Composition comprising citrus leaf extract as active ingredient for preventing or treating obesity

Abstract

The present invention relates to an anti-obesity composition comprising a citrus leaf extract as an active ingredient. The composition comprising the citrus leaf extract according to the present invention has a high content of hesperidin, inhibits adipocyte differentiation and adipocyte synthesis, and exhibits a body weight loss effect even in an obese animal model induced by a high-fat diet. In addition, the composition comprising the citrus leaf extract of the present invention has no cytotoxicity and thus can be safely used for the human body. Accordingly, the composition comprising the citrus leaf extract according to the present invention is expected to be very advantageously used as a pharmaceutical composition, a functional food composition, and a feed composition for preventing or alleviating obesity.

Description

A composition for the prevention or treatment of obesity containing citrus leaf extract as an active ingredient

[0001] The present invention relates to a pharmaceutical composition for preventing or treating obesity comprising a citrus leaf extract as an active ingredient; a food composition for preventing or improving obesity; a feed composition; and a method for preventing or treating obesity comprising the step of administering said composition to an individual other than a human.

[0002] Obesity typically refers to a body mass index (BMI; weight (kg) / height (m²)) of 25 or higher, indicating a condition characterized by an excessive amount of adipose tissue in the body. Obesity occurs when energy intake exceeds energy expenditure and results from a complex interplay of genetic and environmental factors. The obesity rate in Korea was 31.1% in 2011, but it is projected to increase to 46.4% by 2025. Complications caused by obesity, such as diabetes, hypertension, hyperlipidemia, fatty liver, myocardial disease, nephropathy, and stroke, result in a decline in the quality of life.

[0003] Although there are various causes of obesity, considering the lifestyle patterns of urban dwellers characterized by lack of exercise and increasing stress, it is predicted that there will be a continuous increase in the long term; therefore, materials capable of preventing obesity are deemed to have high market potential.

[0004] Currently, various methods are being used to treat obesity, including lifestyle modifications such as dietary therapy, exercise therapy, and behavioral therapy, as well as drug therapy and surgical treatment (Mason, EE. 1992. Methods for voluntary weight loss and control. Obes Surg 2: 275-276). Drug therapy is limited to the use of agents that regulate digestion or appetite. Representative obesity drugs approved for long-term use by the US FDA to date include norepinephrine, sibutramine (Reductil), and orlistat (Xenical). Recently, drug treatments with glucagon-like peptide-1 (GLP-1) hormone-like efficacy, such as diabetes medications like Mounjaro (tirzepatide), Saxenda (liraglutide), or Wegovy (semaglutide), are being prescribed. However, these drugs also carry risks of side effects such as increased blood pressure, insomnia, and dizziness, as well as cardiovascular symptoms such as myocardial infarction and stroke. Furthermore, their use is limited due to their indistinct efficacy and significant practical constraints on their prescription and use, making it necessary to develop more effective compositions for the treatment or improvement of obesity.

[0005] Currently, most appetite suppressants and anti-obesity treatments distributed domestically are manufactured and sold as finished products using imported raw materials. Therefore, it is considered that developing anti-obesity agents with fewer side effects using natural products as novel biological indicators would be beneficial in terms of economic feasibility and competitiveness.

[0006] There is a shortage of health functional food ingredients with body fat reduction capabilities based on domestic resources that can be utilized as useful materials in the field. Therefore, it is necessary to develop these into functional food ingredients based on the evaluation of the body fat-reducing physiological activity of domestic resources and the elucidation of their physiological mechanisms.

[0007] Domestic functional ingredients for health functional foods are classified into notified ingredients, which are ingredients or components notified by the Minister of Food and Drug Safety, and individually recognized ingredients, which are ingredients or components separately recognized by the Minister of Food and Drug Safety. Among the functional ingredients listed in the "Health Functional Food Codex," there are approximately 85 types of notified ingredients, such as nutrients (vitamins and minerals, dietary fiber, etc.), and 175 types of individually recognized ingredients (Ministry of Food and Drug Safety, 2012).

[0008] Therefore, for the advancement of the food industry and regional industries, it is necessary to develop health functional food materials supported by the elucidation of scientific mechanisms using domestic and regional specialty resources.

[0009] Meanwhile, another byproduct of the citrus industry is citrus leaves. These leaves are generated as waste resources during pruning operations performed to increase productivity and marketability before shipment; currently, the branches and leaves produced during this process are all shredded and discarded on the ground.

[0010] The purpose of pruning is to secure good fruiting branches to obtain high-quality fruit in large quantities, to remove bad branches, diseased branches, and obstructive branches, to ensure good light and ventilation, and to adjust the shape and form of the tree for ease of management, thereby increasing economic income by obtaining good quality fruit.

[0011] Although the timing and amount of pruning vary by farm, it is mostly carried out around March or April, and approximately 10–20% of the leaves per tree are shredded and discarded after pruning. In July 2022, the Ministry of Food and Drug Safety issued a notice of administrative notice regarding the "Draft Partial Amendment to the Standards and Specifications for Food," which includes the addition of citrus leaves (Citrus unshiu S. Markovcich / Citrus reticulata Blanco) to the list of raw materials that can be used in food on a limited basis; therefore, it is necessary to proactively promote research on citrus leaves.

[0012] Accordingly, the inventors of the present invention completed the present invention by continuing to conduct experiments to determine the efficacy using natural resources and discovering that tangerine leaf extract exhibits an anti-obesity effect.

[0013]

[0014] Therefore, the technical problem to be solved by the present invention is to provide a pharmaceutical composition for the prevention or treatment of obesity.

[0015] Another technical problem to be solved by the present invention is to provide a composition for inhibiting adipocyte differentiation.

[0016] Another technical problem to be solved by the present invention is to provide a functional food composition for the prevention or improvement of obesity.

[0017] Another technical problem to be solved by the present invention is to provide a feed composition for preventing or improving obesity.

[0018] Another technical problem to be solved by the present invention is to provide a method for preventing or treating obesity, comprising the step of administering the above composition to an individual.

[0019] To solve the above-mentioned technical problem, the present invention provides a pharmaceutical composition for the prevention or treatment of obesity characterized by including a citrus leaf extract as an active ingredient.

[0020] The present invention provides a composition for inhibiting adipocyte differentiation comprising citrus leaf extract as an active ingredient.

[0021] Preferably, the citrus leaf extract is a citrus leaf alcohol extract, characterized by being extracted at 45 to 55°C for 1 hour 30 minutes to 2 hours 30 minutes using 30 to 80% alcohol as an extraction solvent.

[0022] Preferably, the citrus leaf extract is included in an amount of 1 to 80 weight percent based on the total weight of the composition.

[0023] The present invention provides a functional food composition for preventing or improving obesity, characterized by including a citrus leaf extract as an active ingredient.

[0024] The present invention provides a feed composition for preventing or improving obesity, characterized by including a citrus leaf extract as an active ingredient.

[0025] The present invention provides a method for preventing or treating obesity, comprising the step of administering the above composition to an individual.

[0026] As such, the composition containing the citrus leaf extract according to the present invention has a high hesperidin content, inhibits adipocyte differentiation and adipocyte synthesis, and exhibits a weight-loss effect even in an animal model of obesity induced by a high-fat diet. Furthermore, the composition containing the citrus leaf extract of the present invention has no cytotoxicity and can be safely used in the human body. Therefore, the composition containing the citrus leaf extract according to the present invention is expected to be highly useful as a pharmaceutical composition for the prevention or improvement of obesity, a functional food composition, and a feed composition.

[0027] The following drawings attached to this specification illustrate preferred embodiments of the present invention and serve to further enhance understanding of the technical concept of the present invention together with the aforementioned description; therefore, the present invention should not be interpreted as being limited only to the matters described in such drawings.

[0028] Figure 1 is an HPLC chromatogram of citrus leaf extract.

[0029] Figure 2 shows the results of confirming the cell stability of the citrus leaf sample.

[0030] Figure 3 shows the results of analyzing the fat accumulation inhibitory effect of citrus leaf samples.

[0031] Figure 4 shows the results of analyzing the lipid differentiation inhibitory effect of citrus leaf samples.

[0032] Figure 5 shows the results of analyzing the effect of reducing the expression level of SREBP-1c by citrus leaf samples.

[0033] Figure 6 shows the results of analyzing the effect of reducing PPARγ expression levels by citrus leaf samples.

[0034] Figure 7 shows the results of measuring fat cell synthesis using a tangerine leaf sample.

[0035] Figure 8 shows the measurement results of the lipid decomposition effect by citrus leaf samples.

[0036] Figure 9 shows the results of measuring the mRNA expression level of Leptin in citrus leaf samples.

[0037] Figure 10 shows the results of measuring the mRNA expression level of Adiponectin in citrus leaf samples.

[0038] Figure 11 shows the results of measuring the mRNA expression level of AMPK by citrus leaf samples.

[0039] Figure 12 shows the results of measuring the mRNA expression level of PGC-1α in citrus leaf samples.

[0040] Figure 13 shows the results of measuring the mRNA expression level of UCP in citrus leaf samples.

[0041] Figure 14 shows the results of measuring the change in body weight of experimental animals by citrus leaf samples.

[0042] Figure 15 shows the results of measuring changes in dietary intake of experimental animals by citrus leaf samples.

[0043] Figure 16 shows the results of measuring changes in body composition of experimental animals by citrus leaf samples.

[0044] Figure 17 shows the results of measuring the change in epididymal fat weight of experimental animals by citrus leaf samples.

[0045] Figure 18 shows the results of measuring the change in kidney fat weight of experimental animals by citrus leaf samples.

[0046] Figure 19 shows the results of observing histological changes in the adipose tissue of experimental animals using citrus leaf samples.

[0047] Figure 20 shows the results of measuring the blood liver enzyme AST content of experimental animals using citrus leaf samples.

[0048] Figure 21 shows the results of measuring the blood liver enzyme ALT content of experimental animals using citrus leaf samples.

[0049] Figure 22 shows the results of measuring the blood liver enzyme GGT content of experimental animals using citrus leaf samples.

[0050] Figure 23 shows the results of measuring the triglyceride content in the blood of experimental animals using tangerine leaf samples.

[0051] Figure 24 shows the results of measuring the total cholesterol content in the blood of experimental animals using tangerine leaf samples.

[0052] Figure 25 shows the results of measuring the LDL cholesterol content in the blood of experimental animals using citrus leaf samples.

[0053] Figure 26 shows the results of measuring the HDL cholesterol content in the blood of experimental animals using tangerine leaf samples.

[0054] Figure 27 shows the results of measuring the Adiponectin content in the blood of experimental animals using citrus leaf samples.

[0055] Figure 28 shows the results of measuring the Leptin content in the blood of experimental animals using citrus leaf samples.

[0056] Figure 29 shows the results of the analysis of the expression levels of PPARγ and SREBP-1c in experimental animals using citrus leaf samples.

[0057] Figure 30 shows the results of the analysis of the expression level of fat synthesis enzymes in experimental animals using citrus leaf samples.

[0058] Figure 31 shows the results of the analysis of lipase expression levels in experimental animals using citrus leaf samples.

[0059] Figure 32 shows the results of measuring the mRNA expression level of AMPK, a regulator of energy metabolism, in experimental animals fed with citrus leaf samples.

[0060] Figure 33 shows the results of measuring the mRNA expression level of PGC-1α, a regulator of energy metabolism, in experimental animals fed with citrus leaf samples.

[0061] Figure 34 shows the results of measuring the mRNA expression level of UCP, a regulator of energy metabolism, in experimental animals using citrus leaf samples.

[0062] The present invention will be explained in more detail below.

[0063] The present invention provides a pharmaceutical composition for the prevention or treatment of obesity characterized by including a citrus leaf extract as an active ingredient.

[0064] The term "obesity" in this invention refers to the accumulation of excess energy in adipose tissue and surrounding tissues (liver, muscle, heart, etc.), and describes a phenomenon involving the proliferation of fat cells and the accumulation of an excessive amount of lipids within the fat cells. Obesity, one of the chronic diseases of modern people, has emerged as one of the most serious health problems worldwide due to changes in dietary and lifestyle habits resulting from industrialization and improved income levels, and was classified as a disease by the World Health Organization (WHO) in 1996. Furthermore, obesity is directly or indirectly linked to the development of adult diseases such as diabetes, hypertension, hyperlipidemia, and heart disease, as well as various types of cancer, and can lead to various metabolic syndromes.

[0065] In the present invention, the term "adipocytic differentiation" is used interchangeably with "adipogenesis," and specifically refers to the process in which preadiopocytes mature into adipocytes, resulting in the formation of adipose tissue. The complex comprising the curcuminoid compound and stevioside of the present invention may inhibit such adipocyte differentiation and thereby reduce intracellular lipid accumulation.

[0066] According to one embodiment of the present invention, the citrus leaf extract is a citrus leaf alcohol extract, preferably characterized by being extracted at 45 to 55°C for 1 hour 30 minutes to 2 hours 30 minutes using 30 to 80% alcohol as an extraction solvent.

[0067] In the present invention, the term "active ingredient" refers to a component that exhibits the desired activity alone or can exhibit activity together with a carrier that is inactive itself.

[0068] According to one embodiment of the present invention, the citrus leaf extract is characterized by being included in an amount of 1 to 80 weight% based on the total weight of the composition. At this time, if the content is less than 1 weight%, the obesity improvement effect, which is the objective effect of the present invention, cannot be obtained, and if it exceeds 80 weight%, the effect may be inefficient as it is not proportional to the increase in content, and there is a problem that the stability of the formulation is not ensured.

[0069] The composition of the present invention may be a pharmaceutical composition for the prevention or treatment of obesity.

[0070] The composition of the present invention may further include a suitable carrier, excipient, or diluent commonly used in addition to the active ingredient.

[0071] The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier in addition to citrus leaf extract. The pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is one that is commonly used in formulations and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0072] In the present invention, the term "prevention" refers to any act of suppressing obesity or delaying its onset by administering the above-mentioned composition.

[0073] In the present invention, the term "treatment" refers to any act in which symptoms caused by obesity are improved or beneficially altered by the administration of the above-mentioned composition.

[0074] The prevention or treatment of the above obesity can be achieved by inhibiting adipocyte differentiation, inhibiting lipid accumulation, or reducing fat content, etc., upon administration of the above composition.

[0075] The pharmaceutical composition of the present invention can be administered orally or parenterally.

[0076] Suitable dosages of the pharmaceutical composition of the present invention may be prescribed in various ways depending on factors such as the formulation method, mode of administration, patient's age, body weight, gender, pathological condition, diet, time of administration, route of administration, excretion rate, and response sensitivity. The dosage of the pharmaceutical composition of the present invention is generally within the range of 0.001 to 1000 mg / kg, preferably 0.05 to 200 mg / kg, and more preferably 0.1 to 100 mg / kg for adults. In addition, in the case of a topical preparation, it is preferable to apply an amount of 1.0 to 3.0 ml once to five times a day for at least one month for adults. However, the above dosages do not limit the scope of the present invention.

[0077] The pharmaceutical composition of the present invention may be prepared in a unit dose form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily carried out by a person skilled in the art. The formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or may be in the form of an excipient, powder, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0078] Additionally, the composition of the present invention can exhibit preventive, improving, or therapeutic effects on obesity as well as fatty acid or lipid-related cardiovascular diseases directly or indirectly related to obesity by inhibiting adipocyte differentiation and inhibiting fat production.

[0079] The composition of the present invention may be a functional food composition for preventing or improving obesity.

[0080] The term "functional food" as used in the present invention refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc., using raw materials or ingredients that possess functional properties useful to the human body. Here, "functionality" means obtaining beneficial effects for health purposes, such as regulating nutrients or physiological actions regarding the structure and function of the human body. The health functional food of the present invention can be manufactured by methods commonly used in the industry, and can be manufactured by adding raw materials and ingredients commonly added in the industry. Furthermore, unlike general pharmaceuticals, it has the advantage of being made from food ingredients, thus avoiding side effects that may occur with long-term use of pharmaceuticals, and offers excellent portability.

[0081] The food composition according to the present invention may additionally include, in addition to citrus leaf extract as an active ingredient, ingredients that are typically added during food manufacturing, such as protein, carbohydrates, fats, nutrients, seasonings, and flavorings.

[0082] Examples of the above carbohydrates are monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, oligosaccharides, etc.; and polysaccharides, e.g., dextrin, cyclodextrin, etc., which are conventional sugars and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavoring agents, natural flavoring agents [taumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)] and synthetic flavoring agents (saccharin, aspartame, etc.) may be used.

[0083] For example, when the food composition of the present invention is prepared as a drink, in addition to the citrus leaf extract of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, Eucommia ulmoides extract, jujube extract, licorice extract, etc. may be additionally included.

[0084] The composition of the present invention may be a feed composition for preventing or improving obesity.

[0085] The feed composition of the present invention may have an effect of preventing or improving obesity, as described above.

[0086] In the present invention, the term "feed" refers to any natural or artificial prescribed food, single meal, etc., or the components of said single meal, intended for or suitable for animals to eat, consume, and digest.

[0087] The types of the above feed are not particularly limited, and feeds commonly used in the relevant technical field may be used. Non-limiting examples of the above feed include plant-based feeds such as grains, root vegetables, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal, or grain by-products; and animal-based feeds such as proteins, inorganic substances, oils and fats, minerals, oils and fats, single-cell proteins, zooplankton, or food waste. These may be used individually or in a mixture of two or more types.

[0088] The above feed composition may include a feed additive. The feed additive of the present invention corresponds to a supplementary feed under the Feed Management Act.

[0089] The composition of the present invention is not particularly limited to any individual intended for the prevention or treatment of obesity and is applicable to any individual. For example, any non-human animal such as monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cattle, sheep, pigs, goats, etc., as well as birds and fish, may be used, and the composition may be administered orally or parenterally. The preferred dosage of the composition of the present invention depends on the individual's condition and body weight, the severity of the disease, the form of the drug, the route of administration, and the duration, but can be appropriately selected by a person skilled in the art. For example, it may be administered orally, rectally or intravenously, intramuscularly, subcutaneously, intrathecally, or intracerebrovascularly, but is not limited thereto.

[0090] Another aspect of the present invention provides a method for preventing or treating obesity, comprising the step of administering a composition of the present invention to an individual other than a human.

[0091] In the present invention, the term "individual" refers to any animal other than humans that has developed or may develop obesity, and the individual can be efficiently treated by administering the pharmaceutical composition of the present invention to an individual suspected of obesity.

[0092] In the present invention, the term "administration" means introducing the pharmaceutical composition of the present invention to an individual suspected of obesity by any appropriate method. The route of administration may be various oral or parenteral routes as long as it can reach the target tissue, as described above.

[0093] As such, the composition containing the citrus leaf extract according to the present invention has a high hesperidin content, inhibits adipocyte differentiation and adipocyte synthesis, and exhibits a weight-loss effect even in an animal model of obesity induced by a high-fat diet. Furthermore, the composition containing the citrus leaf extract of the present invention has no cytotoxicity and can be safely used in the human body. Therefore, the composition containing the citrus leaf extract according to the present invention is expected to be highly useful as a pharmaceutical composition for the prevention or improvement of obesity, a functional food composition, and a feed composition.

[0094]

[0095] Hereinafter, the present invention will be described in detail with reference to examples and the like to aid in understanding the invention. However, the embodiments according to the present invention may be modified in various different forms, and the scope of the present invention should not be interpreted as being limited to the following embodiments. The embodiments of the present invention are provided to more completely explain the invention to those with average knowledge in the art.

[0096]

[0097] 1. Analysis of Indicator Components in Citrus Leaves

[0098] A. Preparation of alcohol extract

[0099] The collected tangerine leaves were washed with running water, dried using a hot air dryer at 60°C for 48 hours, and then ground. 600 mL of 70% ethanol was added to 60 g of the ground tangerine leaves and extracted for 24 hours, after which the extract was filtered using filter paper.

[0100] The filtered extract was concentrated under reduced pressure at 40°C using a rotary vacuum concentrator, and the concentrated solution was freeze-dried using a freeze dryer to obtain a citrus leaf extract. The obtained extract was stored at freezing temperature (-20°C) and used after being diluted to the required concentration according to the experiment.

[0101]

[0102] B. Preparation of Standard Solutions

[0103] To analyze the flavonoid content in citrus leaves, a total of 19 flavonoid standards as shown in Table 1 below were used. For each standard, a mixed stock solution of 19 types with a concentration of 1.0 mg / mL was prepared by adding DMSO:EtOH (1:1) solvent, and then the standard solution was prepared and used in the experiment by diluting it to the concentration required for the experiment with the same solvent.

[0104]

[0105]

[0106] C. Preparation of test solution

[0107] The citrus leaf extract was dissolved in DMSO:EtOH (1:1) to a concentration of 5 mg / mL and filtered using a Syringe Filter (PTFE, 0.45 μm).

[0108]

[0109] D. HPLC Analysis

[0110] Waters HPLC (2695 Alliance system) was used to analyze flavonoid components in citrus leaf extract. For the HPLC analysis, distilled water containing 0.1% phosphoric acid and acetonitrile (ACN) were used as the mobile phase, and the analysis was performed using the technical elution method. The HPLC analysis conditions for analyzing the flavonoid content in citrus leaf extract are shown in Table 2.

[0111] The correlation coefficients of the respective flavonoid calibration curves for 19 flavonoid standard solutions under HPLC analysis conditions were examined, and all showed excellent correlations (R²) of 0.999 or higher. 2 ) represented the value.

[0112]

[0113]

[0114] 2. Establishment of Optimized Extraction Conditions for Citrus Leaves

[0115] To establish the optimal extraction conditions for tangerine leaves, variable conditions for solvent, temperature, time, and number of times were applied as shown in Table 3, and the variable conditions with excellent hesperidin content and extraction efficiency were selected first according to the sequential order of each step to establish the optimal extraction conditions for tangerine leaves.

[0116]

[0117]

[0118] (1) Analysis of indicator components in citrus leaves

[0119] As a result of analyzing 19 types of flavonoid components using HPLC, two indicator components were detected in citrus leaves. Figure 1 is the HPLC chromatogram of the citrus leaf extract. As shown here, hesperidin (66.98 mg / g) was identified as the major component of the citrus leaf extract, so hesperidin was established as an indicator component for establishing the standardization of citrus leaves as raw materials, and the study was conducted.

[0120]

[0121] (2) Establishment of optimized extraction conditions for citrus leaves

[0122] A. Content of Indicator Components by Solvent Conditions

[0123] To establish optimized extraction conditions for tangerine leaves, extraction conditions were tested using alcohol solvents at 60°C for 3 hours. As shown in Table 5 below (※ Total yield of indicator components: yield of freeze-drying (g) × extract hesperidin content (mg / g)), higher alcohol concentrations resulted in higher hesperidin content. However, it was confirmed that the extraction yield tended to decrease as the alcohol concentration increased.

[0124]

[0125] Based on the detected hesperidin content, the yield of the extract was multiplied by the content of the indicator component to determine the yield of the indicator component. As a result, the 50% alcohol extract showed the best hesperidin yield. Therefore, the optimized extraction solvent condition for alcohol solvent extraction was set to 50% alcohol.

[0126]

[0127] B. Content of Indicator Components by Temperature Condition

[0128] To establish optimized extraction conditions for tangerine leaves according to temperature conditions, tangerine leaves were extracted using a 50% alcohol solvent in the range of 30 to 70℃.

[0129] The results are shown in Table 6 below. As shown in Table 6, the extraction yields were similar, but the highest yield and indicator component content were observed at 50℃.

[0130]

[0131] In addition, the hesperidin content according to extraction conditions showed higher content as the temperature increased, but when comparing the yield of total indicator components, a relatively superior yield was observed under the 50℃ condition.

[0132]

[0133] As shown in Table 7 above, relatively similar yields were observed under extraction conditions of 30–70°C, but extraction conditions by time were conducted based on 50°C.

[0134]

[0135] C. Content of indicator components by extraction time

[0136] To establish optimized extraction conditions for each extraction time, the extraction yield and indicator component content of tangerine leaf extract were analyzed over time under the above-mentioned optimized conditions (50% alcohol, 50℃) over a period of 1 to 5 hours.

[0137]

[0138] Experimental results showed that the extraction yield remained relatively constant after 2 hours, and the content of indicator components was almost similar between 1 and 3 hours, showing a decreasing trend after 4 hours. When the total yield of indicator components was applied, the highest yield was observed at 3 hours of extraction, but similar content was observed at 2 hours, so the optimal extraction time was set to 2 hours.

[0139]

[0140] D. Content of indicator components by extraction frequency

[0141] Experiments were conducted by repeatedly applying optimized solvent, temperature, and time extraction conditions to the residue remaining after extraction. As a result, the content of the indicator component decreased by approximately 43% during the second extraction, so the number of extractions was set to one to establish the optimized extraction conditions.

[0142] As a result of the analysis of the total yield of indicator components, the content of indicator components decreased by more than 79.6% when extracted twice and by more than 96.5% when extracted three times, so the final number of repeated extractions was set to one (Tables 10 and 11).

[0143]

[0144]

[0145] M. Setting Optimized Extraction Conditions for Tangerine Leaves

[0146] To establish raw material standardization conditions based on indicator components, 50% alcohol was set as the most suitable extraction solvent condition.

[0147] To establish raw material standardization conditions based on indicator components, the extraction temperature condition was set to 50℃ alcohol as the most suitable temperature condition.

[0148] Based on the indicator components, the extraction time condition for establishing raw material standardization conditions was set to 2 hours as the most efficient condition.

[0149] As a result of analyzing the content of indicator components according to the number of extractions under three conditions, it was confirmed that most indicator components are extracted in a single extraction.

[0150] Therefore, the optimal raw material standardization condition for tangerine leaves was set as extracting once for 2 hours in a 50% alcohol, 50℃ extraction bed as the most suitable extraction condition.

[0151]

[0152] 3. Evaluation of Anti-obesity Efficacy and Mechanism of Action of Citrus Leaf Extract Using 3T3-L1 Cells

[0153] 1) Experimental Method

[0154] A. Test cell line

[0155] The 3T3-L1 adipocytes used in the experiment were obtained from the American Type Culture Collection (ATCC), and the reagents required for cell culture were purchased from Gibco (USA). The cell culture medium consisted of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% bovine serum (BS) and 1% antibiotics-antimycotic, and was used for the experiment by subculturing every 2 to 3 days in a 37°C cell incubator with 5% CO2.

[0156]

[0157] B. Confirmation of sample safety

[0158] To analyze the cellular stability of the samples, the formazan detection method using XTT reagent was performed. For the analysis, cells were placed in a 96-well plate at a density of 2×10⁶ 4 Each well was dispensed at a concentration of cells / well and subjected to a 24-hour stabilization period. Subsequently, each sample was treated at different concentrations and cultured for 24 hours. After culture, 10 μl of XTT reagent was added per 100 μl to allow the reaction, and absorbance (450 nm, 600 nm) was measured using a microplate reader. Cell viability was calculated using the following Equation 1.

[0159]

[0160]

[0161] C. Induction of Adipogenesis and Analysis of Adipogenesis Inhibitory Efficacy of Samples

[0162] 2×10 5Cells / ml were cultured in a 6-well plate until a cell density of 100% was reached, after which the medium was changed and cultured for an additional 48 hours at 37°C under 5% CO2 conditions. To induce adipogenesis, 3T3-L1 cells were cultured in DMEM medium containing 10% FBS, 175 nM insulin, 0.25 uM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 2 mM L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin; after 2 days, the medium was replaced with DMEM medium containing 10% FBS, 175 nM insulin, 2 mM L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin, and cultured. Sample treatment was carried out concurrently with the differentiation induction process.

[0163]

[0164] D. Analysis of the fat accumulation inhibitory efficacy of samples through Oil Red O staining

[0165] Oil Red O staining was performed to visually evaluate the lipid accumulation inhibitory ability of the samples. The cultured cells were fixed by removing the medium, washing twice with PBS, reacting with 4% formaldehyde (Sigma, USA) at room temperature for 20 minutes, replacing the medium, and reacting at room temperature for 1 hour. Subsequently, the cells were washed twice with 60% isopropanol for 1 minute each, treated with Oil Red O solution (Sigma, USA) for 10 minutes, and stained, after which adipocyte differentiation was analyzed under a microscope. For quantitative evaluation, the stained reagent treated with isopropanol was eluted and recovered, and the absorbance was measured at 570 nm using a microplate reader (Infinite 200, Tecan Co, Switzerland).

[0166]

[0167] E. Analysis of the adipogenesis inhibitory efficacy of the sample through AdipoRed analysis

[0168] AdipoRed assay was performed to evaluate the adipogenesis inhibitory ability of the samples. The culture medium was removed from the cells, and after washing twice with PBS, 0.2 mL of PBS was added. 6 μL of AdipoRed reagent was added and stained for 15 minutes, after which the supernatant was removed. The degree of staining was measured by fluorescence using a microplate reader at citation 485 nm and emission 530 nm.

[0169]

[0170] B. Measurement of expression levels of key factors related to lipid metabolism in adipocytes

[0171] Total RNA was extracted from adipocytes using Trizol and its concentration measured with Nanodrop. Stable cDNA was synthesized using the iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA). Subsequently, real-time PCR was performed using the SYBR green RT-PCR kit and the designed primers. To ensure consistent data interpretation, expression levels were calculated relative to β-actin. The primers used are listed in Table 12 below.

[0172]

[0173]

[0174] Statistical analysis

[0175] All experimental results were calculated as mean ± standard deviation (Mean ± SD) using the statistical program (SPSS ver. 27.0, SPSS Inc., Chicago, IL, USA). For statistical analysis regarding the statistical significance test between experimental groups, one-way analysis of variance test (ANOVA) was performed, and post-hoc testing was conducted using Ducan's multiple range test when significance was minimal or p < 0.05.

[0176]

[0177] 2) Result

[0178] (1) Cell experiment

[0179] a. Confirmation of cell stability of the sample

[0180] As a result of evaluating the cytotoxicity of three samples—roasted tangerine peel, tangerine peel, and tangerine leaves—on 3T3-L1, it was confirmed that no cytotoxicity was exhibited up to 0–500 μg / mL. Figure 2 shows the results of confirming the cell stability of the tangerine leaf samples.

[0181] It was decided to conduct the experiment at a low concentration of 0 to 200 μg / mL for future economic feasibility.

[0182]

[0183] B. Analysis of the fat accumulation inhibitory efficacy of the sample

[0184] To evaluate the efficacy of the sample, ORL 25 μg / mL was used as a positive control. The inhibitory effect on fat accumulation was evaluated using Oil Red O staining, which stains all hydrophobic phases of the cells. Significance was verified using Duncan's multiple range test.

[0185] Figure 3 shows the results of analyzing the lipid accumulation inhibitory efficacy of tangerine leaf samples. As shown here, the roasted tangerine peel sample significantly reduced lipid accumulation at 200 μg / mL, the tangerine peel sample significantly reduced lipid accumulation at 100–200 μg / mL, and the tangerine leaf sample significantly reduced lipid accumulation at 150–200 μg / mL. Based on 200 μg / mL, the effectiveness was found to be in the order of roasted tangerine peel = tangerine peel ≥ tangerine leaf.

[0186]

[0187] C. Analysis of the adipogenesis inhibitory efficacy of the sample

[0188] To evaluate the efficacy of the sample, ORL 25 μg / mL was used as a positive control. The inhibitory effect on fat accumulation was analyzed using the AdipoRed assay, which measures triglycerides by staining.

[0189] Figure 4 shows the results of analyzing the adipogenesis inhibitory effect of tangerine leaf samples. As shown here, it was confirmed that roasted tangerine peel, tangerine peel, and tangerine leaf samples all exhibited a significant adipogenesis inhibitory effect at 200 μg / mL. Specifically, at 200 μg / mL, the effectiveness was found to be in the order of tangerine leaf = tangerine peel ≥ roasted tangerine peel.

[0190]

[0191] D. Elucidation of mechanisms through mRNA expression analysis in cell models

[0192] To evaluate the efficacy of the sample, ORL 25 μg / mL was used as a positive control.

[0193]

[0194] a) Measurement of Adipogenesis

[0195] Adipogenesis was measured by determining the mRNA expression levels associated with adipocyte differentiation.

[0196] Figure 5 shows the results of analyzing the effect of SREBP-1c expression reduction by tangerine leaf samples. As seen here, when evaluating the expression of SREBP-1c, which is involved in the early stages of adipocytes and co-regulates mRNA related to fat accumulation, roasted tangerine peel was significantly reduced at 200 μg / mL, tangerine peel at 150–200 μg / mL, and tangerine leaves at 100–200 μg / mL.

[0197] Figure 6 shows the results of analyzing the effect of citrus leaf samples on reducing PPARγ expression. As seen here, when evaluating the expression of PPARγ, which is involved in the final stage of adipocyte differentiation, roasted tangerine peel and tangerine peel showed a significant decrease at 150–200 μg / mL, and citrus leaves showed a significant decrease at 100–200 μg / mL.

[0198] As a result, based on 200 μg / mL, the effectiveness was found to be in the order of tangerine leaves ≥ roasted tangerine peel = tangerine peel.

[0199]

[0200] b) Measurement of Lipogenesis (Adipocyte Synthesis)

[0201] Adipocyte synthesis was measured by determining the mRNA expression levels of FAS and ACC, enzymes involved in lipid synthesis, and SCD1 and DGAT1, enzymes involved in triglyceride formation.

[0202] Figure 7 shows the results of measuring adipocyte synthesis using tangerine leaf samples. As shown here, when the expression level of FAS was measured, it was significantly decreased in roasted tangerine peel and tangerine peel at 150–200 μg / mL, and in tangerine leaves at 50–200 μg / mL. When the expression level of ACC was measured, it was significantly decreased in roasted tangerine peel and tangerine leaves at 150–200 μg / mL, and in tangerine peel at 200 μg / mL. When the expression level of SCD1 was measured, it was significantly decreased in tangerine peel and tangerine leaves at 150–200 μg / mL, and in roasted tangerine peel at 200 μg / mL. When the expression level of DGAT1 was measured, it was significantly decreased in tangerine peel and tangerine leaves at 200 μg / mL, and in roasted tangerine peel at 100–200 μg / mL.

[0203] As a result, all three samples were evaluated to inhibit adipocyte synthesis when mRNA analysis was performed on the group treated with 200 μg / mL.

[0204]

[0205] c) Measurement of Lipolysis

[0206] Lipid degradation within adipocytes was assessed by measuring the mRNA expression levels of the lipolytic enzymes HSL and ATGL, as well as PPARα and CPT-1, which are involved in the beta-oxidation of fatty acids.

[0207] Figure 8 shows the results of measuring the lipid degradation effect of tangerine leaf samples. As shown here, when HSL expression was measured, roasted tangerine peel and tangerine peel showed a significant increase from 100–200 μg / mL, while tangerine leaves showed a significant increase from 50 μg / mL. When ATGL expression was measured, roasted tangerine peel showed a significant increase from 150–200 μg / mL, while tangerine peel and tangerine leaves showed a significant increase from 100–200 μg / mL. When SCD1 expression was measured, roasted tangerine peel, tangerine peel, and tangerine leaves all showed a significant increase at 200 μg / mL. When CPT-1 expression was measured, roasted tangerine peel, tangerine peel, and tangerine leaves all showed a significant increase at 200 μg / mL.

[0208] As a result, all three samples were evaluated to have increased lipid degradation when mRNA analysis was performed on the group treated with 200 μg / mL.

[0209]

[0210] D) Measurement of Energy Metabolism

[0211] The energy metabolism of adipocytes can be divided into energy intake and consumption homeostasis regulated by Leptin, Adiponectin, and AMPK secreted by adipocytes, and energy consumption metabolism resulting from heat production with increased expression of PGC-1α and UCP within mitochondria.

[0212] Leptin is a hormone secreted by fat cells when body fat accumulation increases; it reduces appetite, thereby decreasing energy intake and increasing energy expenditure.

[0213] Figure 9 shows the results of measuring the mRNA expression levels of Leptin from tangerine leaf samples. Upon confirming the mRNA expression of Leptin, it was found that roasted tangerine peel, tangerine peel, and tangerine leaves all significantly reduced Leptin at concentrations of 150–200 μg / mL.

[0214] In addition, Adiponectin is a hormone involved in the catabolic process of fat that promotes beta-oxidation and inhibits fat synthesis; since its levels decrease when body fat accumulates, it can be used as an indicator of body fat accumulation.

[0215] Figure 10 shows the results of measuring the mRNA expression levels of Adiponectin in tangerine leaf samples. As a result of confirming the mRNA expression of Adiponectin, it was confirmed that the expression significantly increased in roasted tangerine peel at 200 μg / mL, and in tangerine peel and tangerine leaves at 150–200 μg / mL.

[0216] In addition, AMPK catalyzes the process of ATP generation and acts as an intermediate mediator in the regulation of energy metabolism through the action of Leptin and adiponectin.

[0217] Figure 11 shows the results of measuring the mRNA expression levels of AMPK in tangerine leaf samples. Upon confirming the mRNA expression of AMPK, it was found that roasted tangerine peel and tangerine leaves showed a significant increase at 150–200 μg / mL, and tangerine peel at 200 μg / mL.

[0218] As a result, based on 200 μg / mL, the effectiveness was found to be in the order of tangerine leaves ≥ roasted tangerine peel = tangerine peel.

[0219] PGC-1α is a mitochondria-induced heat generation indicator. Figure 12 shows the results of measuring the mRNA expression levels of PGC-1α in citrus leaf samples. When confirming the expression, it was observed that the levels of roasted tangerine peel, tangerine peel, and citrus leaf samples all increased significantly at 150–200 μg / mL.

[0220] UCP is a protein that functions to consume energy by generating heat instead of ATP in cells. Figure 13 shows the results of measuring the mRNA expression levels of UCP in tangerine leaf samples. When confirming the expression, it was observed that the levels of roasted tangerine peel, tangerine peel, and tangerine leaf samples all increased significantly at 150–200 μg / mL.

[0221] As a result, based on 200 μg / mL, the effectiveness was found to be in the order of roasted tangerine peel ≥ tangerine leaves = tangerine peel.

[0222]

[0223] 4. Evaluation of Anti-obesity Efficacy and Mechanism of Action of Citrus Leaf Extract in a High-Fat Diet-Induced Obesity Animal Model

[0224] (1) Experimental method

[0225] A. Experimental Animals and Rearing Environment

[0226] Male C57BL / 6 mice, 4 weeks old and in a Specific-pathogen-free (SPF) state, were obtained from Orient Bio Co., Ltd. and used in the experiment after undergoing a one-week acclimatization period. During the acclimatization period, the experimental diet consisted of standard solid feed (Purina Lab Rodent Chow #38057, Purina Co., Seoul Korea), and filtered drinking water was provided daily and consumed freely.

[0227] During the rearing period, the environment was maintained with a temperature of 23±1℃, humidity of 50±5%, noise of 60 phone or less, lighting hours of 07:00-19:00 (12 hours per day), illuminance of 150-300 lux, and ventilation of 10-12 times per hour. Veterinary quarantine was performed on the general health status of all animals upon entry.

[0228] This experiment was conducted in compliance with Chonnam National University's animal experiment ethics regulations (Approval No. CNU IACUC-YB-2023-98).

[0229]

[0230] B. Establishment of an animal model of obesity induced by a high-fat diet and composition of the group

[0231] The establishment of an animal model of obesity was carried out by feeding a high-fat diet, and the anti-obesity efficacy of the sample was demonstrated by concurrently administering the sample orally. For the high-fat diet, the Rodunt Diet with 60% Kcal Fat (#D12492, Research diets INC.) was purchased from Saeron Bio (Korea) and used.

[0232] Group composition was determined by measuring the weight of each individual so that the weights of the groups would be similar, and the composition of the groups is as shown in Table 13 below.

[0233]

[0234] - CON ; General feed feeding and vehicle oral administration

[0235] - HFD: High-fat diet feeding and oral administration of vehicle

[0236] - ORL: Positive control group, high-fat diet and oral administration of Orlistat (obesity treatment drug)

[0237] - The sample groups of the three types of samples were fed a high-fat diet and administered orally according to sample concentrations.

[0238]

[0239] C. Weekly Biomarker Measurement

[0240] Weekly biomarker measurement involved weekly body weight. Weekly body weight was measured twice a week at a fixed time, and dietary intake was measured daily by assessing the remaining amount the day after feeding.

[0241]

[0242] D. Measurement of body fat and histological changes using DXA

[0243] To measure changes in body fat of experimental animals, measurements were conducted using iNsiGHT VET DXA at ​​the Korea Food Industry Cluster Promotion Agency in Iksan, Jeonbuk on the start date of the experiment, the day before the end of the experiment, and the day before dissection. After anesthetizing the experimental animals with CO2 and isoflurane, measurements were taken using iNSiGHT DXA (osteosis, South Korea).

[0244]

[0245] E. Autopsy and tissue extraction

[0246] Inhalation anesthesia was administered for the autopsy, and blood was collected after the animal was anesthetized. The liver, epididymal fat, retroabdominal fat, and renal fat were excised, and the weight of the excised tissues was measured.

[0247]

[0248] B. Blood analysis

[0249] After allowing the collected blood to coagulate for 30 minutes, the serum was recovered by centrifuging at 3,000 rpm for 10 minutes. The recovered serum was analyzed for Total cholesterol (TC), Triglyceride (TG), High-density Lipoprotein cholesterol (HDL-C), Low-density Lipoprotein cholesterol (LDL-C), Aspartate Aminotransferase (AST), Alanine Aminotransgerase (ALT), and Gamma-Glutamyl Transferase (GGT).

[0250]

[0251] G. Enzyme Immunoassay

[0252] Enzyme immunoassay was used to determine the expression levels of Leptin and Adiponectin in isolated serum. Commercially available assay kits (Adiponectin Human ELISA Kit, #KMP0041, Leptin mouse ELISA kit, #KMC2281) were used, and expression levels were measured according to the manufacturer's measurement method.

[0253]

[0254] Ah. Histopathological analysis

[0255] After dissection, the adipose tissue was fixed in a 10% formalin solution. After dehydration with xylene, it was embedded in a paraffin solution. 10 μm sections were stained with hematoxylin and eosin and observed under a light microscope.

[0256]

[0257] J. Measurement of expression levels of key lipid metabolism factors using animal adipose tissue

[0258] Total RNA was extracted from animal adipose tissue using Trizol. The tissue, frozen via liquid nitrogen, was ground using a mortar and pestle, and Trizol was added. The Trizol layer was separated after centrifugation at 4°C and 16,000 xg for 10 minutes. 200 μL of chloroform was added, the mixture was homogenized, and centrifuged at 4°C and 16,000 xg for 10 minutes. The supernatant was collected, 400 μL of isopropanol was added, and the mixture was centrifuged at 4°C and 16,000 xg for 5 minutes. The supernatant was removed, 75% ethanol was added, and the mixture was centrifuged at 12,000 xg for 10 minutes. The supernatant was completely removed, and the extracted RNA was dissolved in DEPC and its concentration measured using a Nanodrop. Stable cDNA was synthesized using the iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA). Subsequently, real-time PCR was performed using the SYBR green RT-PCR kit and the designed primers. β-actin was used as the housekeeping gene for consistent data interpretation. The primers used were identical to those used in the cell experiments.

[0259]

[0260] Statistical analysis

[0261] All experimental results were calculated as mean ± standard error (Mean ± SE) using the statistical program (SPSS ver. 27.0, SPSS Inc., Chicago, IL, USA). Statistical analysis regarding the statistical significance between experimental groups was performed using ANOVA (one-way analysis of variance test) and Duncan's post-hoc test, and a p-value of < 0.05 was determined to be significant (SPSS V22, SPSS Inc., Chicago, IL, USA).

[0262]

[0263] (2) Result

[0264] A. Change in body weight of experimental animals

[0265] To confirm the effect on body weight gain in an animal model of obesity induced by a high-fat diet, a high-fat diet was administered for 8 weeks.

[0266] The sample was administered orally at a set time every day, and Table 14 below shows the change in body weight twice a week during the experiment period.

[0267] Figure 14 shows the results of measuring the change in body weight of experimental animals by citrus leaf samples.

[0268]

[0269]

[0270] B. Measurement of changes in dietary intake of experimental animals

[0271] Daily dietary intake was checked and measured in an animal model of obesity induced by a high-fat diet.

[0272] Figure 15 shows the results of measuring changes in dietary intake of experimental animals by citrus leaf samples. As can be seen here, it was significantly reduced in the HFD group compared to the CON group, and it can be confirmed that dietary intake decreased significantly in the group administered the sample compared to the HFD group.

[0273]

[0274] C. Measurement of tissue weight changes in experimental animals

[0275] As shown in Table 15 below, when the weight of liver tissue was measured, it was significantly increased in the HFD group compared to the CON group, and significantly decreased in the ORL, PP-M, PP-H, PM, LL, LM, and LH groups compared to the HFD group.

[0276] When kidney weight was measured, it was significantly increased in the HFD group compared to the CON group, and significantly decreased in the ORL, PP-L, PP-M, PP-H, PM, LL, LM, and LH groups compared to the HFD group.

[0277] When the weight of the spleen was measured, it was significantly increased in the HFD group compared to the CON group, and significantly decreased in the ORL, PP-M, PP-H, PM, LM, and LH groups compared to the HFD group.

[0278]

[0279]

[0280] D. Measurement of changes in body composition of experimental animals using DXA

[0281] To measure the body composition of experimental animals, we visited the Korea Food Industry Cluster Promotion Agency in Iksan, Jeonbuk, and conducted measurements using iNsiGHT VET DXA.

[0282] Figure 16 shows the results of measuring changes in body composition of experimental animals using tangerine leaf samples. As can be seen here, changes in the experimental animals were observed from the start of the experiment to before dissection. Specifically, when fat mass was measured after the experiment, it significantly increased in the HFD group compared to the CON group, and fat mass relative to body weight significantly decreased in the ORL, PP-H, and LH groups compared to the HFD group.

[0283]

[0284] E. Measurement of changes in adipose tissue weight in experimental animals after dissection

[0285] To confirm changes in the weight of adipose tissue in an animal model of obesity induced by a high-fat diet, the weights of organs and adipose tissue were measured after dissection.

[0286] Figure 17 shows the results of measuring changes in epididymal fat weight in experimental animals using citrus leaf samples. As can be seen here, epididymal fat significantly increased in the HFD group compared to the CON group, and significantly decreased in the ORL, PP-H, and LH groups compared to the HFD group.

[0287] Figure 18 shows the results of measuring the change in renal fat weight of experimental animals in response to tangerine leaf samples. As can be seen here, renal fat significantly increased in the HFD group compared to the CON group, and significantly decreased in the ORL, PP-H, PM, and LH groups compared to the HFD group.

[0288]

[0289] B. Histological changes in adipose tissue after dissection

[0290] Histological changes were confirmed through H&E staining after collecting adipose tissue from an animal model induced by a high-fat diet.

[0291] Figure 19 shows the results of observing histological changes in the adipose tissue of experimental animals treated with citrus leaf samples. As seen here, hypertrophy of adipocytes was observed in the HFD group compared to the CON group, and it was found that the size of adipocytes significantly decreased in the sample-treated groups.

[0292]

[0293] G. Confirmation of serum biochemical indicators of experimental animals after dissection

[0294] To confirm liver damage and changes in blood lipid content in samples from an obese animal model induced by a high-fat diet, blood was taken after dissection and changes in blood levels were measured.

[0295]

[0296] a) Measurement of blood liver enzymes

[0297] An enzymatic approach to the liver was used to diagnose and monitor the course.

[0298] AST, ALT, and GGT are the most common serum enzymes used to evaluate liver function. AST and ALT are used as indicators of liver dysfunction, and total body fat mass is closely correlated with AST, ALT, and GGT.

[0299] Figure 20 shows the results of measuring the blood liver enzyme AST content in experimental animals using citrus leaf samples. Figure 21 shows the results of measuring the blood liver enzyme ALT content in experimental animals using citrus leaf samples. As can be seen here, when AST and ALT were measured, they significantly increased in the HFD group, and the levels significantly decreased with the administration of the samples.

[0300] Figure 22 shows the results of measuring the blood liver enzyme GGT content of experimental animals using citrus leaf samples. As can be seen here, GGT was significantly increased in the HFD group and significantly decreased in the ORL, PP-M, PP-H, PM, LL, LM, and LH groups.

[0301]

[0302] b) Measurement of blood lipid levels

[0303] Figure 23 shows the results of measuring the triglyceride content in the blood of experimental animals using tangerine leaf samples. As can be seen here, when the triglyceride content in the blood was measured, it significantly increased in the HFD group compared to the CON group, and significantly decreased in the ORL, PP-H, PM, LM, and LH groups.

[0304] Figure 24 shows the results of measuring the total cholesterol content in the blood of experimental animals using tangerine leaf samples. As can be seen here, when the total cholesterol content in the blood was measured, it significantly increased in the HFD group compared to the CON group, and significantly decreased in the ORL, PP-M, PP-H, PM, LL, LM, and LH groups.

[0305] Figure 25 shows the results of measuring the LDL cholesterol content in the blood of experimental animals using tangerine leaf samples. Figure 26 shows the results of measuring the HDL cholesterol content in the blood of experimental animals using tangerine leaf samples. As shown here, when measuring the HDL cholesterol content in the blood, it was significantly decreased in the HFD group compared to the CON group, and significantly increased in the PP-SL, PP-L, PP-M, PP-H, PM, and LH groups. When calculating LDL cholesterol based on these values, the values ​​that were significantly increased in the HFD group compared to the CON group were significantly decreased in the PP-M, PP-H, PM, LL, LM, and LH groups.

[0306]

[0307] A. Confirmation of energy metabolism hormones in the blood of experimental animals after dissection

[0308] Changes in energy metabolism-related hormones were measured for samples in an animal model of obesity induced by a high-fat diet.

[0309] Figure 27 shows the results of measuring the Adiponectin content in the blood of experimental animals using citrus leaf samples. As can be seen here, Adiponectin, which is an indicator of obesity, showed a significantly decreased level in the HFD group, but a significantly increased level in the ORL, PP-M, PP-H, PM, LM, and LH groups.

[0310] Figure 28 shows the results of measuring the Leptin content in the blood of experimental animals using citrus leaf samples. As can be seen here, the Leptin levels, which were significantly increased in the HFD group compared to the CON group, were significantly decreased in the ORL, PP-L, PP-M, PP-H, PM, LM, and LH groups.

[0311]

[0312] Elucidation of the mechanism through analysis of mRNA expression in experimental animals after dissection

[0313] The mechanism of the anti-obesity effect caused by the sample was elucidated by confirming changes in mRNA expression for the sample in an obese animal model induced by a high-fat diet.

[0314] a) Adipogenesis

[0315] Figure 29 shows the results of the analysis of PPARγ and SREBP-1c expression levels in experimental animals using citrus leaf samples. As shown here, when the mRNA expression level of PPARγ was measured, it was significantly decreased in the ORL, PP-M, PP-H, PM, and LH groups. When the mRNA expression level of SREBP-1c was measured, it was significantly decreased in the ORL, PP-H, PM, and LH groups.

[0316] From this, it can be confirmed that all three samples affect adipocyte differentiation.

[0317]

[0318] b) Lipogenesis

[0319] Figure 30 shows the results of the analysis of lipid synthesis enzyme expression levels in experimental animals using tangerine leaf samples. As shown here, when the mRNA expression level of the lipid synthesis enzyme FAS was measured, it was significantly decreased in the ORL, PP-M, PP-H, PM, LM, and LH groups. When the mRNA expression level of the lipid synthesis enzyme ACC was measured, it was significantly decreased in the ORL, PP-M, PP-H, PM, and LH groups. When the mRNA expression level of the triglyceride synthesis enzyme DGAT1 was measured, it was significantly decreased in the ORL, PP-M, PP-H, PM, and LH groups. When the mRNA expression level of the triglyceride synthesis enzyme SCD1 was measured, it was significantly decreased in the ORL, PP-H, PM, and LH groups.

[0320] From this, it can be confirmed that all three samples affect fat synthesis.

[0321]

[0322] c) Lipolysis

[0323] Figure 31 shows the results of the analysis of lipase expression levels in experimental animals using citrus leaf samples. As shown here, when the mRNA expression level of the lipase HSL was measured, it significantly increased in the ORL, PP-H, PM, LM, and LH groups. When the mRNA expression level of the lipase ATGL was measured, it significantly increased in the ORL, PP-H, PM, LL, LM, and LH groups. When the mRNA expression level of PPARα, which is related to the beta-oxidation of fatty acids, was measured, it significantly increased in the ORL, PP-H, PM, and LH groups. When the mRNA expression level of CPT-1, which is related to the beta-oxidation of fatty acids, was measured, it significantly increased in the ORL, PP-M, PP-H, PM, LM, and LH groups.

[0324] From this, it can be confirmed that all three samples affect fat breakdown.

[0325]

[0326] D) Energy metabolism

[0327] Figure 32 shows the results of measuring the mRNA expression level of AMPK, a regulator of energy metabolism, in experimental animals fed with citrus leaf samples. As shown here, when the mRNA expression level of AMPK, a regulator of energy metabolism, was measured, it significantly increased in the ORL, PP-M, PP-H, and LH groups.

[0328] Figure 33 shows the results of measuring the mRNA expression of PGC-1α, a regulator of energy metabolism, in experimental animals fed with citrus leaf samples. As shown here, when the mRNA expression of PGC-1α was measured, it significantly increased in the ORL, PP-H, PM, and LH groups.

[0329] Figure 34 shows the results of measuring the mRNA expression level of UCP, a regulator of energy metabolism, in experimental animals fed with citrus leaf samples. As can be seen here, when the mRNA expression level of UCP was measured, it significantly increased in the ORL, PP-H, PM, and LH groups.

[0330] From this, it was confirmed that all three samples can affect the regulation of energy metabolism.

[0331]

[0332] Specific parts of the present invention have been described in detail above. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0333] As such, the composition containing the citrus leaf extract according to the present invention has a high hesperidin content, inhibits adipocyte differentiation and adipocyte synthesis, and exhibits a weight-loss effect even in an animal model of obesity induced by a high-fat diet. Furthermore, the composition containing the citrus leaf extract of the present invention has no cytotoxicity and can be safely used in the human body. Therefore, the composition containing the citrus leaf extract according to the present invention is expected to be highly useful as a pharmaceutical composition for the prevention or improvement of obesity, a functional food composition, and a feed composition.

Claims

1. A pharmaceutical composition for the prevention or treatment of obesity characterized by containing citrus leaf extract as an active ingredient.

2. In Paragraph 1, A pharmaceutical composition for the prevention or treatment of obesity, characterized in that the above-mentioned citrus leaf extract is a citrus leaf alcohol extract.

3. In Paragraph 2, A pharmaceutical composition for the prevention or treatment of obesity, characterized in that the above-mentioned citrus leaf alcohol extract is prepared by extracting at 45 to 55°C for 1 hour 30 minutes to 2 hours 30 minutes using 30% to 60% alcohol as an extraction solvent.

4. In Paragraph 1, The above composition is a pharmaceutical composition for the prevention or treatment of obesity, characterized by having an inhibitory effect on adipocyte differentiation.

5. In Paragraph 1, The above composition is a pharmaceutical composition for the prevention or treatment of obesity, characterized by having an inhibitory effect on the synthesis of fat cells.

6. In Paragraph 1, The above composition is a pharmaceutical composition for the prevention or treatment of obesity, characterized by having lipid-degrading efficacy.

7. A functional food composition for preventing or improving obesity, characterized by containing tangerine leaf extract as an active ingredient.

8. A feed composition for preventing or improving obesity, characterized by containing tangerine leaf extract as an active ingredient.

9. A method for preventing or treating obesity, comprising the step of administering a composition according to any one of claims 1 to 6 to an individual other than a human.

Images