KR20230092444A - Composition for preventing or treating obesity or diabetes mellitus comprising lotus root extract as an active ingredient - Google Patents

Abstract

The present invention relates to: a method for producing a lotus root extract with excellent efficacy in improving and treating obesity or diabetes; and a pharmaceutical composition for preventing or treating obesity or diabetes containing the extract. Specifically, the present invention relates to: a method for producing a lotus root extract with efficacy in improving or treating obesity or diabetes including a soaking step; and a pharmaceutical composition and a health functional food for preventing or treating obesity or diabetes containing the lotus root extract prepared by the method as an active ingredient. The lotus root extract prepared by the method of the present invention has both an anti-obesity effect (lipid production inhibitory activity/inhibition of adipogenic differentiation and metabolic regulator gene expression) and an anti-diabetic effect (α-glucosidase inhibitory effect/increased expression of proteins related to cell metabolism) and has no side effects as an extract derived from natural herbal medicine to be usefully used in medicine and health functional foods to effectively prevent, improve, and treat obesity or diabetes.

Description

Composition for preventing or treating obesity or diabetes comprising lotus root extract as an active ingredient}

The present invention relates to a method for preparing a lotus root extract excellent in improving and treating obesity or diabetes mellitus, and a composition for preventing or treating obesity or diabetes containing the extract.

There are about 20 billion fat cells in the human body, which play a role in accumulating or releasing energy in the body of mammals. In these cells, there is a complex control principle for the accumulation and release of energy. When the supply of energy exceeds the demand for energy, it is stored as neutral fat in the fat cell, and when the energy is depleted, it is broken down into free fatty acid and glucose. is used Obesity occurs when excessive energy accumulation occurs due to an imbalance in this process, and it is believed to be caused by a phenomenon in which the size or number of fat cells increases.

Obesity, which has about 30-40% of modern people, is known as a strong risk factor that can cause high blood pressure, coronary artery disease, type 2 diabetes, and various types of cancer. In particular, obesity and diabetes have a very close relationship in the prevalence mechanism.

Obesity generally shows symptoms of decreased insulin sensitivity when body fat increases, and in particular, accumulation of abdominal fat is related to glucose intolerance. Obesity is one of many causes of insulin resistance, and type 2 diabetes does not occur in some cases with severe obesity. However, there is a close correlation between obesity and insulin resistance in patients with type 2 diabetes, so the more obese the more insulin resistance.

Dyslipidemia in type 2 diabetes usually improves when blood sugar is controlled, but some patients do not. This case is called insulin resistance syndrome or central obesity syndrome. The most important feature of insulin resistance syndrome is central or visceral obesity. Central and visceral obesity secondary to insulin resistance, accompanied by hyperinsulinemia, hypertension, and impaired glucose tolerance.

As such, the induction of secondary diabetes by obesity is currently emerging as an important issue, and for the purpose of simultaneously improving this, insulin sensitivity improving agents that can reduce insulin resistance, namely Thiazolidinedione-based drugs and biguanides ( There are Xenical (Orlistat) and Reductil (Sibutramine) as anti-obesity drugs whose therapeutic effects have been announced after conducting long-term clinical trials of Biguanide for obese patients with metabolic syndrome. However, these drugs have anti-obesity effects by suppressing appetite and fat absorption rather than promoting fat burning and decomposition, so they are not necessary and sufficient conditions to solve insulin resistance, so obesity and diabetes can be completely solved. Not only are there no, but serious side effects have been reported, so there is no clear guarantee of safety yet. Therefore, there is a need to develop a new material that is safer for the human body while exhibiting efficacy equivalent to or higher than that of conventional materials.

Accordingly, while the inventors of the present invention were developing a new therapeutic agent capable of effectively treating obesity or diabetes without side effects using natural herbal medicines, they established optimal conditions for preparing an extract excellent in treating obesity or diabetes using lotus root, and prepared by the above method. The present invention was completed by confirming that the lotus root extract could effectively improve and treat obesity or diabetes without side effects.

Republic of Korea Patent Publication 10-2002-0018844

An object of the present invention is to provide a pharmaceutical composition for preventing or treating obesity or diabetes comprising a lotus root extract as an active ingredient.

In addition, an object of the present invention is to provide a health functional food composition for preventing or improving obesity or diabetes comprising a lotus root extract as an active ingredient.

In addition, an object of the present invention is to provide a method for preparing a lotus root extract having an improvement or treatment effect on obesity or diabetes.

In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating obesity or diabetes comprising a lotus root extract as an active ingredient.

In one embodiment of the present invention, the lotus root extract may be extracted by water or organic solvent extraction, but is not limited thereto.

In one embodiment of the present invention, the lotus root extract may inhibit adipocyte differentiation and adipogenesis, but is not limited thereto.

In one embodiment of the present invention, the lotus root extract may inhibit the expression of adipogenesis regulators C/EBP-α and PPAR-γ, but is not limited thereto.

In one embodiment of the present invention, the lotus root extract may be to inhibit α-glucosidase, but is not limited thereto.

In one embodiment of the present invention, the lotus root extract may increase p-AMPK expression, but is not limited thereto.

In addition, the present invention provides a health functional food composition for preventing or improving obesity or diabetes comprising a lotus root extract as an active ingredient.

In addition, the present invention comprises the steps of immersing the lotus root in distilled water; crushing; filtering; And it provides a method for producing a lotus root extract having an anti-obesity or anti-diabetic effect comprising the step of concentrating under reduced pressure.

In one embodiment of the present invention, the lotus root may be fermented by Pectinex enzyme treatment, but is not limited thereto.

The lotus root extract prepared by the method of the present invention has both an anti-obesity effect (adipogenesis inhibitory activity / fat differentiation and metabolic regulator gene expression suppression) and an antidiabetic effect (α-glucosidase inhibitory effect / cellular metabolism-related protein expression increase). And, as an extract prepared from natural herbal medicines, it can be usefully used in medicines and health functional foods for effectively preventing, improving, and treating obesity or diabetes without side effects.

1 is a diagram showing the results of component analysis of lotus root extract according to the production method of the present invention through HPLC chromatogram.
Figure 2 is a view showing the tryptophan (tryptophan) component analysis results of the lotus root extract according to the production method of the present invention through HPLC chromatogram.
Figure 3 is a diagram showing the results of analysis of the gallocatechin component of the lotus root extract according to the production method of the present invention through HPLC chromatogram.
Figure 4 is a diagram showing the results of analysis of the catechin component of the lotus root extract according to the production method of the present invention through HPLC chromatogram.
Figure 5 is a diagram showing the results of component analysis of the lotus root extract according to the manufacturing method by improving the pretreatment method (adding a soaking process) of the present invention through HPLC chromatogram.
Figure 6 is a diagram showing the adipogenesis inhibitory activity through the Oil red O staining results of the lotus root extract according to the manufacturing method of the present invention.
Figure 7 is a diagram showing the fat differentiation and metabolic regulator gene expression inhibitory effect of the lotus root extract according to the production method of the present invention.
8 is a diagram showing the α-glucosidase inhibitory effect of lotus root extract according to the manufacturing method of the present invention.
9 is a diagram showing the expression results of glucose-related proteins (GLUT4 and p-AKT) and cell metabolism-related proteins (p-AMPK) of the lotus root extract according to the manufacturing method of the present invention.

The present invention is characterized by providing a pharmaceutical composition for preventing or treating obesity or diabetes using lotus root extract, which is a natural herb.

The lotus root used in the present invention is an underground stem of a lotus flower with an empty hole, a hard tissue, a low-calorie food, and contains tannin, iron, asparagine, arginine, tyrosine, lecithin, vitamin C, etc. Tannin has anti-inflammatory action, stops coughing and thirst, and has a strong action to restore lost energy. It is rich in blood circulation, cleans blood vessels, improves skin metabolism, contains lecithin, a type of unsaturated fatty acid, which suppresses excessive production of cholesterol in the blood, and has a sedative effect to calm the mind and nerves. It is known to have a function that helps to get rid of fatigue and get a good night's sleep. Diabetic patients feel weak, dry mouth, thirst, poor blood circulation, and poor skin. Lotus root can help alleviate these symptoms.

In the present invention, the pharmaceutical composition may be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the active ingredients of the present invention, and the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, and expanding agents. , lubricants, lubricants or flavoring agents may be used.

The pharmaceutical composition may be preferably formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration.

Formulations of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops, or injectable solutions. For example, for formulation in the form of a tablet or capsule, the active ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inactive carrier such as ethanol, glycerol, or water. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. In compositions formulated as liquid solutions, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added if necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare formulations for injections such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets. Furthermore, using a method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field, it can be preferably formulated according to each disease or component.

The active ingredient of the present invention may be included in an amount of 0.1 to 30% by weight based on the total weight of the composition.

In addition, the active ingredient of the present invention can be administered in a therapeutically effective amount in combination with one or more therapeutic agents (pharmaceutical combination). For example, a synergistic effect may occur when the active ingredient of the present invention is used in combination with a chemotherapeutic agent. When the active ingredient of the present invention is administered together with other therapies, the dosage of the co-administered compound will of course vary depending on the type of co-drug used, the specific drug used, the condition to be treated, and the like.

In addition, the present invention can provide a method for preventing or treating obesity or diabetes comprising administering the composition of the present invention to a mammal.

As used herein, the term "mammal" refers to a mammal that is a subject of treatment, observation or experimentation, and preferably refers to a human.

As used herein, the term "therapeutically effective amount" refers to an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, which is considered by a researcher, veterinarian, physician or other clinician, which An amount that induces relief of the symptoms of the disease or disorder being treated is included. It is obvious to those skilled in the art that the therapeutically effective dosage and frequency of administration of the active ingredient of the present invention will vary depending on the desired effect. Therefore, the optimal dose to be administered can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the content of the active ingredient and other ingredients contained in the composition, the type of formulation, and the patient's age, weight, and general health condition , Gender and diet, administration time, administration route and secretion rate of the composition, treatment period, can be adjusted according to various factors including drugs used simultaneously. In the treatment method of the present invention, in the case of adults, the active ingredient of the present invention can be administered at a dose of 0.001 mg/kg to 1000 mg/kg when administered once to several times a day, but particularly limiting the range It is not.

Furthermore, the composition according to the present invention is composed of natural herbal medicines prescribed in oriental medicine and does not cause side effects and toxicity in the body and can be safely used even when taken for a long time, so the lotus root extract of the present invention is suitable for preventing or improving obesity or diabetes It can also be used as a food composition.

Therefore, the present invention can provide a health functional food for preventing or improving obesity or diabetes comprising the lotus root extract prepared by the method of the present invention as an active ingredient.

The food composition of the present invention can be easily utilized as a food that is effective in preventing and improving obesity or diabetes, for example, as a main ingredient, supplementary ingredient, food additive, functional food or beverage of food.

As used herein, the term "food" means a natural product or processed product containing one or more nutrients, and preferably means a product that can be directly eaten through a certain degree of processing, and as a general meaning , foods, food additives, functional foods and beverages.

Foods to which the food composition according to the present invention can be added include, for example, various foods, beverages, chewing gum, tea, vitamin complexes, and functional foods. In addition, in the present invention, food includes special nutritional food (eg, formula milk, infant food, baby food, etc.), processed meat product, fish meat product, tofu, jelly, noodles (eg, ramen, noodles, etc.), bread, health supplement food, seasoning Foods (eg, soy sauce, soybean paste, gochujang, mixed paste, etc.), sauces, confectionery (eg, snacks), candies, chocolates, chewing gum, ice cream, dairy products (eg, fermented milk, cheese, etc.), other processed foods, kimchi, It includes, but is not limited to, pickled foods (various types of kimchi, pickled vegetables, etc.), beverages (eg, fruit drinks, vegetable drinks, soy milk, fermented beverages, etc.), and natural seasonings (eg, ramen soup, etc.). The food, beverage or food additive may be prepared by a conventional manufacturing method.

In addition, the above “functional food” refers to a food group or food composition that has added value so that the function of the food acts for a specific purpose by using physical, biochemical, or bioengineering methods, etc., to control biological defense rhythms and prevent diseases It means a food designed and processed to sufficiently express the body's regulatory functions related to recovery and recovery, and specifically, it may be a health functional food. The functional food may include food additives that are acceptable in food science, and may further include appropriate carriers, excipients, and diluents commonly used in the manufacture of functional foods.

Furthermore, foods containing the food composition of the present invention other than those described above are various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and fillers (cheese, chocolate, etc.), pectins acids and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. and can be used.

In the food containing the food composition of the present invention, the amount of the active ingredient according to the present invention may be included in 0.001% to 90% by weight of the total weight of the food, preferably 0.1% to 40% by weight. In the case of beverages, it may be included in a ratio of 0.001 g to 2 g, preferably 0.01 g to 0.1 g, based on 100 mL, but long-term intake for health and hygiene purposes or health control purposes In the case of, it may be less than the above range, and the active ingredient may be used in an amount greater than the above range because there is no problem in terms of safety, so it is not limited to the above range.

In addition, the present invention conducted research to develop an optimal manufacturing process with excellent obesity or diabetes treatment effects using such lotus root, and as a result, the lotus root extract prepared through the process of precipitating or fermenting the lotus root in distilled water It was confirmed that the treatment effect of obesity or diabetes was excellent.

In the present invention, the process for preparing the lotus root extract may be prepared according to a conventional method for obtaining an extract from a natural product known in the art, that is, extraction using a conventional solvent under conditions of conventional temperature and pressure. can For example, extraction may be performed using at least one solvent selected from the group consisting of water, alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.

In addition, the method of extracting the extract using a solvent may be extracted through various methods such as hot water extraction, cold needle extraction, reflux extraction, ultrasonic extraction, but is not limited thereto.

The prepared extract may then be filtered or concentrated or dried to remove the solvent, and both filtration, concentration and drying may be performed. For example, filter paper or a vacuum filter may be used for filtration, a vacuum concentrator may be used for concentration, and a freeze-drying method may be used for drying, but is not limited thereto.

Preferably, the lotus root extract is immersed in distilled water for 1 hour or 2 hours; crushing; filtering the extract; And it may be obtained through the step of concentrating under reduced pressure.

In addition, in the method for preparing the lotus root extract of the present invention, the lotus root may be characterized in that it is fermented by Pectinex enzyme treatment.

The Pectinex is a complex enzyme containing polygalacturonase derived from Aspergillus aculeatus.

Hereinafter, the components and technical features of the present invention will be described in more detail through the following examples. However, the following examples are merely illustrative of the content of the present invention, but the scope of the invention is not limited by the examples.

Example 1. Production of lotus root extract having activity in treating obesity or diabetes

1-1. Manufacture of raw lotus root juice

After adding water to the raw lotus root, it was pulverized and filtered first with a non-woven fabric, followed by filtration under reduced pressure to prepare an extract.

1-2. Manufacturing of non-fermented lotus root extract

The lotus root was pulverized, extracted at low temperature at 40° C. for 4 hours, filtered, and then concentrated to prepare a powder.

1-3. Manufacturing of fermented lotus root extract

The lotus root was pulverized, extracted at low temperature at 40° C. for 4 hours, treated with Pectinex enzyme and cellulose, filtered, and concentrated to prepare a powder.

Example 2. Analysis of components of lotus root extract according to manufacturing method

Component analysis of the raw lotus root extract, non-fermented lotus root extract and fermented lotus root extract prepared in Example 1 was performed.

After precisely taking L-tyrosine, dissolve it in 20% methanol, dilute it appropriately, take a certain amount of the used standard solution and raw lotus root, non-fermented and fermented lotus root extract powder, and then add 20% methanol to a concentration of 20 mg/mL The prepared test solution was used, and specific analysis conditions are shown in Table 1 below.

detector UV absorbance photometer 210 nm column YMC tri-art C18 (5 μm, 4.6 × 250 mm) column temperature 35℃ injection volume 20 µL Lee Dong-sang Mobile Phase A: 0.1% H ₃ PO ₄ in H ₂ O
Mobile phase B: ACN time (minutes) Mobile phase A (%) Mobile phase B (%) 0 97 3 10 97 3 15 95 5 40 95 5 60 92 8 70 92 8 85 90 10 95 88 12 115 88 12 116 97 3 120 97 3 flow rate 0.7 mL/min

As a result, referring to FIG. 1, as a result of analyzing the main components according to the lotus root pretreatment method, it was confirmed that components A, B and C including tyrosine were commonly contained.

When comparing raw lotus root juice with non-fermented and fermented lotus root at the same concentration, it was confirmed that the absorption of A, B, and C components was generally higher in raw lotus root juice, and tyrosine was extracted at a lower temperature than raw lotus root. It was confirmed that the absorption was relatively high in non-fermented and fermented lotus roots.

In Example 3 below, in order to confirm unidentified components other than tyrosine among the four main components of raw lotus root, non-fermented and fermented lotus root, additional separation of specific components from the lotus root extract was performed.

Example 3. Separation of main components of lotus root

3-1. Tryptophan component separation experiment

As for the experimental method, about 1 Kg of dried lotus root was taken, 10 L of primary distilled water was added, and ultrasonic extraction was performed twice for 2 hours. The extract was first filtered using a non-woven fabric and then centrifuged at 2,500 rpm for 5 minutes to remove suspended matter. The supernatant obtained after centrifugation was filtered under reduced pressure for the second time using No. 4 filter paper, and then concentrated to obtain a water extract (280 g). The water extract was suspended in 1 L of distilled water, and then extracted twice for 3 to 5 hours by adding 1 L of ethyl acetate, and then concentrated to obtain an ethyl acetate fraction (0.85 g). Normal butanol was added to the water layer remaining after the fractionation, and fractionation was performed twice for 3 to 5 hours, and then concentrated to obtain a normal butanol fraction (9.11 g). The normal butanol fraction was subjected to first Diaion HP-20 column chromatography according to the following method.

First, Diaion gel was washed and filled in a column, and then completely equilibrated with distilled water. After loading the sample, water, 20% methanol, and 40% methanol were sequentially eluted, and then concentrated under reduced pressure to obtain each fraction. ODS-A column chromatography was performed on the 20% methanol fraction among the three fractions as follows. After loading the sample on a column (2.5 × 25 cm) equilibrated with 40% methanol, it was eluted with the same solvent conditions to secure an aliquot. After confirming the high-content fraction of the target component through TLC analysis, it was concentrated to finally secure 4.2 mg of tryptophan (FIG. 2).

Specific analysis conditions are shown in Table 2 below.

HPLC Agilent 1200 infinity system Column YMC triart C18 (250×4.6 m.m.I.D, 5μm) Temperature 35℃ Detector UV210nm Injection volume 20μL Flow 0.7 mL/min Mobile phase - Solvent A(%) : 0.1%H ₃ PO ₄ in H ₂ O
- Solvent B (%) : ACN Gradient
Time(min) Solvent A (%) Solvent B (%) 0 97 3 10 97 3 15 95 5 40 95 5 60 92 8 70 92 8 85 90 10 95 88 12 115 88 12 116 97 3 120 97 3

As a result, as a result of comparing the NMR results of the secured compound with the records of the reference literature (Journal of the faculty of pharmacy, 2003, 23(2), 85-94), the component was identified as tryptophan.

3-2. Gallocatechin component separation experiment

As for the experimental method, about 1 Kg of dried lotus root was taken, 10 L of primary distilled water was added, and ultrasonic extraction was performed twice for 2 hours. The extract was first filtered using a non-woven fabric and then centrifuged at 2,500 rpm for 5 minutes to remove suspended matter. The supernatant obtained after centrifugation was filtered under reduced pressure for the second time using No. 4 filter paper, and then concentrated to obtain a water extract (280 g). After suspending the water extract in 1 L of distilled water, 1 L of ethyl acetate was added to extract twice for 3-5 hours, and then concentrated to obtain an ethyl acetate fraction (0.85 g). Normal butanol was added to the water layer remaining after the fractionation, and fractionation was performed twice for 3-5 hours, and then concentrated to obtain a normal butanol fraction (9.11 g). The ethyl acetate fraction was subjected to first ODS-A column chromatography according to the following method. After loading the sample on a column (2.5 × 27 cm) equilibrated with 40% methanol, the sample was eluted with the same solvent conditions to secure an aliquot. After confirming and separating the target component through TLC analysis, it was concentrated to obtain a total of four fractions. For further purification from ODS-A purification fraction 1, sephadex column chromatography was performed. First, after loading the sample on an equilibrated column (1.5 × 30 cm), the solvent was constantly eluted using 40% methanol under the same solvent conditions. Each aliquot was concentrated to obtain 10.6 mg of gallocatechin after confirming the high-content fraction through TLC analysis (FIG. 3).

Specific analysis conditions are shown in Table 3 below.

HPLC Agilent 1200 infinity system Column YMC triart C18 (250×4.6 m.m.I.D, 5μm) Temperature 35℃ Detector UV210nm Injection volume 20μL Flow 0.7 mL/min Mobile phase - Solvent A(%) : 0.1%H ₃ PO ₄ in H ₂ O
- Solvent B (%) : ACN Gradient
Time(min) Solvent A (%) Solvent B (%) 0 97 3 10 97 3 15 95 5 40 95 5 60 92 8 70 92 8 85 90 10 95 88 12 115 88 12 116 97 3 120 97 3

As a result, as a result of comparing the NMR results of the secured compound with the records of the reference literature (Molecules, 2020, 25, 4917-4931), the component was identified as gallocatechin.

3-3. Catechin component separation experiment

As for the experimental method, about 1 Kg of dried lotus root was taken, 10 L of primary distilled water was added, and ultrasonic extraction was performed twice for 2 hours. The extract was first filtered using a non-woven fabric and then centrifuged at 2,500 rpm for 5 minutes to remove suspended matter. The supernatant obtained after centrifugation was filtered under reduced pressure for the second time using No. 4 filter paper, and then concentrated to obtain a water extract (280 g). After suspending the water extract in 1 L of distilled water, 1 L of ethyl acetate was added to extract twice for 3-5 hours, and then concentrated to obtain an ethyl acetate fraction (0.85 g). Normal butanol was added to the water layer remaining after the fractionation, and fractionation was performed twice for 3-5 hours, and then concentrated to obtain a normal butanol fraction (9.11 g). The ethyl acetate fraction was subjected to first ODS-A column chromatography according to the following method. After loading the sample on a column (2.5 × 27 cm) equilibrated with 40% methanol, the sample was eluted with the same solvent conditions to secure an aliquot. After confirming and separating the target component through TLC analysis, it was concentrated to obtain a total of four fractions. Sephadex column chromatography was performed for further purification from ODS-A purification fraction 4. First, after loading the sample on an equilibrated column (1.5 × 30 cm), the solvent was constantly eluted using 50% methanol under the same solvent conditions. After separating the high-content fraction through TLC analysis, it was concentrated to obtain a total of two fractions, of which 10.0 mg of catechin was finally obtained from the second fraction (FIG. 4).

Specific analysis conditions are shown in Table 4 below.

HPLC Agilent 1200 infinity system Column YMC triart C18 (250×4.6 m.m.I.D, 5μm) Temperature 35℃ Detector UV210nm Injection volume 20μL Flow 0.7 mL/min Mobile phase - Solvent A(%) : 0.1%H ₃ PO ₄ in H ₂ O
- Solvent B (%) : ACN Gradient
Time(min) Solvent A (%) Solvent B (%) 0 97 3 10 97 3 15 95 5 40 95 5 60 92 8 70 92 8 85 90 10 95 88 12 115 88 12 116 97 3 120 97 3

As a result, as a result of comparing the NMR results of the obtained compound with the records of the reference literature (Molecules, 2020, 25, 4917-4931), the component was identified as catechin.

Example 4. Lotus root component analysis according to pretreatment improvement (adding soaking process)

i) Fresh lotus root juice obtained by adding water to raw lotus root and then pulverizing and filtering (1st filtration: non-woven fabric / 2nd filtration: reduced pressure filtration) and ii) immersing raw lotus root in distilled water for 1 or 2 hours and grinding After filtration (first filtration: non-woven fabric / second filtration: filtration under reduced pressure), extracts concentrated under reduced pressure and iii) fresh lotus roots were cut into 1.0 to 1.5 cm, soaked in distilled water for 1 or 2 hours, pulverized, and filtered (first After filtration: non-woven fabric / secondary filtration: filtration under reduced pressure), component analysis was performed on the extract concentrated under reduced pressure at a temperature of 45-50 ° C.

After precisely taking L-tyrosine, tryptophan, gallocatechin and catechin, dissolving them in 20% methanol, diluting them appropriately, and adding 20% methanol to the standard solution and the prepared extract. A test solution prepared at a concentration of 20 mg/mL was used, and specific analysis conditions are shown in Table 5 below.

detector UV absorbance photometer 210 nm column YMC tri-art C18 (5 μm, 4.6 × 250 mm) column temperature 35℃ injection volume 20 µL Lee Dong-sang Mobile Phase A: 0.1% H ₃ PO ₄ in H ₂ O
Mobile phase B: ACN time (minutes) Mobile phase A (%) Mobile phase B (%) 0 97 3 10 97 3 15 95 5 40 95 5 60 92 8 70 92 8 85 90 10 95 88 12 115 88 12 116 97 3 120 97 3 flow rate 0.7 mL/min

As a result, as shown in FIG. 5, as a result of comparing the changes in the components of the lotus root powder prepared after immersing the raw lotus root in water for 1 hour or 2 hours in the form of cutting and uncutting, it was cut to 1.0 to 1.5 cm and then cut for 2 hours. It was found that certain components significantly increased in the retention time (RT) of immersed lotus root from 20 minutes to 35 minutes.

Example 5. Evaluation of anti-obesity efficacy of raw lotus root, non-fermented and fermented lotus root extracts

5-1. Fat differentiation inhibitory ability measurement experiment (Oil red O staining)

3T3-L1 cells were cultured at 37°C and 5% CO ₂ conditions after replacing the medium with BCS-Media every 2-3 days. 3T3-L1 preadipocytes became confluent to differentiate into adipocytes, and after 2 days, the differentiation induction medium was replaced and marked as Day 0. Differentiation induction medium was used for 48 hours from Day 0 to Day 2, including 0.5 mM IBMX, 1 μM DEX, and 167 nM insulin in DMEM containing 10% BCS and 1% penicillin-streptomycin. After 2 days of differentiation, the medium was replaced with post-differentiation medium containing 167 nM insulin along with FBS-Media at intervals of 2 days from Day 2 to Day 7, and differentiation of cells was terminated on Day 7.

Oil Red-O staining is a method for measuring intracellular fat produced by staining differentiated 3T3-L1 cells with Oil Red-O reagent. Oil Red-O staining experiments were performed to examine the effects of raw lotus root, non-fermented and fermented lotus root extracts on adipocyte differentiation and adipogenesis in 3T3-L1 cells.

The medium of the differentiated cells was removed, the cells were washed with D-PBS (DPBS, SH30028.03), fixed with 10% Formalin for 2 hours, and then washed with 40% Isopropyl alcohol (2-Propanol, 67-63-0). After drying, the washed cells were stained with Oil-red O Solution, and the size of intracellular fat was observed under an optical microscope (AE 31, Motic, USA). In addition, after extracting fat using 100% isopropanol for quantitative analysis, 200 μL of each was transferred to a 96 well plate and the absorbance was measured at 520 nm with a microplate reader.

As a result of the experiment, as shown in Figure 6a, the differentiation and adipogenesis inhibitory effects of adipocytes were found in both raw lotus root, non-fermented and fermented lotus root extracts, and as shown in Figure 6b, Oil Red- It was confirmed that the accumulated fat content by O staining was the lowest in the raw lotus root extract.

5-2. Confirmation of suppression of production of fat differentiation regulatory genes (C/EBP-α and PPAR-γ) according to polymerase chain reaction method (RT-PCR)

In order to investigate the effect of raw lotus root, non-fermented and fermented lotus root extracts on adipocytes in 3T3-L1 cells, C/EBP-α and PPAR-γ, which are factors related to adipogenesis, were confirmed at the mRNA level. C/EBP-α and PPAR-γ are early factors for differentiation from 3T3-L1 pre-adipocytes to adipocytes, and this is a method for measuring the ability to inhibit adipogenesis produced in cells. 3T3-L1 pre-adipocytes were treated with raw lotus root, non-fermented and fermented lotus root extracts at a concentration of 200 μg/mL by ratio, differentiated into adipocytes for 7 days, and then total RNA using RNAiso (Takara, Tokyo, Japan) separated. The isolated total RNA was quantified to 1 μg, added to a buffer reaction solution (Maxime RT PreMix; Intron, Washington, USA) to which Oligo-dT and DEPC were added, reacted at 45 ° C for about 60 minutes, and then treated at 95 ° C for 5 minutes. cDNA was synthesized. Reaction mixture containing this template cDNA and Taq polymerase (WizPureTM FX-PCR 2X Master; Wizbiosolutions, Seongnam, Korea) and each primer sequence, 5'-GACCACTCGCATTCCTTT (Forward), 5'-CCACAGACTC GGCACTCA (Reverse); C/EBPα, 5'-CACTCCCACTCTTCCACCT-3' (Forward), 5'-CTTGCTCAGTGTCCTTGCTG-3' (Reverse); PPAR-γ, 5'-GTTAGCCATGTGGTA GGAGACA (Forward), 5'-CCCAGCCGTTAGTGAAGAGT (Reverse); After PCR amplification with GAPDH, it was analyzed by 1.5% agarose gel electrophoresis.

As a result of the experiment, as shown in FIG. 7a, it was confirmed that all of the raw lotus root, non-enzymatic and enzyme-treated lotus root extract inhibited the expression of factors involved in fat metabolism (C/EBP-α and PPAR-γ).

Example 6. Evaluation of antidiabetic efficacy of raw lotus root, non-fermented and fermented lotus root extracts

6-1. α-glucosidase inhibitory activity confirmation test

α-glucosidase inhibitory activity is measured by measuring the amount of p -nitrophenyl cleaved through the reaction between the enzyme α-glucosidase and the substrate p -nitrophenyl-α-D-glucopyranoside. 6.8) was dissolved to 1 unit/mL and used as an enzyme solution, and p -nitrophenyl-α-D-glucopyranoside was dissolved to 2 mM in 0.1 M sodium phosphate buffer (PH 6.8) and used as a substrate solution. After adding 10 μL of the sample dissolved in DMSO and 50 μL of the enzyme solution, reacting at room temperature for 5 minutes, the absorbance was measured at 405 nm using a microplate reader. Next, 50 μL of the substrate solution was added and reacted at room temperature for 5 minutes, and absorbance was measured at 405 nm.

As a result of the experiment, as shown in FIG. 8, the IC ₅₀ values of raw lotus root, non-fermented and fermented lotus root extracts were 0.41, 0.64 and 0.96 μg/mL, and in the case of acarbose, a positive control group, it was 7.56 μg/mL, and raw lotus root , high α-glucosidase inhibitory activity of non-fermented and fermented lotus root extracts was confirmed.

6-2. Measurement of protein expression related to glucose uptake and cell metabolism

Using C2C12 myoblast cells, which are muscle cells, we identified Glut4, Akt, and AMPK, which are factors that regulate glucose uptake in the body in relation to sugar metabolism. First, when C2C12 cells became 100% confluent, they were cultured for 4 days after replacing them with normal DMEM medium supplemented with 2% horse serum. C2C12 cells were washed twice with PBS and lysed in lysis buffer (50 mM Tris-HCl [pH 7.4], 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM sodium orthovanadate. , 1 mM NaF, 1 µg/mL aprotinin, 1 µg/mL leupeptin, and 1 µg/mL pepstatin), and left on ice for about 5 minutes, followed by centrifugation at 13,000 rpm for 20-30 minutes. SDS sample buffer was added to the obtained supernatant and then boiled at 100° C. for 5 minutes to induce protein denaturation. Next, proteins were separated using SDS PAGE, transferred to a PVDF membrane, and western blotting analysis was performed using each antibody.

As a result of the experiment, as shown in FIG. 9a, as a result of analyzing the effects of raw lotus root, non-fermented and fermented lotus root extracts on AMPK, Akt activation and GLUT4 membrane transition related to glucose absorption promotion, it was confirmed that fermented lotus root extract was the most effective, As shown in Figure 9b, when the expression level of each gene factor was quantified, it was confirmed that p-AMPK activity increased in all of the raw lotus root, non-fermented and fermented lotus root extracts.

Claims (9)

A pharmaceutical composition for preventing or treating obesity or diabetes comprising a lotus root extract as an active ingredient. According to claim 1,
The extract is a pharmaceutical composition for preventing or treating obesity or diabetes, characterized in that extracted by water or organic solvent extraction method. According to claim 1,
The pharmaceutical composition for preventing or treating obesity or diabetes, characterized in that the composition inhibits adipocyte differentiation and adipogenesis. According to claim 1,
The composition is a pharmaceutical composition for the prevention or treatment of obesity or diabetes, characterized in that inhibits the expression of fat differentiation regulators C / EBP-α and PPAR-γ. According to claim 1,
The composition is a pharmaceutical composition for preventing or treating obesity or diabetes, characterized in that inhibits α-glucosidase. According to claim 1,
The composition is a pharmaceutical composition for preventing or treating obesity or diabetes, characterized in that it increases the expression of p-AMPK. A health functional food composition for preventing or improving obesity or diabetes comprising lotus root extract as an active ingredient. immersing the lotus root in distilled water;
crushing;
filtering; and
Method for producing a lotus root extract having an anti-obesity or anti-diabetic effect comprising the step of concentrating under reduced pressure. According to claim 8,
The method for producing a lotus root extract having an anti-obesity or anti-diabetic effect, characterized in that the lotus root is fermented by Pectinex enzyme treatment.

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