group," "differentiation + NBP (10 μg / ml) group," and "differentiation + NBP (50 μg / ml) group" provided in Example 2-2 were stained with oil red for fat droplets. The cell morphology of each group was observed under a microscope and photographed. The results are also shown in FIG. Figure 29 Next, the absorbance of each group of cells at a wavelength of 490 nm was measured and quantified into relative absorbance values. The results are shown in Figure 30 .

[0132] On the other hand, the oil droplets and triglycerides in the adipose-derived stem cells (undifferentiated) provided in Example 2-1, and the "differentiation" group, "differentiation + NBP (2 μg / ml) group", "differentiation + NBP (10 μg / ml) group", and "differentiation + NBP (50 μg / ml) group" provided in Example 2-2 were quantified, and the results are shown in FIG. Figure 31 and Figure 32 .

[0133] Depend on Figures 29 to 32 As shown in the figure, cells in the "differentiated" group, untreated with n-butyl phenyl peptide, exhibited high levels of oil droplet accumulation and triglycerides. However, compared to the "differentiated" group, cells in the "differentiated + NBP (2 μg / mL) group," "differentiated + NBP (10 μg / mL) group," and "differentiated + NBP (50 μg / mL)" groups treated with n-butyl phenyl peptide exhibited significantly reduced oil droplet accumulation and triglyceride levels. These results demonstrate that n-butyl phenyl peptide can effectively inhibit oil droplet accumulation and reduce triglyceride levels in adipocytes.

[0134] Example 3: Hepatocyte Experiment

[0135] Mouse hepatocytes were divided into three groups and treated as follows:

[0136] (1) “Uninduced” group: cells were cultured in DMEM / F12 medium at 37°C and 5% carbon dioxide for 24 hours.

[0137] (2) "Fatty acid induced" group: The same procedure as the "uninduced" group was performed, but fatty acids were added to the culture medium to induce the formation of oil droplets in mouse hepatocytes.

[0138] (3) “Fatty acid induction + n-butyl phenyl peptide” group: The same procedure as the “fatty acid induction” group was performed, but n-butyl phenyl peptide was further added to the culture medium, wherein the concentration of n-butyl phenyl peptide in the culture medium was 20 μg / ml.

[0139] Afterwards, the three groups of cells were stained with oil red dye for fat droplets, and the cell morphology of each group was observed under a microscope and photographed. The results are shown in Figure 33 .Depend on Figure 33 It can be seen that compared with the "fatty acid induced" group cells without n-butyl phenyl peptide treatment, the oil droplets in the "fatty acid induced + n-butyl phenyl peptide" group cells were significantly reduced. This result shows that n-butyl phenyl peptide can effectively reduce the fat content of hepatocytes and reduce the accumulation of lipid oil droplets in hepatocytes.

[0140] From the results of the above examples, it can be seen that n-butylphenyl peptide can be used to inhibit white fat accumulation, promote the conversion of white fat into brown fat, inhibit weight gain, inhibit obesity, reduce the liver fat content of an individual, reduce the fat content of hepatocytes, increase the antioxidant activity of the liver, reduce the levels of triglycerides, glucose, total cholesterol and low-density lipoprotein cholesterol in the blood, prevent or treat obesity, prevent metabolic syndrome associated with obesity, prevent or treat fatty liver, improve liver lesions caused by fatty liver, and / or reduce liver damage in an individual.

Claims

1. A method of using n-butylphthalide in the preparation of a composition, characterized in that: The composition is used to promote fat browning.

2. The use according to claim 1, characterized in that The composition is provided in an oral dosage form.

3. The use according to claim 1 or 2, characterized in that The composition is a pharmaceutical composition, a food composition or a feed composition.

4. The use according to claim 3, characterized in that The food composition is a health food, a health food, a functional food, a nutritional supplement or a special nutritional food.

5. A method of using n-butylphthalide in the preparation of a composition, characterized in that: The composition is used for at least one of the following: increasing an individual's oxygen consumption rate, increasing an individual's energy consumption rate, and increasing an individual's brown fat ratio.

6. The use according to claim 5, characterized in that The composition is provided in an oral dosage form.

7. The use according to claim 5 or 6, characterized in that The composition is a pharmaceutical composition, a food composition or a feed composition.

8. The use according to claim 7, characterized in that The food composition is a health food, a health food, a functional food, a nutritional supplement or a special nutritional food.

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