KR20240108014A - Functional food composition for improving metabolic syndrome having complex extract of tangerine peel and beet - Google Patents

Abstract

A functional food composition containing a natural complex extract and having a synergistic effect on metabolic syndrome is disclosed. The present invention provides a functional food composition for improving metabolic syndrome containing a complex extract of tangerine peel extract and beet extract as an active ingredient.

Description

Functional food composition for improving metabolic syndrome containing complex extract of tangerine peel and beet}

The present invention relates to a functional food composition for improving metabolic syndrome, and more specifically, to a functional food composition for improving metabolic syndrome containing natural product extracts.

Along with economic development, our country's lifestyle has developed into that of advanced Western countries. As dietary habits change and physical activity decreases, body fat in the body increases and the number of obese people increases due to metabolic imbalance, resulting in diabetes, hyperlipidemia, and blood clots. The incidence of so-called metabolic syndrome, which is a combination of cardiovascular disease and disability, is continuously increasing.

Although products with high commercial potential for improving obesity are being launched, their safety is problematic due to side effects, and the demand for products based on natural products is increasing, reflecting consumer sentiment that prefers natural products. Natural products have relatively fewer side effects and toxicity than synthetic materials. In Korea, a variety of knowledge about traditional medicine based on natural products has been accumulated, making it advantageous for the development of obesity improvement products using natural products.

Tangerines are rich in vitamin C and are known to be good for the skin as well as improving metabolic diseases, and beets are known to help with diet and relieve constipation. However, the synergistic effect of tangerines and beets on metabolic syndrome has not yet been reported.

[Prior patent literature]

- Korean Patent No. 10-2052610 (registered on November 29, 2019)

The present invention seeks to provide a functional food composition containing a natural complex extract that has a synergistic effect on metabolic syndrome.

In order to solve the above problems, the present invention provides a functional food composition for improving metabolic syndrome containing a complex extract of tangerine peel extract and beet extract as an active ingredient.

In addition, the extract provides a functional food composition for improving metabolic syndrome, wherein the extract is a methanol extract.

In addition, the complex extract provides a functional food composition for improving metabolic syndrome, wherein the concentration of the tangerine peel extract is 20 to 80 μg/mL, and the concentration of the beet extract is 100 to 300 μg/mL.

The present invention can provide a functional food composition for improving metabolic syndrome that contains a complex extract of tangerine peel extract and beet extract as an active ingredient and exerts a synergistic effect on metabolic syndrome.

Figure 1 is an HPLC spectrum shown upon injection of 25 μg/mL of Betaine in an experimental example of the present invention.
Figure 2 is an HPLC spectrum shown when 200 μg/mL of beet extract was injected in an experimental example of the present invention.
Figure 3 is a graph showing the quantitative results of the amount of betaine contained in 200 μg/mL through a calibration curve in an experimental example of the present invention.
Figure 4 is an HPLC spectrum shown when 25 μg/mL of hesperidin was injected in an experimental example of the present invention.
Figure 5 is an HPLC spectrum shown when 50 μg/mL of tangerine peel extract was injected in an experimental example of the present invention.
Figure 6 is a graph showing the quantitative results of the amount of hesperidin contained in 50 μg/mL through a calibration curve in an experimental example of the present invention.
Figure 7 is a graph showing the test results for setting the highest concentration capable of treating 3T3-L1 cells through a toxicity test of the extract in an experimental example of the present invention.
Figure 8 is a photograph and graph showing the results of measuring the decrease in adipocyte differentiation through the Oil red O assay in an experimental example of the present invention.
Figure 9 is a graph showing the results of evaluating the possibility of improving metabolic syndrome when the liver is treated with a single natural product in human application in an experimental example of the present invention.
Figure 10 is a graph showing the results of evaluating the cytotoxicity of the complex extract in an experimental example of the present invention.
Figure 11 is an HPLC spectrum showing the betaine peak upon injection of the mixed extract in an experimental example of the present invention.
Figure 12 is an HPLC spectrum showing the hesperidin peak upon injection of the mixed extract in an experimental example of the present invention.
Figure 13 is a graph showing the results of measuring the decrease in mRNA related to fat metabolism using RT-PCR in an experimental example of the present invention.

Hereinafter, the present invention will be described in detail through preferred embodiments. Prior to this, the terms or words used in this specification and claims should not be construed as limited to their usual or dictionary meanings, and the inventor should appropriately define the concept of terms in order to explain his or her invention in the best way. Based on the principle of definability, it must be interpreted with meaning and concept consistent with the technical idea of the present invention. Therefore, the configuration of the embodiments described in this specification is only one of the most preferred embodiments of the present invention and does not represent the entire technical idea of the present invention, so various equivalents and modifications can be substituted for them at the time of filing the present application. You must understand that there may be. In addition, throughout the specification, when a part is said to "include" a certain element, this means that, unless specifically stated to the contrary, it does not exclude other elements but may further include other elements.

The present invention discloses a functional food composition for improving metabolic syndrome comprising a complex extract of tangerine peel extract and beet extract as an active ingredient.

In the present invention, there is no limitation on the form or nature of the extract, and it may be a solution, a concentrate, or a solid or powder obtained by removing the solvent used in preparing the extract.

The extract can be prepared using a solvent extraction method. The extraction solvent used to prepare the extract using the solvent extraction method is preferably methanol, and it is more preferable to use ultrasound during extraction. Ultrasonic waves can be used for 10 to 100 minutes, preferably 20 to 50 minutes.

In the present invention, a synergistic effect in improving metabolic syndrome appears when the concentrations of the tangerine peel extract and the beet extract constituting the complex extract are 20 to 80 μg/mL and 100 to 300 μg/mL, respectively, and preferably 30 to 70 μg/mL and It was confirmed through experiments that the synergistic effect in improving metabolic syndrome was maximized at 150 to 250 μg/mL.

The composition according to the invention is provided in the form of a food composition.

The food composition includes all types of functional foods, nutritional supplements, health foods, food additives, etc. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

For example, as a health food, the composition itself can be prepared and consumed in the form of tea, juice, and drinks, or can be consumed by granulating, encapsulating, and powdering.

In addition, functional foods include beverages (including alcoholic beverages), fruits and their processed foods (e.g. canned fruit, bottled foods, jam, marmalades, etc.), fish, meat, and their processed foods (e.g. ham, sausages, corned beef, etc.) , bread and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, retort food. , can be manufactured by adding extracts to frozen foods, various seasonings (e.g. soybean paste, soy sauce, sauce, etc.).

Additionally, in order to use the composition of the present invention in the form of a food additive, it can be prepared and used in the form of a powder or concentrate.

The preferred content of the extract in the food composition of the present invention may be 0.001 to 50%, preferably 0.1 to 50%, and more preferably 1 to 20%, based on the total weight of the food composition.

Hereinafter, the present invention will be described in more detail through specific production examples and experimental examples.

Manufacturing example

Dried beet (root) and tangerine peel were pulverized and sonicated for 30 minutes each using 80% (v/v) methanol to obtain beet extract and tangerine peel extract, which were mixed to prepare a composite extract.

Detection of indicator substances in beets and tangerine peels

The indicator substances of beets and tangerines were set to betaine and hesperidin, which are known to be effective in improving metabolic syndrome, respectively, and detection and quantification of the indicator substances were performed using HPLC. For HPLC analysis, each of the prepared beet extract and tangerine peel extract was centrifuged at 3,000 rpm for 5 minutes, the supernatant was filtered using Whatman No.1 filter paper, and the extract was evaporated using a vacuum rotary evaporator (Buchi R-100, Germany). ) and dried using a freeze dryer. Afterwards, 20 μL of the extract was analyzed using HPLC.

Figure 1 is an HPLC spectrum that appears when 25 μg/mL of Betaine is injected, and Figure 2 is an HPLC spectrum that appears when 200 μg/mL of beet extract is injected.

Referring to Figures 1 and 2, since the retention time of the peak generated from pure betaine, which is an indicator substance, and the peak generated from the beet extract are the same, detection of betaine in the beet extract was confirmed, and the concentration contained in 200 μg/mL was confirmed through the calibration curve. The amount of betaine was quantified, and the results are shown in Figure 3 and Table 1 below. Figure 3 is a calibration curve created based on the correlation between the concentration of the betaine indicator substance and the peak area generated by HPLC, and Table 1 shows the peak generated when 200 μg/mL of beet extract was injected and the betaine content calculated from the calibration curve.

No. Area Conc.(μg/mL) One 292.9500 53.92 2 291.2053 53.6 3 293.7813 54.08 Ave. 292.6455 53.87 CV(%) 0.4 0.5

Referring to Figure 3 and Table 1, it was confirmed that 53.87 μg, or about 26.9%, per 200 μg of beet extract was betaine.

Figure 4 shows the HPLC spectrum when 25 μg/mL of hesperidin was injected, and Figure 5 shows the HPLC spectrum when 50 μg/mL of tangerine peel extract was injected.

Referring to Figures 4 and 5, since the retention time of the peak generated from pure hesperidin, which is an indicator substance, and the peak generated from the tangerine peel extract are the same, it was confirmed that hesperidin was detected in the tangerine peel extract, and the concentration contained in 50 μg/mL was confirmed through the calibration curve. The amount of hesperidin was quantified, and the results are shown in Figure 6 and Table 2 below. Figure 6 is a calibration curve created based on the correlation between the concentration of the hesperidin indicator substance and the peak area generated by HPLC, and Table 2 shows the hesperidin content calculated from the peak generated when 50 μg/mL of tangerine peel extract was injected and the calibration curve.

No. Area Conc. (μg/mL) One 21.3299 2.734 2 21.5843 2.766 3 21.696 2.78 Ave. 21.5367 2.760 CV(%) 0.9 0.9

Referring to Figure 6 and Table 2, it was confirmed that 2.760 μg per 50 μg of tangerine peel extract, that is, about 5.5%, was hesperidin.

Evaluation of the potential to improve metabolic syndrome of a single extract

Betaine and hesperidin were detected from dried beets and dried tangerine peel, respectively, which are dried natural products. Since each indicator substance is known to be effective in improving metabolic syndrome, when each natural product is treated with cells and applied to the human body, it is associated with improvement in metabolic syndrome. Whether there were any changes in these indicators was confirmed through cell experiments and simple human application tests.

(1) Setting the highest concentration that can be treated with 3T3-L1 cells through toxicity testing of the extract

To determine whether the natural product inhibits the differentiation of 3T3-L1 cells, known as preadipocytes, into adipocytes, a toxicity test (MTT assay) was performed as follows: 100 μL per well was dispensed into a 96 well plate for 24 hours. Cultured for an hour (37°C, 5% CO ₂ incubator). Natural products prepared at specific concentrations (three concentrations were treated by serial dilution starting from the maximum extraction concentration of tangerine peel extract 500 μg/mL and beet extract 1 mg/mL) were added at 10 μL per well and reacted in an incubator for 24 hours. . 10 μL of EZ-Cytox was added to each well, reacted for 1 hour, shaken gently, and absorbance was measured at 450 nm. For the toxicity test, it was confirmed whether the absorbance decreased with the addition of natural products, and the test results are shown in Figure 7.

Referring to Figure 7, the tangerine peel extract and the beet extract seemed to be non-toxic at 125 μg/mL and 500 μg/mL, respectively, and the experiment was conducted by setting each concentration as the cell treatment concentration.

(2) Measurement of decreased adipocyte differentiation through Oil red O assay

To determine whether adipocyte differentiation is inhibited, which is one of the effects of improving metabolic syndrome, Oil red O assay was performed as follows: After cell culture, the medium was removed, treated with 1 mL of 10% formalin for 5 minutes, and then treated with 1 mL of 10% formalin. Cells were fixed with % formalin. Afterwards, the well was washed with isopropanol, dried completely, and treated with Oil red O. After dissolving Oil red O bound to the lipid droplet with 100% isopropanol, the absorbance at 490 nm was measured to compare the absorbance of cells treated with natural product extract and untreated cells, and the measurement results are shown in Figure 8.

Referring to Figure 8, 3T3-L1 cells, which are pre-adipocytes, are differentiated into adipocytes, and the differentiated adipocytes are stained with Oil red O reagent, and the degree of staining can be measured through absorbance, but differentiation does not proceed. If not, it can be seen that there is no staining and no absorbance is detected. However, the differentiated cells were stained with Oil Red O, so many red-stained areas were visible under the microscope, and absorbance was also detected. In contrast, the amount of staining in 3T3-L1 cells treated with beet extract or tangerine peel extract was significantly reduced during differentiation, which can be seen as inhibiting differentiation into adipocytes.

(3) Evaluation of the possibility of improving metabolic syndrome when treated with a single natural product in simple human application

The human application test was conducted with a total of 45 subjects, and the subjects were divided into three groups of 15 each. In the test, overweight adult men and women with a BMI of 30 kg/m ² or more were administered 15 g/day of the prepared tangerine peel extract (test group 1) and tangerine peel extract (test group 2) for a total of 12 weeks. They were ingested (the control group was an untreated group), and body weight changes were checked after 6 and 12 weeks, and the results are shown in Figure 9.

Referring to Figure 9, over a total of 12 weeks of testing, the tangerine peel extract treatment group (Test Group 1) showed a significant change in weight loss, and the beet extract treatment group (Test Group 2) showed a weight loss, but it was not statistically significant. didn't In addition to showing the effect of suppressing adipocyte differentiation in the above cell experiment, the liver also showed effectiveness in weight loss in human trials, indicating that tangerine peel extract and beet extract are effective in improving metabolic syndrome.

Evaluation of the potential of complex extracts to improve metabolic syndrome

In order to determine whether there are complementary effects when taken in combination with tangerine peel extract and beet extract, which have shown positive effects in improving metabolic syndrome, the mixing ratio of the complex natural product was set through a cytotoxicity test, and the effectiveness at the time of mixing was determined through a cytotoxicity test. Whether the substance decreased or increased was analyzed through HPLC, and whether it increased or decreased metabolic syndrome-related markers in vitro was determined through experiments.

(1) Evaluation of cytotoxicity of complex extract

Each was mixed at the maximum concentration within the limits of 125 μg/mL, the maximum concentration of the tangerine peel extract treatment, and 500 μg/mL, the maximum concentration of the beet extract, and while the maximum concentration of one substance was fixed, the concentration of the other substance was reduced to 1. The mixture was mixed and diluted at /5 and 1/10, and treated with cells. Cell toxicity was evaluated using the same method as described above, and the results are shown in Figure 10.

Referring to Figure 10, assuming that the non-treatment group (Negative control) had a 100% survival rate, there was no combination in all treatment groups that reduced the survival rate compared to the untreated group. In order to set the optimal formulation, the formulation with the highest survival rate in the toxicity test was set as the optimal mixing ratio, which was a mixture of 125 μg/mL of tangerine peel extract and 500 μg/mL of beet extract, i.e., a mass ratio of 1:4. was set as optimal, and subsequent experiments were conducted.

(2) Quantification of indicator substances in complex extracts

200 μg/mL of beet extract, which was the detection concentration of betaine and hesperidin, and 50 μg/mL of tangerine peel extract were mixed to adjust the mixing ratio to 4:1, and each indicator substance was detected and quantified according to the HPLC analysis method described above.

Figure 11 is an HPLC spectrum showing betaine peak upon injection of mixed extract.

Referring to Figures 1 and 11, when compared to the peak generated from pure betaine, which is an indicator substance, the peak appeared at the same time as that detected in the mixed extract, confirming it as betaine, and calculating the concentration by substituting it into the calibration curve, and the result It is shown in Table 3 below.

No. Area Conc.(μg/mL) One 430.3153 79.22 2 428.8660 78.95 3 427.2553 78.66 Ave. 428.8122 78.94 CV(%) 0.4 0.4

Referring to Table 3, compared to about 54 μg/mL of betaine in the above-mentioned 200 μg/mL beet extract, about 79 μg/mL was detected in the mixed extract, which means that when mixing the tangerine peel extract and the beet extract, This suggests that a synergistic effect may occur, suggesting that even more positive effects can be expected.

Figure 12 is an HPLC spectrum showing the hesperidin peak upon injection of the mixed extract.

Referring to FIGS. 4 and 12, when compared to the peak generated from pure hesperidin, which is an indicator substance, the peak appeared at the same time as that detected in the mixed extract, confirming it as hesperidin, and calculating the concentration by substituting the result into the calibration curve. It is shown in Table 4 below.

No. Area Conc.(μg/mL) One 16.3244 2.112 2 15.7600 2.042 3 16.4936 2.133 Ave. 16.1927 2.10 CV(%) 2.4 2.3

Referring to Table 4, compared to about 2.7 μg/mL of hesperidin in the above-mentioned 50 μg/mL tangerine peel extract, about 2.1 μg/mL was detected in the mixed extract, showing a tendency to decrease, but the numerical significance was not significant. It is judged that there is no such thing.

(4) Measurement of decrease in fat metabolism-related mRNA using RT-PCR

When the extract was treated alone or in combination with 3T3-L1 cells, RT-PCR was performed to confirm at the mRNA level whether there were any changes in markers related to the improvement of metabolic syndrome as follows: Extract-treated cells and up to 10 ⁷ cells of untreated cells were centrifuged at 13.5K rpm for 3 minutes to remove the medium, and an appropriate RLT PLUS solution was administered to the cells depending on the amount of cells. Afterwards, the cell lysate was placed in a gDNA eliminator column and gDNA was removed using a centrifuge at 10,000 rpm for 30 seconds. Afterwards, 70% ethanol was added and mixed well, then placed in an RNeasy spin column and centrifuged at 10,000 xg for 1 minute. After removing the separated liquid, RW1 buffer was added to the same column and centrifuged at 10,000 xg for 1 minute. Afterwards, RPE buffer was added to the same column and centrifuged at 10,000 RPE buffer was added to the same column and centrifuged at 10,000 xg for 1 minute. Finally, the RNeasy spin column was placed in a new 1.5 mL tube, centrifuged at 10,000 xg for 1 minute, and the amount of RNA in the separated solution was quantified. The RNA was mixed with 5X PrimeScriptIV cDNA Synthesis Mix, Template RNA, and RNase Free water and reacted at 42°C for 20 minutes to make cDNA. cDNA was added with Ultra Pure water and forward and reverse primers (SREBP-1, C/EBPα, PPARγ, FAS, AMPK, β-actin) and gene expression was tested using RT-PCR, and the results are shown in Figure 13. .

Referring to Figure 13, the identified markers showed a significant decrease in all markers of PPARγ, ACCase, Adiponectin, C/EBα, and FAS. In particular, although it was not statistically significant, it was confirmed that when the complex extract was treated, the effect was greater than that of the single extract.

Hereinafter, application examples of food compositions and pharmaceutical compositions containing the extract according to the present invention as an active ingredient will be described, but are not limited thereto.

Application Example 1: Preparation of liquid composition

Table 5 below illustrates the composition of a liquid composition containing the complex extract according to the present invention described above as an active ingredient. The liquid composition is prepared by adding and dissolving each of the following ingredients in purified water according to a typical liquid composition manufacturing method, adding an appropriate amount of lemon flavor, mixing the following ingredients, adding purified water to adjust the total volume to 100 ml, and then filling and sterilizing it in a brown bottle. can be manufactured.

ingredient content complex extract 200 mg Iseonghwadang 10g Mannitol 5g Purified water Appropriate amount

Application example 2: Manufacturing health functional foods

Table 6 below shows the composition of a health functional food containing the extract according to the present invention described above as an active ingredient. In the composition below, the composition ratio of the vitamin and mineral mixture indicates that ingredients relatively suitable for health functional food are mixed as a preferred application example, but the mixing ratio may be modified arbitrarily, and each component is prepared according to the usual health functional food manufacturing method. After mixing, granules are prepared and can be used to manufacture a health functional food composition according to a conventional method.

ingredient content ingredient content complex extract 1000 mg folic acid 50 μg vitamin mixture Appropriate amount Calcium Pantothenate 0.5 mg Vitamin A Acetate 70 ㎍ mineral mixture Appropriate amount Vitamin E 1.0 mg Ferrous sulfate 1.75 mg Vitamin B1 0.13 mg zinc oxide 0.82 mg Vitamin B2 0.15 mg Magnesium Carbonate 25.3 mg Vitamin B6 0.5 mg Potassium Phosphate Monobasic 15 mg Vitamin B12 0.2 μg Potassium Phosphate Dibasic 55 mg Vitamin C 10 mg Potassium citrate 90 mg biotin 10 μg calcium carbonate 100 mg Nicotinic acid amide 1.7 mg Magnesium chloride 24.8 mg

Application example 3: Healthy beverage production

Table 7 below shows the composition of a functional health drink containing the extract according to the present invention described above as an active ingredient. Health drink production follows a typical health drink production method, for example, mixing the following ingredients, stirring and heating at 85°C for about 1 hour, filtering the resulting solution, placing it in a sterilized 2L container, sealing and sterilizing it. It can be stored refrigerated and used. The following composition ratio is a mixture of ingredients that are relatively suitable for beverages of choice as a preferred example, but the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as demand class, demand country, and intended use.

ingredient content complex extract 1000 mg citric acid 1000 mg oligosaccharide 100g plum concentrate 2g taurine 1g Purified Water Mix Total 900 ml

The preferred embodiments of the present invention described above have been disclosed to solve technical problems, and those skilled in the art will be able to make various modifications, changes, additions, etc. within the spirit and scope of the present invention. , such modifications and changes should be regarded as falling within the scope of the patent claims below.

Claims (3)

A functional food composition for improving metabolic syndrome comprising a complex extract of tangerine peel extract and beet extract as an active ingredient. According to paragraph 1,
A functional food composition for improving metabolic syndrome, wherein the extract is a methanol extract. According to paragraph 1,
The complex extract is a functional food composition for improving metabolic syndrome, characterized in that the concentration of the tangerine peel extract is 20 to 80 μg/mL, and the concentration of the beet extract is 100 to 300 μg/mL.