PHARMACEUTICAL COMPOSITION COMPRISING MARMELO EXTRACT FOR PREVENTING OR TREATING OBESITY

Abstract

The present invention relates to a composition for treatment and/or food usable in prevention, improvement and/or treatment of related disease such as reduction of body weight and/or body fat, lowering blood glucose, lowering cholesterol in blood, and the like, which comprises a marmelo extract as an active ingredient, and a method for preparation thereof.

Claims

1. A method for preventing, improving or treating obesity, or reducing body weight or body fat, comprising administering a pharmaceutically effective amount of marmelo extract to a subject in need of prevention, improvement or treatment of obesity.

2. The method for preventing, improving or treating obesity, or reducing body weight or body fat according to claim 1, wherein the administering is administering the marmelo extract in an amount of 1 to 400 mg/kg, based on the body weight of the subject to be administered.

3. The method for preventing, improving or treating obesity, or reducing body weight or body fat according to claim 1, wherein the administering is administering the marmelo extract in an amount of 0.0048 to 1.92 g/day, based on the body weight 60 kg of the subject to be administered.

4. The method for preventing, improving or treating obesity, or reducing body weight or body fat according to claim 1, wherein the subject is a primate, a rodent, a mammal, a bird, a reptile, an amphibian or a vertebrate.

5. The method for preventing, improving or treating obesity, or reducing body weight or body fat according to claim 1, wherein the marmelo extract is administered by oral administration, intravenous administration, intramuscular administration, subcutaneous administration or intraperitoneal administration.

6. The method for preventing, improving or treating obesity, or reducing body weight or body fat according to claim 1, wherein the marmelo extract is, an extract obtained by extracting marmelo with 10 to 70 (v/v) % alcohol.

7. The method for preventing, improving or treating obesity, or reducing body weight or body fat according to claim 1, wherein the marmelo of the marmelo extract is, in a solid form remaining after removing marmelo juice after compressing marmelo.

8. The method for preventing, improving or treating obesity, or reducing body weight or body fat according to claim 6, wherein the alcohol extraction is performed by adding 1 to 10 volume times of alcohol at 50 to 100° C. and extracting for 1 to 24 hours.

9. A method for preventing, improving or treating metabolic disease, or lowering blood glucose or lowering cholesterol in blood, comprising administering a pharmaceutically effective amount of marmelo extract to a subject in need of prevention, improvement or treatment of metabolic disease.

10. The method for preventing, improving or treating metabolic disease, or lowering blood glucose or lowering cholesterol in blood according to claim 9, wherein the administering is administering the marmelo extract in an amount of 1 to 400 mg/kg, based on the body weight of the subject to be administered.

11. The method for preventing, improving or treating metabolic disease, or lowering blood glucose or lowering cholesterol in blood according to claim 9, wherein the administering is administering the marmelo extract in an amount of 0.0048 to 1.92 g/day, based on the body weight 60 kg of the subject to be administered.

12. The method for preventing, improving or treating metabolic disease, or lowering blood glucose or lowering cholesterol in blood according to claim 9, wherein the subject is a primate, a rodent, a mammal, a bird, a reptile, an amphibian or a vertebrate.

13. The method for preventing, improving or treating metabolic disease, or lowering blood glucose or lowering cholesterol in blood according to claim 9, wherein the marmelo extract is administered by oral administration, intravenous administration, intramuscular administration, subcutaneous administration or intraperitoneal administration.

14. The method for preventing, improving or treating metabolic disease, or lowering blood glucose or lowering cholesterol in blood according to claim 9, wherein the marmelo extract is, an extract obtained by extracting marmelo with 10 to 70 (v/v) % alcohol.

15. The method for preventing, improving or treating metabolic disease, or lowering blood glucose or lowering cholesterol in blood according to claim 9, wherein the marmelo of the marmelo extract is, in a solid form remaining after removing marmelo juice after compressing marmelo.

16. The method for preventing, improving or treating metabolic disease, or lowering blood glucose or lowering cholesterol in blood according to claim 14, wherein the alcohol extraction is performed by adding 1 to 10 volume times of alcohol at 50 to 100° C. and extracting for 1 to 24 hours.

17. The method for preventing, improving or treating metabolic disease, or lowering blood glucose or lowering cholesterol in blood according to claim 9, wherein the metabolic disease is diabetes, hyperlipidemia, arteriosclerosis, hyperinsulinemia, or metabolic syndrome.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0094] FIG. 1a and FIG. 1B are graphs showing the result of analyzing the chlorogenic acid content in the marmelo extract by HPLC chromatography. FIG. 1a shows the chlorogenic acid standard product, and FIG. 1B shows the result of Sample c (Table 4 of Example 2.2).

[0095] FIG. 2 is a graph showing the weekly body weight change according to the diet type. Group 1 refers to the control diet group, and Group 2 refers to the high-fat diet group, and Group 3 refers to the high-fat diet group administered with mdB-44.

[0096] FIG. 3 is a photograph observing the histological change of the epididymis fat tissue using an optical microscope (Carl Zeiss).

[0097] FIG. 4 is a graph showing the fat accumulation inhibitory effect by confirming the fat accumulation amount according to the extraction method of the marmelo extract. The first—is without the addition of an extract and no differentiation, the second—is for differentiation and culture without adding an extract, EE is a marmelo extract extracted with ethanol without additional processing, and mdB-44 is a marmelo extract with additional processing.

MODE FOR INVENTION

[0098] Hereinafter, the present invention will be described in more detail by examples. However, the following examples are intended to illustrate the present invention only, but the scope of the present invention is not limited by the following examples.

Example 1. Preparation of Marmelo Extract

[0099] Marmelo fruits (LICORICE EXTRACT (Tashkent, Republic of Uzbekistan)) were weighed, selected, washed and foreign substances were removed. Then, the marmelo was compressed with a compressor at a pressure of 1˜5 kg/cm.sup.2 to remove marmelo juice (liquid in a juice form generated by compressing marmelo fruits) and isolate solids. The solids were extracted with 30˜50 (v/v) % spirit of 4˜6 volume times at 65˜80° C. for 5˜6 hours twice. The extract was filtered with a 10 μm standard filter, and vacuum concentrated to 65 brix or more using a vacuum evaporator (EYELA, Tokyo, Japan) to obtain a marmelo concentrate. The concentrate was powdered by a spray drier. The marmelo extract in a concentrate or powder phase prepared as above was used for the following example.

Example 2. Analysis of Chlorogenic Acid Content of Marmelo Extract

[0100] 2.1 HPLC Analysis Method

[0101] The chlorogenic acid (CGA) content of the marmelo extract prepared in Example 1 was analyzed using LC-20A high-performance liquid chromatography (HPLC) system (Shimadzu, Japan). The HPLC analysis conditions were shown in the following Table 1 (analysis conditions), and Table 2 (moving bed gradient condition).

TABLE-US-00001 TABLE 1 Items Conditions Column Capcell Pak C.sub.18 column (Shiseido, 5 μm, 250 × 4.6 mm) Column Temperature 40° C. Flow 1.4 mL/min Detector Diode Array Detector (DAD) 330 nm Injection Volume 5 μL Mobile Phase A: phosphoric acid:water = 0.5:99.5(v/v), B: phosphoric acid:AcN = 0.5:99.5(v/v) Run time 40 min

TABLE-US-00002 TABLE 2 Time (Min) Solvent A (%) Solvent B (%) 0 95 5 7 95 5 27 70 30 28 10 90 30 10 90 31 95 5 40 95 5

[0102] Chlorogenic acid standard product 5 mg was weighed and dissolved in methanol 50 mL. 5 mL was taken in this solution and moved to a volume flask, and 5 mL methanol was added and 20 mL distilled water was added to use it as a standard solution stock solution. The solution of the stock solution was used as a standard stock solution, and was diluted step by step to prepare a calibration curve to make a standard solution for chlorogenic acid (final concentration: 0.00833, 0.00416, 0.00208, 0.00104, 0.01666 mg/mL), and the corresponding information was shown in the following Table 3. In addition, the marmelo extract 30 mg in a powder state of Example 1 was weighed and dissolved by adding 30% methanol 5 mL and filtered with a 0.45 um filter and used as a test solution (final concentration 30 mg/5 mL) (marmelo extract). The corresponding information was shown in the following Table 3.

TABLE-US-00003 TABLE 3 Sample Pretreatment concentration (Concentration) Chlorogenic acid standard 0.00833, 0.00416, 0.00208, solution 0.00104, 0.01666 mg/mL in 30% MeOH Marmelo extract 30 mg/5 mL in 30% MeOH (HPLC grade)

[0103] 2.2 Experimental Result

[0104] By the HPLC analysis method of 2.1, an experiment was performed three times, and the obtained values were shown in Table 4, FIGS. 1a (chlorogenic acid Standard product) and 1b (Sample c).

TABLE-US-00004 TABLE 4 Sample name Content (mg/g) Sample a 0.82 Sample b 0.7 Sample c 0.73

[0105] (The Samples a, b and c mean test solutions including the marmelo extract used for each experiment, when performing the experiment 3 times)

[0106] As shown in Table 4, FIG. 1a and FIG. 1B, the chlorogenic acid content in the marmelo extract prepared in Example 1 was shown as 0.82 mg/g in Sample a, 0.7 mg/g in Sample b, and 0.73 mg/g in Sample c, and therefore, it was confirmed that the distribution of the chlorogenic acid content was in the range of ±20% based on the case of Sample c, which had an intermediate value. The result shows that the marmelo extract obtained in Example 1 comprises chlorogenic acid in a certain range (for example, 0.75(mg/g)±20%) and this result shows that the chlorogenic acid content comprised in the extract is in an equal range even after performing the extraction several times.

Example 3. Preparation of Test Material and Experimental Animal

[0107] 3.1 Test Material Preparation and Animal Experiment Approval

[0108] As a test material, the marmelo extract in a powder form prepared in Example 1 (powder: hereinafter, ‘mdB-44’) was used, and all the animal experiments in the following examples were performed in accordance with the animal experiment regulations under the approval of the Animal Experiment Ethics Committee of Hallym University (Hallym 2019-67).

[0109] 3.2 Test Group and Test Material Administration

[0110] Specific pathogen free 5-week-old, male C57BL/6 mice were purchased from Doo Yeol Biotech and used. After one week of quarantine and adaptation, healthy animals without weight loss were selected and used for the experiment. The experiment animals were bred in a breeding environment set at a temperature of 23±3° C., a relative humidity of 50±10%, a ventilation frequency of 10˜15 times/hour, an illumination time of 12 hours (08:00˜20:00), and an illumination intensity of 150˜300 Lux. Adaptation period experimental animals were allowed to freely ingest solid feed for laboratory animals (Cargill Agripurina, Co., Ltd.) and drinking water. After a one-week adaptation period, healthy animals were selected and were classified into the control diet group (CD) (Group 1), high-fat diet (HFD) control group (Group 2), high-fat diet+200 mg/kg body weight (BW) mdB-44 (experimental animals of Example 3.1) administration group (Group 3) by randomized block design. The energy ratio (kcal %) (protein:carbohydrate:fat) of the control diet for the whole test period was 20:70:10, and the energy ratio (kcal %) (protein:carbohydrate:fat) of the high-fat diet was 20:20:60, and both diets were purchased from Research Diets, Inc. (New Brunswick, N.J., USA). The composition of the control diet group and high-fat diet was shown in Table 5, and the experiment design was shown in Table 6.

TABLE-US-00005 TABLE 5 Control diet High-fat diet Control diet (CD) (10 High-fat diet (HFD) (60 (CD) (10 kcal % fat) (HFD) (60 kcal % fat) kcal % fat) each kcal % fat) each each composition each composition composition calorie content composition calorie content content (g) (kcal) content (g) (kcal) Casein, 80 Mesh 200 800 200 800 L-Cystine 3 12 3 12 Corn starch 315 1260 0 0 Maltodextrin 10 35 140 125 500 Sucrose 350 1400 68.8 275.2 Cellulose, BW200 50 0 50 0 Soybean oil 25 225 25 225 Lard* 20 180 245 2205 Mineral mix 10 0 10 0 Dicalcium phosphate 13 0 13 0 Calcium carbonate 5.5 0 5.5 0 Potassium citrate, 16.5 0 16.5 0 1H.sub.2O Vitamin mix 10 40 10 40 V10001 Choline bitartrate 2 0 2 0 Total 1055.05 4057 773.85 4057 *(Lard: calculating cholesterol as 0.95 mg/g)

TABLE-US-00006 TABLE 6 Group 1 Group 2 Group 3 Control group Negative control Experimental (normal) group group Experimental diet CD HFD HFD Test material — — mdB-44 200 (mg/kg BW) Number of 10 10 10 animals (head)

[0111] During the entire period of the test, drinking water was freely ingested, and 200 mg of the test material of Example 3.1 was dissolved in the drinking water per 1 kg and orally administered at a certain time for 8 weeks (Group 3). In the following example using the corresponding experimental animal, statistical processing was expressed as mean±standard error. The collected result was analyzed using GraphPad Prism 5.0 (GraphPad software) program. To compare the difference between the test material administration group and control group, Student's t-test and one-way analysis variance (ANOVA) were used. It was judged to be statistically significant only when p<0.05 or more.

Example 4. Body Weight and Dietary Intake Measurement

[0112] The body weight of the experiment animals prepared in Example 3 at week 0 and week 8, and dietary intake was measured at 2-day intervals for 8 weeks. The amount ingested during the entire period of the test was calculated to calculate the 1-day dietary intake and 1 day energy intake. Food Efficiency Ratio (FER) was calculated as in Equation 1 below by dividing the weight gain during the test period by the amount of food consumed during the same period.

Food Efficiency Ratio=body weight gain (g)/dietary intake (g)  [Equation 1]

[0113] The measured body weight result and the total body weight gain and daily body weight gain according thereto were shown in Table 7 and the dietary intake and food efficiency ratio measured for 8 weeks were shown in Table 8, and in particular, the body weight result was shown in FIG. 2.

TABLE-US-00007 TABLE 7 Group 1 Group 2 Group 3 Experiment CD HFD HFD diet Test material — — mdB-44 200 (mg/kg BW) Week 0 23.14 ± 0.35 23.06 ± 0.24   23.10 ± 0.27 .sup.  Week 8 31.20 ± 0.67 41.91 ± 0.91*** 36.45 ± 0.91.sup.### Total body 8.1 ± 0.7 18.9 ± 0.9*** 13.4 ± 1.1.sup.##  weight gain (g) Daily body 0.146 ± 0.013  0.343 ± 0.016*** .sup. 0.243 ± 0.020.sup.## weight gain (g/day) (The result in Table 8 is expressed as mean ± standard error, and means *p < 0.05, **p < 0.01, ***p < 0.001, .sup.#p < 0.05, .sup.##p < 0.01, .sup.###p < 0.001; Group 1 (normal control group); Group 2 (negative control group); Group 3: experimental group, (mdB-44 administration group)

TABLE-US-00008 TABLE 8 Group 1 Group 2 Group 3 Experiment diet CD HFD HFD Test material (mg/kg BW) — — mdB-44 200 Total diet intake (g) 145.4 ± 1.4  129.1 ± 1.2***  112.7 ± 1.5.sup.###  Daily diet intake (g/day) 2.64 ± 0.03 2.35 ± 0.02***  2.05 ± 0.03.sup.### Daily energy intake (kcal/day) 10.18 ± 0.10  12.30 ± 0.11***  10.73 ± 0.15.sup.### Food efficiency ratio (body 0.055 ± 0.005 0.146 ± 0.007*** 0.119 ± 0.009.sup.#  weight gain/diet intake) (The numerical value is expressed as mean ± standard error, and means *p < 0.05, **p < 0.01, ***p < 0.001, .sup.#p < 0.05, .sup.##p < 0.01, .sup.###p < 0.001)

[0114] As shown in Tables 7 and 8 above, the experimental animals of all the test groups showed a normal change in body weight as their body weight continued to increase during the test period. Compared to the control diet group (Group 1), the body weight of the high-fat diet control group (Group 2) was significantly increased. On the other hand, in Group 3, the test group to which the marmelo extract was administered, compared to the high-fat diet control group (Group 2), the body weight of the experimental animals increased by high-fat diet supply (body weight gain) was significantly reduced. Through this, it was confirmed that even with a high-fat diet, when the marmelo extract was consumed in parallel, weight gain was suppressed.

[0115] In addition, the dietary intake of Group 3, the test group to which the marmelo extract was administered was significantly reduced, compared to the negative control group (Group 2), but the food efficiency ratio (body weight gain/dietary intake) was also reduced, and this means that weight loss is not due to a decrease in dietary intake alone.

Example 5. Body Fat Measurement

[0116] After anesthetizing the experimental animals 1 day before completing the experimental animal test of Example 3.2, body fat percentage was evaluated by measuring body components using dual-energy X-ray absorptiometry (DEXA, PIXImus™, GE Lunar), and the result was shown in Table 9.

TABLE-US-00009 TABLE 9 Group 1 Group 2 Group 3 Experiment diet CD HFD HFD Test material — — mdB-44 200 (mg/kg BW) Fat (%) 29.58 ± 0.84 42.84 ± 1.66*** 37.97 ± 1.46.sup.# (The numerical value is expressed as mean ± standard error, and means *p < 0.05, **p < 0.01, ***p < 0.001, .sup.#p < 0.05, .sup.##p < 0.01, .sup.###p < 0.001)

[0117] In the body fat percentage, compared to 29.58±0.84% of the control diet group (Group 1), the body fat percentage was significantly increased as 42.84±1.66% in the high-fat diet control group (Group 2), and compared to the high-fat diet control group (Group 2), the body fat percentage was significantly reduced in the mdB-44 200 mg/kg BW administration group (Group 3).

Example 6. Blood and Fat Analysis

Example 6.1 Blood Collecting and Tissue Extraction

[0118] After completion of the test of the experimental animal of Example 3, the experimental animal was sacrificed. Before sacrifice, after anesthetizing using an anesthetic prepared by diluting tribromoethanol with tert-amyl alcohol, orbital blood was collected. Blood was placed in a serum separate tube (Becton Dickinson), left at a room temperature for 30 minutes, and then centrifuged at 5,000 rpm for 10 minutes to separate serum, and stored at −70° C. until analysis. After blood collection, the experimental animal was sacrificed and white fat tissue (epididymal fat, visceral fat, retroperitoneal fat, inguinal fat) was extracted, and then rinsed with cold physiological saline, and excess water was removed with filter paper, and the weight was measured. Epididymal fat tissue was divided into thirds and part was fixed in 4% paraformaldehyde (PFA) and then embedded in paraffin to perform tissue staining, for a part, total RNA was isolated and then real-time PCR was performed, and for a part, protein was isolated and then western blot was performed, and it was stored at ˜70° C. for following further analysis.

[0119] 6.2 Tissue (Fat Tissue) Weight Measurement

[0120] The weight of the fat tissue in Example 6.1 was measured, and the result was shown in Table 10.

TABLE-US-00010 TABLE 10 Group 1 Group 2 Group 3 Experiment diet CD HFD HFD Test material (mg/kg BW) — — mdB-44 200 Epididymal fat 1.030 ± 0.070 2.471 ± 0.079*** 2.023 ± 0.109.sup.##  Retroperitoneal fat 0.447 ± 0.037 1.184 ± 0.063*** 0.954 ± 0.074.sup.#  Visceral fat 0.603 ± 0.050 1.603 ± 0.114*** 0.975 ± 0.073.sup.### Inguinal fat 0.210 ± 0.031 0.544 ± 0.033*** 0.490 ± 0.052 .sup.  White fat total weight 2.29 ± 0.14 5.80 ± 0.21*** 4.44 ± 0.21.sup.### (The numerical value is expressed as mean ± standard error, and means *p < 0.05, **p < 0.01, ***p < 0.001, .sup.#p < 0.05, .sup.##p < 0.01, .sup.###p < 0.001)

[0121] Body fat is classified into white fat tissue and brown fat tissue according to its shape and action. White fat tissue mainly stores excess energy in the body as fat, and brown fat tissue functions to produce heat and as the weight of white fat increases, the body weight increases. The weight of all of the epididymal fat, retroperitoneal fat, visceral fat and inguinal fat which are white fat tissue, was significantly increased in the high-fat diet control group (Group 2) compared to the control diet group (Group 1). On the other hand, in Group 3 to which the marmelo extract was administered, compared to the high-fat diet control group (Group 2), the body fat of all the tested items was reduced.

[0122] 6.3 Blood Glucose-Related Numerical Value Analysis

[0123] The content of glucose in blood collected in Example 6.1 was measured using a blood biochemical analyzer (KoneLab 20 XT, Thermo Fisher Scientific), and the insulin content was measured using a measurement kit purchased from Millipore Corporation according to the method suggested by the manufacturer.

[0124] Since insulin resistance is the most important factor in metabolic syndrome, measurement of blood insulin concentration is very useful for measuring insulin sensitivity and resistance, and in the present study, insulin resistance (HOMA-IR) and insulin sensitivity (QUICKI) were calculated using the blood insulin concentration and fasting blood glucose. Insulin resistance (homeostatic model assessment for insulin resistance, HOMA-IR) and insulin sensitivity (quantitative insulin sensitivity check index, QUICKI) were calculated using the fasting blood glucose and blood insulin content by the following Equations 2 and 3, and the measured result was shown in Table 11.

Insulin resistance (HOMA-IR)=[insulin (mU/L)×glucose (mg/dL)]/405  [Equation 2]

Insulin sensitivity (QUICKI)=1/[log insulin (mU/L)+log glucose (mg/dL)]  [Equation 3]

TABLE-US-00011 TABLE 11 Group 1 Group 2 Group 3 Experiment diet CD HFD HFD Test material — — mdB-44 200 (mg/kg BW) Glucose (mg/dL) 149.0 ± 10.8  196.8 ± 7.8**   185.2 ± 7.5 .sup.  Insulin (ng/mL) 2.62 ± 0.21 9.12 ± 0.97*** 5.67 ± 0.81.sup.# HOMA-IR 22.9 ± 2.2  107.1 ± 12.4***  62.3 ± 9.3.sup.##  QUICKI 0.253 ± 0.002 0.218 ± 0.004*** 0.230 ± 0.003.sup.#

[0125] Glucose was significantly increased in the high-fat diet control group (Group 2), compared to the control diet group (Group 1), and it was reduced in Group 3, compared to the high-fat diet control group (Group 2).

[0126] The blood insulin content was significantly increased as 9.12±0.97 ng/mL in the high-fat diet control group (Group 2), compared to 2.62±0.21 ng/mL in the control diet group (Group 1), while it was significantly reduced as 5.67±0.81 ng/mL in the mdB-44 administration group (Group 3), compared to the high-fat diet control group.

[0127] The insulin resistance was significantly increased as 107.1±12.4 in the high-fat diet control group (Group 2), compared to 22.9±2.2 in the control diet group (Group 1), while it was significantly reduced in the mdB-44 administration group (Group 3). The insulin sensitivity was significantly reduced as 0.218±0.004 in the high-fat diet control group (Group 2), compared to 0.253±0.002 of the control diet group (Group 1), while it was increased in the mdB-44 administration group (Group 3), compared to the high-fat diet control group. Through this, it was confirmed that the marmelo extract administration group inhibited insulin resistance caused by high-fat diet to improve impaired glucose tolerance.

[0128] 6.4 Fat-Related Numerical Value Analysis

[0129] The contents of triglyceride (TG), total cholesterol (CHOL) and HDL-cholesterol (HDL-CHOL) in blood collected in Example 6.1 were measured using a blood biochemical analyzer (KoneLab 20 XT, Thermo Fisher Scientific), and using the blood cholesterol content and HDL-cholesterol content, an atherogenic index was calculated by the following Equation 4.

Atherogenic index=total cholesterol content/HDL-cholesterol content  [Equation 4]

[0130] The obtained result was shown in Table 12.

TABLE-US-00012 TABLE 12 Group 1 Group 2 Group 3 Experiment diet CD HFD HFD Test material — — mdB-44 200 (mg/kg BW) TG (mg/dL)  77.1 ± 3.3 .sup. 90.4 ± 3.4.sup.*  77.3 ± 3.2.sup.# CHOL (mg/dL) 165.7 ± 8.4 188.4 ± 4.7.sup.*  171.8 ± 5.3.sup.# HDL-CHOL 151.6 ± 6.2 126.9 ± 5.8.sup.** 150.9 ± 6.1.sup.# (mg/dL) Atherogenic  1.09 ± 0.04  1.52 ± 0.11.sup.**  .sup. 1.15 ± 0.04.sup.## index

[0131] In general, when a high-fat diet is ingested, the supple of fatty acids increases and the concentration of triglycerides and total cholesterol in blood increases, while the concentration of HDL-cholesterol in blood tends to decrease, which causes cardiovascular and coronary vascular disease.

[0132] The contents of blood triglycerides and blood total cholesterol were significantly increased in the high-fat diet control group (Group 2), compared to the control diet group (Group 1), while it was significantly reduced in the Group 3 to which mdB-44 was administered, compared to Group 2, and in particular, the blood triglyceride numerical value was measured very similarly to the control diet group (Group 1). The blood HDL-cholesterol content was significantly reduced in the high-fat diet control group (Group 2) compared to the control diet group (Group 1), while it was significantly increased in Group 3 to which mdB-44 was administered, compared to Group 2, and was measured very similarly to the control diet group (Group 1). As a result of calculating the atherogenic index (See Equation 4), which is the most effective predictive factor of the occurrence of coronary artery disease using the blood cholesterol content and HDL-cholesterol content, it was significantly increased as 1.52±0.11 in the high-fat diet control group (Group 2), compared to 1.09±0.04 of the control diet group (Group 1), while it was significantly reduced as 1.15±0.04 (Group 3) in the mdB-44 200 mg/kg BW administration group (Group 3), compared to Group 2.

[0133] 6.5 Serum Leptin and Adiponectin Content Analysis

[0134] Adipose tissue is recognized as an endocrine organ that not only stores surplus energy, but also affects the metabolic process of the whole body by synthesizing and secreting various adipokines that play a very important physiological role. Leptin, one of the representative adipokines secreted from adipose tissue, is an important substance that regulates energy intake and storage, insulin sensitivity, and metabolic rate, and it is known that the secretion of leptin increases in case of obesity. Another major adipokine, adiponectin, is known as an important hormone that increases fatty acid oxidation in muscle and at the same time, prevents chronic disease such as diabetes and metabolic syndrome through the role of anti-inflammatory cytokines.

[0135] The leptin and adiponectin contents in blood of the blood collected in Example 6.1 were measured using a measurement kit purchased from R&D Systems according to the method suggested by the manufacturer, and the result was shown in Table 13 below.

TABLE-US-00013 TABLE 13 Group 1 Group 2 Group 3 Experiment diet CD HFD HFD Test material — — mdB-44 200 (mg/kg BW) Adiponectin 10.45 ± 0.26 9.45 ± 0.25* 10.84 ± 0.37.sup.##  (ng/mL) Leptin (ng/mL) 21.14 ± 2.96 116.04 ± 9.49*** 69.53 ± 5.13.sup.###

[0136] The blood leptin content was significantly increased in the high-fat diet control group (Group 2) compared to the control diet group (Group 1), and this was significantly reduced in the mdB-44 administration group (Group 3) compared to Group 2. The blood adiponectin content was significantly reduced in the high-fat diet control group (Group 2) compared to the control diet group (Group 1), and it was increased in the mdB-44 administration group (Group 3), compared to the high-fat diet control group (Group 2). It is known that the increase of the size, number and amount of fat cells in the visceral fat tissue has a positive correlation with the amount of leptin secretion, and a negative correlation with the amount of adiponectin secretion, and therefore, by the above result, the mdB-44 administration group significantly reduced the increased blood leptin and increased the amount of adiponectin secretion in obesity mice to show anti-obesity effect.

[0137] 6.6 Histological Analysis of Epididymal Fat Tissue

[0138] The fixed epididymal fat tissue fixed by removing from the experimental animal in Example 6.1 was embedded in paraffin and a tissue section of 5 μm was prepared from the embedded tissue. After paraffin removal, tissue was hydrated by decreasing % of alcohol sequentially from 100% alcohol to 0% alcohol (H.sub.2O). Then, the tissue was stained (hematoxylin & eosin staining, H&E staining) using Accustain® Hematoxylin and Eosin Stains (Sigma-Aldrich Co.) according to the method suggested by the manufacturer, and the histological change of each tissue was observed using an optical microscope (Carl Zeiss). In addition, the fat cell size and cell number were measured using AxioVision Imaging analysis System for the photograph of epididymal fat tissue obtained by H&E staining. The histological change of the epididymal fat tissue was shown in FIG. 3, and the cell number of the measured fat tissue according to the fat cell size was shown in Table 14.

TABLE-US-00014 TABLE 14 Group 1 Group 2 Group 3 Experiment diet CD HFD HFD Test material — — mdB-44 200 (mg/kg BW) <80 μm 161.0 ± 7.1  1.0 ± 1.0*** 41.7 ± 2.2.sup.### 80~100 μm  16.0 ± 1.7 9.3 ± 1.5* 45.0 ± 3.5.sup.### 100~120 μm  0.0 ± 0.0 28.0 ± 4.0*  12.7 ± 1.2.sup.#  >120 μm  0.0 ± 0.0 12.7 ± 1.2** .sup. 0.0 ± 0.0.sup.## Fat cell number in same 177.0 ± 8.7  51.0 ± 5.7*** 99.3 ± 5.2.sup.##  area Average fat globule size  80.9 ± 0.1 108.5 ± 1.0*** 88.3 ± 0.4.sup.### (μm) (The numerical value is expressed as mean ± standard error, and means *p < 0.05, **p < 0.01, ***p < 0.001, .sup.#p< 0.05, .sup.##p< 0.01, .sup.###p < 0.001)

[0139] As shown in FIG. 3, the size of fat cells was significantly increased in the epididymal fat tissue of the high-fat diet control group (Group 2) compared to the control diet group (Group 1), and in the mdB-44 administration group (Group 3), the size of fat cells showed a tendency to decrease.

[0140] In addition, as shown in Table 14 showing the result of measuring the cell number according to the fat cell size of the epididymal fat tissue, it was confirmed that the size of fat cells was generally small, as in the control diet group (Group 1), the number of cells with a fat cell size of <80 μm was 161.0±7.1, while the number of fat cells with a size of >100 μm was not observed. It was confirmed that the size of fat cells in the high-fat diet control group (Group 2) was large as the number of cells with a fat cell size of 100˜120 μm was 28.0±4.0 and the cell number with a size of >120 μm was 12.0±2.1. As the result of comparing the cell number by fat cell size of the test material administration group (Group 3), compared to the high-fat diet control group (Group 2), the fat cell number with a size of <80 μm and a size of 80˜100 μm was significantly increased, and the cell number with a size of 100˜120 μm was significantly reduced. The fat cell number with a size of >120 μm was not observed in the mdB-44 200 mg/kg BW administration group (Group 3).

[0141] The total number of fat cells relative to the same area was significantly reduced as 51.0±5.7 in the high-fat diet control group (Group 2), compared to 177.0±8.7 of the control diet group (Group 1). For the average fat globule size, compared to 80.9±0.1 μm of the control diet group (Group 1), the fat globule size was significantly increased as 108.5±1.0 μm in the high-fat diet control group (Group 2), and the fat globule was significantly reduced as 88.3±0.4 μm (Group 3) in the mdB-44 200 mg/kg BW administration group (Group 3).

[0142] From the result, it could be confirmed that administration of the marmelo extract showed anti-obesity effect, and in particular, also had an effect of inhibiting hypertrophy of white fat tissue caused by high-fat diet.

[0143] 6.6 Analysis of mRNA Expression of Energy Metabolism-Related Genes in Fat Tissue

[0144] After isolating total RNA using TRIzol® Reagent (Thermo Fisher Scientific) from the epididymal fat tissue obtained in Example 6.1, it was qualified using a micro-volume spectrophotometer (BioSpec-nano, SHIMADZU), and RNA with an OD260/280 value of 1.8 was used in the experiment. After obtaining cDNA using HyperScript™ RT master mix kit (GeneAll Biotechnology) from total RNA (2 μg), real-time PCR was performed using Rotor-Gene 300 PCR (Corbett Research) and Rotor-Gene™ SYBR Green kit (QIAGEN). The primer information used in the experiment was shown in Table 15. The quantitative analysis of expression of genes was performed using Rotor-Gene 6000 Series System Software program (Corbett Research).

TABLE-US-00015 TABLE 15 Sequence SEQ mRNA (5′ .fwdarw. 3′) Genebank No. ID NO C/EBPα-FOR TGGACAAGAACA XM_021168520.2  1 primer GCAACGAGTAC C/EBPα-REV GCAGTTGCCCAT XM_021168520.2  2 primer GGCCTTGAC PPARγ-FOR CAAAACACCAGT XM_021164279.2  3 primer GTGAATTA PPARγ-REV ACCATGGTAATT XM_021164279.2  4 primer TCTTGTGA SREBP-1c-FOR CACTTCTGGAGA NM_011480.4  5 primer CATCGCAAAC SREBP-1c-REV ATGGTAGACAAC NM_011480.4  6 primer AGCCGCATC CPT1β-FOR GTGCTGGAGGTG NM_009948.2  7 primer GCTTTGGT CPT1β-REV TGCTTGACGGAT NM_009948.2  8 primer GTGGTTCC FAS-FOR  AGGGGTCGACCT NM_007988.3  9 primer GGTCCTCA FAS-REV  GCCATGCCCAGA NM_007988.3 10 primer GGGTGGTT aP2-FOR  GGATTTGGTCAC NM_024406.3 11 primer CATCCGGT aP2-REV  TTCACCTTCCTG NM_024406.3 12 primer TCGTCTGC HSL-FOR  CCGTTCCTGCAG XM_030242181.1 13 primer ACTCTCTC HSL-REV  CCACGCAACTCT XM_030242181.1 14 primer GGGTCTAT GAPDH-FOR AGGTTGTCTCCT XM_029478683.1 15 primer GCGACT GAPDH-REV TGCTGTAGCCGT XM_029478683.1 16 primer ATTCATTGTCA ACC1-FOR GGAGATGTACGC AY451393.1 17 primer TGACCGAGAA ACC1-REV ACCCGACGCATG AY451393.1 18 primer GTTTTCA ACL-FOR  TGGATGCCACAG BC056378.1 19 primer CTGACTAC ACL-REV  GGTTCAGCAAGG BC056378.1 20 primer TCAGCTTC

[0145] The effect of the test material on lipogenesis, degradation and energy metabolism-related mRNA expression in the epididymal fat tissue was analyzed, and the result was shown in Table 16.

TABLE-US-00016 TABLE 16 Group 1 Group 2 Group 3 Experiment diet CD HFD HFD Test material — — mdB-44 200 (mg/kg BW) C/EBPα 1.00 ± 0.36  4.10 ± 0.05*** 2.20 ± 0.48.sup.## PPARγ 1.00 ± 0.12  2.25 ± 0.20** 1.11 ± 0.06.sup.## SREBP-1c 1.00 ± 0.12  2.41 ± 0.23** 1.51 ± 0.17.sup.#  CPT1 1.00 ± 0.19 0.37 ± 0.07* .sup. 2.28 ± 0.14.sup.### FAS 1.00 ± 0.52 2.90 ± 0.39* .sup. 0.25 ± 0.10.sup.### aP2 1.00 ± 0.11 1.98 ± 0.38* 0.94 ± 0.13.sup.#  HSL 1.00 ± 0.07  0.45 ± 0.04*** 0.78 ± 0.07.sup.## ACC1 1.01 ± 0.08  3.66 ± 0.42*** 2.82 ± 0.35.sup.  ACL 0.96 ± 0.14 1.49 ± 0.15* 0.75 ± 0.16.sup.#  (The result was calculated as the expression level of each material relative to the GAPDH expression level (expression level of each material/expression level of GAPDH), and is expressed as mean ± standard error, and means *p < 0.05, **p < 0.01, ***p < 0.001, .sup.#p < 0.05, .sup.##p < 0.01, .sup.###p < 0.001)

[0146] Transcriptional activation factors should be activated at the fat cell gene regulatory region for differentiation from preadipocytes to mature fat cells, and transcription factors which regulates differentiation of fat cells include SREBPs (sterol-regulatory element-binding protein), PPARs (Peroxisome Proliferator-Activated Receptor), C/EBPs (CCAAT/enhancer binding protein), and the like. SREBPs include SREBP-1 and SREBP-2, and among them, SREBP-1 is rapidly induced in the early stage of differentiation of preadipocytes to promote adipocyte differentiation and plays a role in promoting fat metabolism by increasing expression of genes related to fat metabolism.

[0147] As shown in Table 16, in the epididymal fat tissue, the SREBP-1c mRNA expression was significantly increased in the high-fat diet control group (Group 2), compared to the control diet group (Group 1), and it was significantly reduced in the mdB-44 200 mg/kg BW administration group (Group 3) compared to the high-fat diet control group (Group 2). C/EBPβ is expressed by induction of hormones (insulin, dexamethasone, etc.) in the early stage of adipocyte differentiation, and when C/EBPβ is activated, the expression of C/EBPα and PPARγ, the transcription factors of adipocytes, is induced in the late stage of differentiation. C/EBPα interacts with PPARγ to have an effect, and C/EBPα increases before expression of specific genes in adipose tissue to regulate energy homeostasis, and PPARγ is a major regulator of adipogenesis and is widely expressed in white adipose tissue. In the epididymal fat tissue, the C/EBPα and PPAR-γ mRNA expression was significantly increased in the high-fat diet control group (Group 2), compared to the control diet group (Group 1), and it was significantly reduced in the mdB-44 200 mg/kg BW administration group (Group 3) compared to the high-fat diet control group (Group 2).

[0148] FAS (fatty acid synthase) is an enzyme involved in fatty acid biosynthesis, and when fatty acids are synthesized in the liver, they are converted into triglycerides and play a role of circulating through blood vessels in the form of VLDL to deliver triglycerides to adipose tissue. The FAS mRNA expression in the epididymal fat tissue was significantly increased in the high-fat diet control group (Group 2), compared to the control diet group (Group 1), and it was significantly reduced in the mdB-44 200 mg/kg BW administration group (Group 3), compared to the high-fat diet control group (Group 2).

[0149] aP2, known as a target gene for lipogenesis, is expressed in fat cells and macrophages, and is known to be involved in inflammation and metabolic reactions. The aP2 mRNA expression in the epididymal fat tissue was significantly increased in the high-fat diet control group (Group 2), compared to the control diet group (Group 1), and the aP2 mRNA expression was significantly reduced in the mdB-44 200 mg/kg BW administration group (Group 3), compared to the high-fat diet control group (Group 2) (Table 16).

[0150] CPT-1 (Carnitine palmitoyl transferase-1) is a rate-limiting stage enzyme that inflows fatty acids into the mitochondria, and in order for fatty acids to flow into cells and be oxidized, a transferase inside the membrane is required, and CPT-1 is one of these transferases, and plays a major role in lipid use. The CPT-1 expression in the epididymal fat tissue was significantly reduced in the high-fat diet control group (Group 2), compared to the control diet group (Group 1), and the CPT-1 mRNA expression was significantly increased in the mdB-44 administration group (Group 3), compared to the high-fat diet control group (Group 2).

[0151] HSL (hormone-sensitive lipase) is distributed in various organs, but is mainly activated in adipose tissue, and becomes active HSL by secretion of hormones such as epinephrine and glucagon, and the active HSL plays a role of breaking down a triglyceride in lipoprotein into one glycerol and three fatty acids. The HSL mRNA expression in the epididymal fat tissue was significantly reduced in the high-fat diet control group (Group 2), compared to the control diet group (Group 1). The HSL mRNA expression was significantly increased in the mdB-44 200 mg/kg BW administration group (Group 3), compared to the high-fat diet control group (Group 2) (Table 16).

[0152] ACC1 is mainly present in adipose tissue, where fatty acid synthesis is important, and ACC2 is mainly present in oxidized tissue such as skeletal muscle and heart to regulate fatty acid synthesis and oxidation. The ACC1 mRNA expression in the epididymal fat tissue was significantly increased in the high-fat diet control group (Group 2), compared to the control diet group (Group 1), and the ACC1 mRNA expression was reduced in the mdB-44 200 mg/kg BW administration group (Group 3), compared to the high-fat diet control group (Group 2).

[0153] ACL is an enzyme involved in the synthesis of cytosolic acetyl-CoA by converting citrate to acetyl-CoA, and is used in important biosynthetic pathways such as fatty acids and cholesterol. The ACL mRNA expression in the epididymal fat tissue was significantly increased in the high-fat diet control group (Group 2), compared to the control diet group (Group 1), and the ACL mRNA expression was significantly reduced in the mdB-44 200 mg/kg BW administration group (Group 3), compared to the high-fat diet control group (Group 2).

[0154] 6.7 Analysis of Protein Expression in Fat Tissue

[0155] In order to investigate the protein expression in the epididymal fat tissue, after adding lysis buffer (Pierce® IP Lysis buffer, Thermo Scientific) to the epididymal fat, it was homogenized with a homogenizer. The homogenized solution was centrifuged at 12,000 rpm for 10 minutes, and then the supernatant was collected to obtain epididymal fat tissue lysates. The protein amount of the tissue lysate was measured using BCA protein assay kit (Thermo Scientific).

[0156] After isolating protein (50 μg) by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), it was moved to polyvinylidene difluoride membrane (Milipore). After blocking the membrane in 5% skim milk-TBST (20 mmol/L Tris.HCl, pH 7.5, 150 mmol/L NaCl, 0.1% Tween 20) for 1 hour, an antibody to be measured was added respectively, and was stirred at 4° C. for 16 hours or at a room temperature for 1 hour. The information of the antibodies used in the experiment was shown in Table 17. Then, horseradish peroxidase (HRP)-linked anti-rabbit IgG or HRP-linked anti-mouse IgG was added and stirred for 1 hour, and each protein band was visualized by the enhanced chemiluminescence method using Luminata™ Forte Western HRP Substrate (Millipore). The protein expression level was quantified using ImageQuant™ LAS 500 imaging systems (GE Healthcare Bio-Sciences AB).

TABLE-US-00017 TABLE 17 Antibody name Detailed information Manufacturer p-AMPK Phospho-AMPKα (Thr172) #2535 Cell Signaling Technology AMPK AMPKa #2532 Cell Signaling Technology β-actin Beta-actin #3700 Cell Signaling Technology

[0157] AMP-activated protein kinase (AMPK) is a factor that improves insulin resistance, and the activated AMPK is known to block ATP consumption pathways such as fat and cholesterol synthesis, while activating ATP production pathways such as glycolysis or fatty acid oxidation. In particular, in relation to lipid metabolism, it has been reported that it plays an important role in reducing body fat due to increased fatty acid oxidation, by inhibiting the activity of acetyl-CoA carboxylase (ACC) enzyme through protein phosphorylation.

[0158] In order to investigate the effect of the test material on AMPK activity in the epididymal adipose tissue, western blot was performed and quantified using the epididymal adipose tissue lysate, and the result was shown in Table 18.

TABLE-US-00018 TABLE 18 Group 1 Group 2 Group 3 Experiment diet CD HFD HFD Test material — — mdB-44 200 (mg/kg BW) p-AMPK/AMPK 1.00 ± 0.0 0.64 ± 0.04* 1.50 ± 0.22.sup.# Ratio (The numerical value is expressed as mean ± standard error, and means *p < 0.05, **p < 0.01, ***p < 0.001, .sup.#p < 0.05, .sup.##p < 0.01, .sup.###p < 0.001)

[0159] As shown in Table 18, as the result of evaluating the AMPK activity by the ratio of p-AMPK/AMPK, the AMPK activity reduced in the high-fat diet control group (Group 2) was significantly increased in the mdB-44 200 mg/kg BW administration group (Group 3).

[0160] As confirmed above, it was confirmed that the marmelo extract administered together with a high-fat diet effectively inhibited the increase in the body weight, liver and fat tissue weight, blood cholesterol, blood insulin and leptin contents, induced by the high-fat diet, and inhibited the size increase of fat cells. This suggests that the marmelo has anti-obesity effect and lipid improvement effect on dietary obesity, and furthermore, it has effects of prevention, treatment and/or improvement on metabolic disease, and suggests the potential to be developed as a health functional material with anti-obesity efficacy and/or effect of prevention, treatment and/or improvement on metabolic disease in the future.

Example 7. Confirmation of Lipid Accumulation Inhibitory Effect According to Extraction Method

[0161] 7.1 Cell Culture

[0162] Mouse-derived preadipocytes, 3T3-L1 cells, were purchased from the Korea Cell Line Bank, and used in the following examples to test the effect of inhibiting fat accumulation. The 3T3-L1 cells were cultured in a humidified CO.sub.2 incubator (5% CO.sub.2/95% air) at 37° C., using a cell culture medium (complete DMEM culture) prepared by adding 10% bovine calf serum (BCS), 100 units/mL penicillin and 100 μg/mL streptomycin to Dulbecco's Modified Eagle's Medium (DMEM, Welgene). When the cells reached about 80% full of the culture dish, the cell monolayer was washed with phosphate buffer saline (PBS, pH 7.4) and then the cells were subcultured by adding trypsin-2.65 mM EDTA, and the medium was changed every 2 days.

[0163] 7.2 Cell Differentiation Induction and Test Material Treatment

[0164] The 3T3-L1 cells cultured in Example 7.1 were inoculated in a 24-well plate at a concentration of 1×10.sup.5 cells/well. After the cells reached confluence state, the cells were cultured by sequentially exchanging the cell culture medium with three types of differentiation-inducing medium (DM), to induce differentiation into adipocytes. Specifically, the differentiation was stimulated for 2 days by exchanging the cell culture medium with a differentiation-inducing medium obtained by adding DMI (1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 5 μg/mL insulin) to 10% FBS-supplemented DMEM medium. After 2 days, the medium was exchanged with fresh differentiation-inducing medium obtained by adding 5 μg/mL insulin to 10% FBS-supplemented DMEM medium, to stimulate differentiation for another 2 days. After stimulation of differentiation for 4 days in total, cells were maintained in 10% FBS-supplemented DMEM medium for about 4 to 8 days to induce differentiation into adipocytes, during which the cell culture medium was exchanged every 2 days.

[0165] In order to test effects of marmelo extract on differentiation into adipocytes, the powder phase-marmelo extract (mdB-44) prepared in Example 1 was added to the differentiation-inducing medium at a concentration of 200 ug/ml. In addition, for comparison, an ethanol extract (EE) of marmelo fruit (LICORICE EXTRACT (Tashkent, Republic of Uzbekistan)) was prepared by the same manner of Example 1 except that the marmelo fruit was not subjected to compression and juice removal, and used for the aforementioned test of adipocyte-differentiation at a concentration of 200 ug/ml. In addition, a control group in which culture and differentiation was induced without adding an extract and a control group in which differentiation was not performed without adding an extract were prepared.

[0166] 7.3 Measurement of Adipocyte Differentiation (Fat Accumulation)

[0167] After performing induction of 3T3-L1 cell differentiation and treatment with the extract as in Example 7.2, the adipocyte-differentiated cells were rinsed with Dulbecco's phosphate buffered saline (Welgene), and fixed by adding 4% paraformaldehyde (Biosesang) at room temperature for 1 hour. After fixing, the cells were stained by treating with Oil Red 0 (Sigma-Aldrich) solution at room temperature for 1 to 2 hours. After observing the degree of staining of adipocytes, the cells were rinsed with distilled water and adipocytes were observed under a microscope. The degree of adipocyte differentiation was analyzed using the image J program, and the results are shown in FIG. 4.

[0168] 7.4 Result

[0169] As shown in FIG. 4, it was confirmed that the extract (mdB-44) extracted from marmelo fruit subjected to compression and juice removal as described in Example 1 exhibits excellent lipid accumulation inhibitory effects compared to the ethanol extract (EE) extracted from marmelo fruit which was not subjected to compression and juice removal.