Composition for lowering blood glucose comprising citrus unshiu leaf extract as active ingredient

Abstract

The present invention relates to a composition for lowering blood glucose, comprising a Citrus unshiu leaf extract. The composition containing a Citrus unshiu leaf extract according to the present invention has a high hesperidin content and excellent α-glucosidase inhibitory activity and sucrase inhibitory activity. In addition, the composition comprising a Citrus unshiu leaf extract, of the present invention, has no cytotoxicity, and thus can be safely used in the human body. Therefore, the composition comprising a Citrus unshiu leaf extract, according to the present invention, is expected to be very effectively used as a pharmaceutical composition, a functional food composition, and a feed composition, for lowering blood glucose or preventing diabetes.

Description

A blood sugar-lowering composition containing tangerine leaf extract as an active ingredient

[0001] The present invention relates to a composition for lowering blood sugar comprising a citrus leaf extract, and more specifically, to a pharmaceutical composition or functional food composition for the prevention or treatment of diseases related to blood sugar lowering or abnormal blood sugar control comprising a citrus leaf alcohol extract as an active ingredient; a feed composition; and a method for the prevention or treatment of diseases related to lowering blood sugar or abnormal blood sugar control, comprising the step of administering the composition to an individual other than a human.

[0002]

[0003] Despite extensive global research on the treatment and prevention of diabetes, the prevalence of the disease continues to steadily increase. With the recent rapid rise in the obese population, coupled with inappropriate lifestyle habits and the entry into an aging society, there are concerns that the number of diabetes patients will surge in the future, making urgent measures at the national level necessary.

[0004] Diabetes, which was broadly classified into insulin-dependent and insulin-independent types, has now been renamed Type 1 and Type 2. Type 2 diabetes (type 2 diabetes melitus), which accounts for more than 90% of diabetes patients, is known to develop due to problems with insulin action (insulin resistance) or defects in insulin secretion, and oral hypoglycemic agents or insulin preparations are used for treatment along with dietary control and exercise therapy.

[0005] Among them, oral hypoglycemic agents include sulfonylureas, which are drugs that promote insulin secretion; metformin and glitazones, which improve insulin resistance; and alpha-glycosidase inhibitors, which inhibit glucose absorption.

[0006] Although oral hypoglycemic agents with three different mechanisms of action are used in clinical practice, their use is restricted due to side effects such as hypoglycemia, hepatotoxicity, weight gain, and lactic acidosis. Additionally, while blood sugar control is possible to some extent with the aggressive use of oral hypoglycemic agents or insulin preparations, the results of two recently completed large-scale clinical trials are very negative (The Diabetes Control and Complications Trial (DCCT) groups: Engl J Med, 2002, 342:381, The United Kingdom Prospective Diabets Study (UKPDS) groups: JAMA, 2002, 287:2542).

[0007] Despite aggressive treatment for diabetes, a significant number of patients have failed to reach their target blood glucose levels. Recently, based on the understanding that drugs acting on only a single mechanism have limitations in lowering blood glucose to normal levels, there is a trend toward marketing drugs formulated by combining agents with different mechanisms.

[0008] For the successful treatment of type 2 diabetes, which has a more complex pathogenesis than type 1 diabetes, there is a growing need to develop drugs that possess two or three mechanisms rather than just one. From this perspective, it is necessary to develop preventive and therapeutic agents for diabetes from natural products containing diverse components.

[0009] With the recent rapid increase in the obese population, there is a growing number of people who do not exhibit characteristic symptoms of diabetes but show problems with the glucose tolerance test. These individuals are referred to as having impaired glucose tolerance (IGT), and the fact is that a significant number of them eventually develop diabetes. Therefore, while early detection is important, developing agents that can block or delay the progression of IGT to diabetes is also a critical issue.

[0010] Glucose absorbed into the body can be burned in peripheral tissues, or blood sugar levels can be regulated through a feedback mechanism in which glucose stimulates beta cells to secrete insulin. The ability of an organism to metabolize glucose normally in this way is called glucose tolerance. Hyperglycemia can be caused by a decrease in this glucose tolerance.

[0011] Among medicinal plants, ginseng is a preparation that exhibits various physiological activities, and its antidiabetic activity has been reported by many researchers. In particular, Sotaniemi and Vuksan (Sotaniemi et al., Diabetes Care, 1995, 18:1373; Vuksan et al., Arch Intern Med, 2000, 60:1009; Vuksan et al., Diabetes Care, 2000, 23:1221) published clinical trial results on the antidiabetic activity of Panax notoginseng and reported the antidiabetic activity of Korean ginseng or Panax notoginseng through animal experiments. However, as recently reported by Vuksan et al., even commercially available ginseng from the same company can show significant differences in antidiabetic activity depending on the experiment (Variable effects of American ginseng: a batch of American ginseng (Panax quinquefolius L.) with a depressed ginsenoside profile does not affect postprandial glycemia : Eur J Clin Nutr, 2003, 57(2):243-8).

[0012] 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).

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] Accordingly, the inventors continued to conduct experiments to determine the efficacy using natural resources, and as a result, discovered that tangerine leaf extract exhibits a blood sugar-lowering effect, thereby completing the present invention.

[0018]

[0019] Therefore, the technical problem to be solved by the present invention is to provide a pharmaceutical composition for lowering blood glucose.

[0020] Another technical problem to be solved by the present invention is to provide a functional food composition for lowering blood sugar.

[0021] Another technical problem to be solved by the present invention is to provide a feed composition for lowering blood sugar.

[0022] Another technical problem to be solved by the present invention is to provide a method for improving blood sugar levels, comprising the step of administering the above composition to an individual.

[0023]

[0024] To solve the above-mentioned technical problem, the present invention provides a pharmaceutical composition for lowering blood sugar characterized by including a citrus leaf extract as an active ingredient.

[0025] 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.

[0026] Preferably, the citrus leaf extract is included in an amount of 1 to 80 weight percent based on the total weight of the composition.

[0027] According to one embodiment of the present invention, the composition may be a composition for lowering blood glucose or for preventing or treating diseases related to abnormal blood glucose control.

[0028] The above-mentioned diseases related to abnormal blood glucose control may be one or more diseases selected from the group consisting of diabetes, diabetic complications, hyperglycemia, obesity, cardiovascular disease, low HDL-cholesterolemia, hyperlipidemia, and inflammation.

[0029] The present invention provides a functional food composition for lowering blood sugar, characterized by including citrus leaf extract as an active ingredient.

[0030] The present invention provides a feed composition for lowering blood sugar, characterized by including citrus leaf extract as an active ingredient.

[0031] The present invention provides a method for improving blood sugar levels, comprising the step of administering the above composition to an individual.

[0032] According to one embodiment of the present invention, a method for preventing or improving a disease related to abnormal blood sugar control is provided, comprising the step of administering the composition to an individual.

[0033]

[0034] As such, the composition containing the citrus leaf extract according to the present invention has a high hesperidin content and exhibits excellent alpha-glucosidase and sucrase inhibitory activities. 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 lowering blood sugar or preventing diabetes, as well as as a functional food composition and a feed composition.

[0035]

[0036] 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.

[0037] Figure 1 is an HPLC chromatogram of citrus leaf extract.

[0038] Figure 2 shows the results of the cell viability and toxicity evaluation of citrus leaf samples.

[0039] Figure 3 shows the results of in-vitro intestinal α-Glucosidase inhibitory activity using an analysis kit of citrus leaf samples.

[0040] Figure 4 shows the results of the α-Glucosidase inhibitory activity of tangerine leaf extract in rats.

[0041] Figure 5 shows the results of the Sucrase inhibitory activity of tangerine leaf extract.

[0042] Figure 6 shows an experimental method for measuring the blood sugar improvement effect using an animal model.

[0043] Figure 7 shows the blood glucose-lowering effect of citrus samples according to sucrose intake in an animal model.

[0044] Figure 8 shows the blood glucose-lowering effect of different citrus samples according to starch intake in an animal model.

[0045] The present invention will be explained in more detail below.

[0046] The present invention provides a blood sugar-lowering composition characterized by including citrus leaf extract as an active ingredient.

[0047] 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.

[0048] 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.

[0049] 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 blood glucose lowering 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.

[0050] In one embodiment of the present invention, the composition of the present invention may be a pharmaceutical composition for lowering blood glucose.

[0051] In another embodiment of the present invention, the composition of the present invention may be a pharmaceutical composition for the prevention or treatment of diseases related to abnormal blood sugar control.

[0052] The disease related to the above-mentioned abnormal blood sugar control may be diabetes or hyperglycemia.

[0053] The composition of the present invention may further include a suitable carrier, excipient, or diluent commonly used in addition to the active ingredient.

[0054] 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).

[0055] In this specification, the term "prevention" refers to any act of suppressing or delaying the progression of a disease related to abnormal blood glucose control through the administration of a composition of the present invention.

[0056] In this specification, the term “treatment” means the inhibition of the development of a disease related to abnormal blood glucose control; the alleviation of a disease related to abnormal blood glucose control; and the elimination of a disease related to abnormal blood glucose control.

[0057] According to one embodiment of the present invention, the disease related to the abnormal blood glucose control includes, but is not limited to, one or more diseases selected from the group consisting of diabetes, diabetic complications, hyperglycemia, obesity, cardiovascular disease, low HDL-cholesterolemia, hyperlipidemia, and inflammation.

[0058] According to one embodiment of the present invention, the diabetic complications are broadly classified into acute complications and chronic complications. Acute complications occur when blood sugar levels temporarily become excessively high or low and are life-threatening fatal complications, while chronic complications result in visual impairment, neurological disorders, renal dysfunction, and cardiac dysfunction when high blood sugar levels persist for a long period. For example, the disease may include one or more diseases selected from the group consisting of diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, and diabetic osteoporosis, but is not limited thereto.

[0059] According to one embodiment of the present invention, the cardiovascular disease includes one or more diseases selected from the group consisting of hypertension, heart disease, stroke, thrombosis, angina pectoris, heart failure, myocardial infarction, coronary artery disease, aneurysm, embolism, atherosclerosis, atherosclerosis, and arteriosclerosis, but is not limited thereto.

[0060] The pharmaceutical composition of the present invention may be administered orally or parenterally, preferably by parenteral administration, and more preferably by topical application.

[0061] 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.

[0062] 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.

[0063] The composition of the present invention may be a functional food composition for lowering blood sugar.

[0064] In another embodiment of the present invention, the composition of the present invention may be a food composition for preventing or improving diseases related to abnormal blood sugar control.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] The composition of the present invention may be a feed composition for lowering blood sugar.

[0070] In another embodiment of the present invention, the composition of the present invention may be a feed composition for preventing or improving diseases related to abnormal blood sugar control.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] The composition of the present invention is not particularly limited and is applicable to any individual intended for the prevention or improvement of diseases related to abnormal blood glucose control. 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. The composition may be administered parenterally, subcutaneously, intraperitoneally, intrapulmonaryly, and intranasally, and for local treatment, may be administered by a suitable method including administration into the lesion if necessary. The preferred dosage of the pharmaceutical composition of the present invention depends on the individual's condition and body weight, the severity of the disease, the drug form, 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.

[0075] Another aspect of the present invention provides a method for improving blood sugar levels, comprising the step of administering a composition of the present invention to an individual.

[0076] Another aspect of the present invention provides a method for improving a disease related to abnormal blood sugar control, comprising the step of administering a composition of the present invention to an individual.

[0077] In the present invention, the term "individual" refers to any animal other than humans that has developed or may develop a disease related to abnormal blood sugar control, and the individual can be efficiently treated by administering the composition of the present invention to an individual suspected of having a disease related to abnormal blood sugar control.

[0078] In the present invention, the term "administration" means introducing the composition of the present invention to an individual suspected of having a disease related to abnormal blood glucose control 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.

[0079] As such, the composition containing the citrus leaf extract according to the present invention has a high hesperidin content and exhibits excellent alpha-glucosidase and sucrase inhibitory activities. 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 lowering blood sugar or preventing diabetes, as well as as a functional food composition and a feed composition.

[0080]

[0081] 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.

[0082]

[0083] 1. Analysis of Indicator Components in Citrus Leaves

[0084] A. Preparation of alcohol extract

[0085] 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% alcohol was added to 60 g of the ground tangerine leaves and extracted for 24 hours, after which the extract was filtered using filter paper.

[0086] 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 in a freezer (-20°C) and used after being diluted to the required concentration according to the experiment.

[0087]

[0088] B. Preparation of Standard Solutions

[0089] 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.

[0090]

[0091]

[0092] C. Preparation of test solution

[0093] 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).

[0094]

[0095] D. HPLC Analysis

[0096] 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 the citrus leaf extract are shown in Table 2.

[0097] When the correlation coefficients of each flavonoid calibration curve for 19 types of flavonoid standard solutions were checked according to HPLC analysis conditions, all showed excellent correlation (R2) values ​​of 0.999 or higher.

[0098]

[0099]

[0100] 2. Establishment of Optimized Extraction Conditions for Citrus Leaves

[0101] 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.

[0102]

[0103] Step | Condition | Variable | Condition | Optimal Condition 1 | By Solvent (Alcohol), % | 0, 30, 50, 70, 100 | 60℃, 3 hours 2 | By Temperature, ℃ | 30, 40, 50, 60, 70 | Optimal Solvent, 3 hours | By Hour, hr | 1, 2, 3, 4, 5 | Optimal Solvent, Optimal Temperature 4 | By Cycle, 1, 2, 3 | Optimal Solvent, Optimal Temperature, Optimal Time

[0104] (1) Analysis of indicator components in citrus leaves

[0105] 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.

[0106]

[0107] (2) Establishment of optimized extraction conditions for citrus leaves

[0108] A. Content of Indicator Components by Solvent Conditions

[0109] 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.

[0110]

[0111] 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, and the 50% alcohol extract showed the best hesperidin yield.

[0112] Therefore, the optimized extraction solvent condition for alcohol solvent extraction was set to 50% alcohol.

[0113]

[0114]

[0115]

[0116] B. Content of Indicator Components by Temperature Condition

[0117] 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℃.

[0118] 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℃.

[0119]

[0120]

[0121] 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.

[0122]

[0123]

[0124] 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.

[0125]

[0126] C. Content of indicator components by extraction time

[0127] 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.

[0128]

[0129]

[0130] As a result of the experiment, the extraction yield showed a relatively constant yield starting from 2 hours, and the content of indicator components was almost the same from 1 to 3 hours and showed a decreasing trend after 4 hours.

[0131] When the total yield of indicator components was applied, the highest yield was observed in 3-hour extraction, but similar content was observed in 2 hours, so the optimal extraction time was set to 2 hours.

[0132]

[0133]

[0134] D. Content of indicator components by extraction frequency

[0135] 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.

[0136] 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).

[0137]

[0138]

[0139]

[0140] E. Setting Optimized Extraction Conditions for Citrus Leaves

[0141] To establish raw material standardization conditions based on indicator components, 50% alcohol was set as the most suitable extraction solvent condition.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146]

[0147] 3. Evaluation of Cell Viability and Toxicity

[0148] An MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide), an analytical method for measuring cellular metabolic activity (mitochondrial activity) as an indicator of cell viability, proliferation, and cytotoxicity, was performed.

[0149] By utilizing the ability of mitochondria dehydrogenase in living cells to convert the yellow water-soluble substrate MTT into the deep blue insoluble formazan, the amount of formazan produced is proportional to the number of living cells.

[0150] After counting the cells, the cells were seeded into each well of a 96-well plate at the desired concentration and cultured for 24 hours. Then, 100 μL of medium containing samples of various concentrations was added, and the cells were cultured for 24 and 48 hours at 37°C under 5% CO₂ conditions. After the culture was finished, 10 μL of MTT (thiazolyl blue tetrazolium bromide) solution, prepared by dissolving it in PBS at a concentration of 5 mg / mL, was added to each well and reacted for 1 hour, after which the medium was removed. Subsequently, 100 μL of DMSO (dimethylsulfoxide) was added to dissolve all the formazan crystals formed in each well, and the absorbance was measured at 540 nm using a micro-plate reader. Cell viability was calculated according to the following Equation 1.

[0151]

[0152]

[0153] Figure 2 shows the results of the cell viability and toxicity evaluation of citrus leaf samples. As shown here, it was confirmed that the citrus leaf extract maintained a high cell viability without exhibiting toxicity to cells, similar to tangerine peel and roasted tangerine peel derived from the same citrus variety, compared to the control group CON. These results indicate that the citrus leaf extract of the present invention can be used as a functional food composition and feed composition that is harmless and safe even when ingested.

[0154]

[0155] 4. Measurement of blood sugar improvement effect

[0156] Diabetes treatment methods are broadly categorized into diet, exercise, and medication, and are determined based on the patient's symptoms. In the treatment of diabetes, the prevention and treatment of various complications arising from the disease are more important than the disease itself; therefore, the focus is on maintaining normal blood sugar levels and preventing complications.

[0157] In terms of preventing complications, the most important thing is to always maintain blood glucose levels at an appropriate level. Currently, insulin medications and various hypoglycemic agents are used, but it is difficult to manage blood glucose levels thoroughly over a long period, and since there are few noticeable symptoms until complications appear, it is very important to take medication to prevent complications along with maintaining blood glucose levels.

[0158] Complications of diabetes are broadly classified into acute and chronic complications. Acute complications occur when blood sugar levels temporarily become excessively high or low and are life-threatening, while chronic complications result from the prolonged persistence of high blood sugar, leading to visual impairment, neurological disorders, renal dysfunction, and cardiac dysfunction.

[0159] Therefore, the development of preventive and therapeutic agents for diabetes is very important and is being researched and developed in various aspects; in this study, the potential of a blood sugar-improving treatment was evaluated using tangerine leaf extract and the indicator component hesperidin.

[0160]

[0161] 1) In-vitro activity

[0162] A. Alpha-glucosidase (α-Glucosidase) Inhibitory Activity Analysis (Analysis Kit)

[0163] α-Glucosidase is an enzyme secreted in the small intestine that promotes carbohydrate metabolism by breaking down α-1,4 bonds to convert polysaccharides and disaccharides into monosaccharides.

[0164] Therefore, α-Glucosidase has a key function in glucose production and is an important factor in controlling postprandial hyperglycemia, a condition in which blood glucose levels rise rapidly after a meal. To measure the degree of inhibition, an α-Glucosidase inhibitor screening kit (ab284520, abcam) was used.

[0165] In this experiment, according to the protocol provided by the manufacturer, 10 μL of a sample of tangerine leaf extract diluted to 10 ng / mL and 10 μL of α-Glucosidase Positive Control / α-Glucosidase diluted to 1X using α-Glucosidase assay buffer were mixed, and the total volume was adjusted to 100 μL using α-Glucosidase assay buffer.

[0166] After that, the reaction was carried out in the dark at room temperature for 15-20 minutes, and 20 μL of a solution mixed with an α-Glucosidase substrate was added. The absorbance was measured at 410 nm at 10-minute intervals for 60 minutes using the Kinetic mode of a SpectraMax ABS Plus Microplate reader (Molecular devices), and the inhibitory activity was calculated by the following Equation 2.

[0167]

[0168]

[0169] * Control: Control group absorbance gradient

[0170] * Sample: Sample treatment absorbance gradient

[0171]

[0172] Figure 3 shows the results of the in-vitro α-Glucosidase inhibitory activity of tangerine leaf extract using an analysis kit. In the analysis kit results used to evaluate the α-glucosidase inhibitory activity of tangerine leaf extract, Acarbose, the positive control, was found to inhibit α-Glucosidase by 88.97±0.43%, and the tangerine leaf extract was found to have a higher inhibitory efficacy of 90.09±0.15% when treated at a concentration of 10 ng / mL.

[0173]

[0174] B. Rat intestinal α-glucosidase inhibitory activity

[0175] Rat intestinal acetone powder was used as the enzyme, and PNP-glycoside (pNPG, p-Nitrophenyl α-D-gluco-pyranoside) was used as the substrate.

[0176] 100 mg of rat intestinal acetone powder was added to 3 mL of 0.9% NaCl solution and sonicated 12 times for 30 seconds in an iced water bath, then centrifuged for 30 minutes (10,000 × g, 4 ℃), and the supernatant was separated and used as the rat α-glucosidase solution.

[0177] 50 mL of sample solution was added to 100 µL of rat alpha-glucosidase solution and incubated at 37°C for 10 minutes, then 50 mL of 5 mM pNPG solution (pNPG, p-Nitrophenyl α-D-gluco-pyranoside) was added and reacted at 37°C for 15 minutes, and the inhibition of rat alpha-glucosidase activity was analyzed by measuring the absorbance using an ELISA reader at 405 nm.

[0178] Figure 4 shows the results of the α-glucosidase inhibitory activity of tangerine leaf extract in rats. As a result of analyzing the α-glucosidase inhibitory activity of tangerine leaf extract, the hot water extract showed inhibitory activity levels of 10.86±1.00% at 1 mg / mL, 21.95±1.38% at 2 mg / mL, 57.74±0.58% at 5 mg / mL, and 91.92±0.54% at 10 mg / mL, respectively, while the alcohol extract showed inhibitory activity levels of 19.24±4.48% at 1 mg / mL, 45.42±0.64% at 2 mg / mL, 99.37±6.66% at 5 mg / mL, and 102.73±5.22% at 10 mg / mL.

[0179] Based on these results, citrus leaf extract showed potential as a natural hypoglycemic agent that inhibits blood sugar elevation by strongly inhibiting the activity of α-glucosidase.

[0180]

[0181] C. Rat Intestinal Sucrase Inhibitory Activity

[0182] 100 mg of rat intestinal acetone powder was added to 3 mL of 0.9% NaCl solution, sonicated 12 times for 30 seconds in an iced water bath, and centrifuged for 30 minutes (10,000 × g, 4℃). The supernatant was separated and used as the solution for evaluating rat intestinal sucrase activity.

[0183] Citrus leaf extract was added to the separated supernatant solution and incubated at 37°C for 10 minutes, after which 200 mM sucrose was added as a substrate and reacted at 37°C for 30 minutes. Subsequently, the reaction solution was added to 1 mL of a solution mixed with Glucose oxidase / peroxidase reagent (Sigma G3660) and O-Dianisidine reagent (Sigma D2679) and incubated at 37°C for 10 minutes. Then, 1 mL of 12N sulfuric acid was added to stop the reaction, and the absorbance was measured at 540 nm using an ELISA reader to analyze the inhibition of activity against sucrose-based sucrase.

[0184] Figure 5 shows the results of the Sucrase inhibitory activity of the tangerine leaf extract. As a result of the analysis of the sucrase inhibitory activity of the tangerine leaf extract, no effective inhibitory activity was observed in the hot water extract under the conditions of this study. However, the alcohol extract showed sucrase inhibitory activity at levels of 42.69±0.94% at 1 mg / mL, 75.76±3.08% at 2 mg / mL, 94.56±1.20% at 5 mg / mL, and 97.96±1.91% at 10 mg / mL.

[0185] Through these results, the citrus leaf extract of the present invention demonstrated potential for application as a natural hypoglycemic agent that inhibits blood sugar elevation by inhibiting the activity of sucrase.

[0186]

[0187]

[0188] As a result of confirming the IC50 for the inhibitory activity of alpha-glucosidase (α-glucosidase) and sucrase in rat intestines, it was confirmed that tangerine leaf extract can inhibit the enzyme activity that produces glucose even in small amounts, and that the inhibition of sucrase activity that produces monosaccharides can have a blood glucose-lowering effect even at an amount of 1.2 mg / mL or less.

[0189]

[0190] 2) In-vivo activity

[0191] A. Animal Experiment Conditions

[0192] The animal experiment conditions were maintained at a temperature of 22°C and a humidity of 50%, and all air in the rearing space (SPF zone) was filtered through a HEPA filter. The rats were fed a solid diet (Teklad) via free feeding, and the lighting and extinguishing times in the rearing room were divided into 12-hour intervals. Four-week-old SD (Sprague-Dawley) rats were purchased and acclimatized for one week; five-week-old SD rats were then divided into at least three groups with at least 10 rats per group, and blood glucose levels were measured after a 24-hour fast, during which only water was supplied.

[0193] For the 0-hour blood glucose measurement, the measurement was performed before oral administration, and measurements were conducted at 30 minutes, 1 hour, 2 hours, and 3 hours. For blood glucose measurement, the tip of the rat's tail was slightly incised using a sterile scalpel, and approximately 0.5 mL of blood was absorbed into a blood glucose strip to measure the value obtained from the meter (Acura View).

[0194] Figure 6 shows an experimental method for measuring the blood sugar improvement effect using an animal model.

[0195] All experimental results were expressed as Mean±SD, and the significance between each mean was analyzed using Duncan's multiple range test with p<0.05 using SPSS 12 (Statistical Package for Social Science, SPSS Inc. Chicago, IL, USA) and Student's t-test.

[0196]

[0197] B. Evaluation of blood glucose changes in SD rats following sucrose intake

[0198] SD rats were fasted for 24 hours prior to the experiment, and citrus leaf extract was added to 2.0 g / kg body weight of sucrose and administered orally in a dose of 1 mL using an oral administration tube. The control group was administered only 2.0 g / kg body weight of sucrose. Blood was collected from the tail vein at 30 minutes, 1 hour, 2 hours, and 3 hours after oral administration, and changes in venous blood glucose concentration were measured using a blood glucose meter (Acura View).

[0199] To observe changes in blood glucose levels after a meal, initial blood glucose levels were measured in mice that had been fasted for 24 hours, and the blood glucose-improving effects of tangerine leaf extract and the major components of the extract, hesperidin and hesperetin, were observed at intervals of 30 minutes, 1 hour, and 2 hours.

[0200] Figure 7 shows the blood glucose-lowering effect of citrus samples according to sucrose intake in an animal model. As shown here, the blood glucose level of the control group administered only sucrose (Sucrose 2.0 g / kg) rose sharply to 241.00 ± 11.42 mg / dL after 30 minutes, whereas the hot water extract of citrus leaves, alcohol extract, hesperidin (indicator component), and hesperetin (component) showed blood glucose levels of 190.88 ± 7.66 mg / dL, 195.63 ± 7.21 mg / dL, 216.25 ± 9.15 mg / dL, and 224.75 ± 13.57 mg / dL, respectively, compared to the control group, and showed a concentration-dependent effect of lowering blood glucose.

[0201] In addition, as a result of investigating pharmacodynamic parameters, the Cmax value, which is the maximum blood glucose concentration of glucose absorbed over 3 hours, was statistically significantly reduced in the sample administration group compared to the control group, as shown in Table 13 below.

[0202]

[0203]

[0204] C. Evaluation of blood glucose changes in SD rats following starch intake

[0205] To observe changes in blood glucose levels after a meal, initial blood glucose levels were measured in mice that had been fasted for 24 hours, and the blood glucose-improving effects of tangerine leaf extract and the major components of the extract, hesperidin and hesperetin, were observed at intervals of 30 minutes, 1 hour, and 2 hours.

[0206] Figure 8 shows the blood glucose-lowering effect of different citrus samples according to starch intake in an animal model. As shown here, in the case of the control group administered only starch (Sucrose 2.0 g / kg), blood glucose levels rose rapidly to 258.88 ± 28.76 mg / dL 30 minutes after administration. However, the hot water extract of citrus leaves, alcohol extract, hesperidin (indicator component), and hesperetin (component) showed blood glucose levels of 215.00 ± 5.48 mg / dL, 204.00 ± 8.74 mg / dL, 223.25 ± 15.74 mg / dL, and 211.83 ± 9.37 mg / dL, respectively, which were lowered compared to the control group and showed a concentration-dependent effect of lowering blood glucose.

[0207] In addition, as a result of investigating pharmacodynamic parameters, the Cmax value, which is the maximum blood glucose concentration of glucose absorbed over 3 hours, was statistically (p<0.05) significantly reduced in the sample administration group compared to the control group, as shown in Table 14 below.

[0208]

[0209]

[0210] 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.

[0211]

[0212] As such, the composition containing the citrus leaf extract according to the present invention has a high hesperidin content and exhibits excellent alpha-glucosidase and sucrase inhibitory activities. 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 lowering blood sugar or preventing diabetes, as well as as a functional food composition and a feed composition.

Claims

1. A pharmaceutical composition for lowering blood sugar characterized by containing tangerine leaf extract as an active ingredient.

2. In Paragraph 1, A pharmaceutical composition for lowering blood sugar, characterized in that the above tangerine leaf extract is prepared by extracting at 45 to 55°C for 1 hour 30 minutes to 2 hours 30 minutes using 30 to 80% alcohol as an extraction solvent.

3. A pharmaceutical composition for the prevention or treatment of diseases related to abnormal blood sugar control, characterized by containing tangerine leaf extract as an active ingredient.

4. In Paragraph 3, A pharmaceutical composition for the prevention or treatment of a disease related to abnormal blood sugar control, characterized in that the disease related to abnormal blood sugar control is one or more diseases selected from the group consisting of diabetes, diabetic complications, hyperglycemia, obesity, cardiovascular disease, low HDL-cholesterolemia, hyperlipidemia, and inflammation.

5. In Paragraph 3, A pharmaceutical composition for the prevention or treatment of diseases related to abnormal blood sugar control, characterized in that the above tangerine leaf extract is prepared by extracting at 45 to 55°C for 1 hour 30 minutes to 2 hours 30 minutes using 30 to 80% alcohol as an extraction solvent.

6. A functional food composition for lowering blood sugar, characterized by containing tangerine leaf extract as an active ingredient.

7. A functional food composition for the prevention or improvement of diseases related to abnormal blood sugar control, characterized by containing tangerine leaf extract as an active ingredient.

8. A feed composition for lowering blood sugar characterized by containing tangerine leaf extract as an active ingredient.

9. A feed composition for the prevention or improvement of diseases related to abnormal blood sugar control, characterized by containing tangerine leaf extract as an active ingredient.

10. A method for improving blood sugar, comprising the step of administering a pharmaceutical composition according to claim 1 or 2 to an individual other than a human.

11. A method for preventing or improving a disease related to abnormal blood sugar control, comprising the step of administering a pharmaceutical composition according to any one of claims 3 to 5 to an individual other than a human.

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