KR20220153712A - A composition for improving, preventing or treating obesity and metabolic diseases comprising polysaccharide fraction isolated from radish leave - Google Patents

Abstract

The present invention relates to a composition for ameliorating, preventing or treating obesity or metabolic diseases, and more specifically, to a composition for ameliorating, preventing or treating obesity or metabolic diseases, which comprises a polysaccharide fraction derived from radish leaves as an active ingredient, thereby reducing the increase of body weight and body fat, and reducing the concentration of endotoxin, triglyceride and LTB4 in serum.

Description

A composition for improving, preventing or treating obesity and metabolic diseases comprising polysaccharide fraction isolated from radish leave}

The present invention relates to a composition for improving, preventing or treating obesity or metabolic diseases by containing a polysaccharide fraction derived from radish greens as an active ingredient.

Due to the westernized diet, the occurrence of metabolic diseases such as obesity, diabetes, hyperlipidemia, non-alcoholic fatty liver, and dyslipidemia due to abnormal lipid and carbohydrate metabolism has rapidly increased in modern society.

The obesity refers to a state in which excessive body fat is accumulated. The pharmacological mechanisms of currently used obesity drugs are largely 1) inhibition of fat absorption, 2) promotion of fat decomposition and heat generation, 3) regulation of appetite and satiety, 4) inhibition of protein metabolism, and 5) regulation of emotions related to food intake. can share Representative anti-obesity drugs include Xenical™, which suppresses fat absorption, and Reductil™, which suppresses appetite by stimulating the sympathetic nervous system. The obesity treatment has been reported to cause temporary weight loss, rapid weight loss, and drug-induced side effects on the heart, blood pressure, and nervous system.

Diabetes is a disease caused by an insufficient secretion of insulin or a failure to function normally, and is characterized by hyperglycemia in which the concentration of glucose in the blood increases and glucose is excreted in the urine. PPAR-γ activators, GLP-1 derivatives, DPP-IV inhibitors, etc. are currently used for diabetes treatment, but these conventional drugs are reported to have side effects of weight gain and toxicity to the liver, kidneys, muscles, heart, etc. have.

The hyperlipidemia is known as a disease that causes an excessive amount of fat components present in the blood and accumulated on the walls of blood vessels to cause inflammation, resulting in cardiovascular diseases such as myocardial infarction, stroke or cerebral infarction. Currently used drugs for hyperlipidemia include 'statin' drugs that have HMG-CoA reductase inhibitory activity, but it has been reported that they have side effects that are toxic to the liver or muscles when used for a long period of time.

The dyslipidemia refers to a state in which total cholesterol, LDL cholesterol or triglyceride in the blood is increased, or HDL cholesterol is decreased, and is known to increase the risk of cardiovascular diseases such as heart attack and stroke. Currently, drugs that can be used as a treatment for dyslipidemia include Bayer's BAY13-9952 (implitapide), a drug that inhibits the activity of MTP (microsomal triglyceride transfer protein), but these drugs have specific mechanisms of action in cells. There is a problem that there is a possibility of causing side effects because it has not been identified.

Currently, development of excellent therapeutic agents for overall obesity or metabolic diseases is incomplete, and therapeutic agents for individual specific diseases are also reported to have various side effects as described above. There is an urgent need for the development of therapeutic agents.

Republic of Korea Patent Publication No. 10-2013-0104692

An object of the present invention is to provide a food composition for improving or preventing obesity or metabolic disease, which contains a polysaccharide fraction derived from radish greens as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating obesity or metabolic diseases comprising a polysaccharide fraction derived from radish greens as an active ingredient.

In order to achieve the above object, the present invention provides a food composition for improving or preventing obesity or metabolic disease, comprising a polysaccharide fraction derived from radish greens as an active ingredient.

The noon-derived polysaccharide fraction contains 65 to 75% by weight of neutral sugar, 20 to 30% by weight of uronic acid, and a KDO analog (2-keto-3-deoxy-D- manno-octulosonic acid) may include 1 to 10% by weight.

The radish-derived polysaccharide fraction may further include 0.5 to 5% by weight of protein.

The noon-derived polysaccharide fraction may further include 0.5 to 10% by weight of polyphenol.

The neutral polysaccharide may include rhamnose, fucose, arabinose, xylose, mannose, galactose, glucose, glucuronic acid, and galacturonic acid.

The neutral polysaccharide is 1 to 15 mol% of rhamnose, 1 to 15 mol% of fucose, 10 to 30 mol% of arabinose, 0.1 to 5 mol% of xylose, 1 to 10 mol% of mannose, 30 to 50 mol% of galactose, 1 to 5 mol% of glucose, 1 to 5 mol% of glucuronic acid, and 5 to 20 mol% of galacturonic acid.

The polysaccharide fraction derived from noen (a) firstly extracted at 80 to 110 ° C. by mixing an extraction solvent with noen green powder; (b) recovering fractions by precipitating the firstly extracted extract with C1 to C4 alcohol; and (c) recovering a fraction having a molecular weight of 10 to 20 kDa or more from the fraction.

The metabolic disease may be any one selected from the group consisting of dyslipidemia, fatty liver, insulin resistance syndrome and diabetes, and the dyslipidemia is any one selected from the group consisting of hyperlipidemia, hypercholesterolemia and hypertriglyceridemia. It may be more than that, and the fatty liver may be non-alcoholic fatty liver.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating obesity or metabolic disease comprising a polysaccharide fraction derived from radish greens as an active ingredient.

The composition of the present invention contains a polysaccharide fraction derived from noen, and the composition inhibits preadipocyte differentiation and fat accumulation, reduces the increase in body weight and body fat, lowers endotoxin, triglyceride and total cholesterol concentrations in serum, , Since it has the effect of suppressing the expression of adipogenesis-related hormones in serum, it can be effectively used for the prevention and treatment of metabolic diseases as well as obesity.

1 is a body composition image of an animal model classified into groups 1 to 5 measured using a body composition analyzer.
Figure 2 measures the body fat mass change (%) of animal models divided into group 1 (NC, negative control group), group 2 (HFD), group 3 (PRG4) and group 5 (WERG50) measured using a body composition analyzer is the graph shown.
Figure 3 is a graph showing the measured weight gain of animal models divided into group 1 (NC, negative control group), group 2 (HFD), group 3 (PRG4) and group 5 (WERG50).
4 is a white epididymal adipose tissue extracted from animal models classified into group 1 (NC, negative control group), group 2 (HFD), group 3 (PRG4) and group 5 (WERG50), and the weight of the white epididymal adipose tissue This is a graph showing the measured results.
5 is a graph showing blood triglyceride concentrations of animal models classified into group 1 (NC, negative control group), group 2 (HFD), group 3 (PRG4), and group 5 (WERG50).
Figure 6 is a graph showing the concentration of LTB4 in animal models divided into group 1 (NC, negative control group), group 2 (HFD), group 3 (PRG4) and group 5 (WERG50).
7 is a graph showing endotoxin concentrations in the blood of animal models classified into group 1 (NC, negative control group), group 2 (HFD), group 3 (PRG4), and group 5 (WERG50).

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition capable of improving, preventing or treating obesity or metabolic diseases by containing a polysaccharide fraction derived from radish greens as an active ingredient.

Radish (Raphanus sativus) is an annual or monthly herb belonging to the mustard family. It has been introduced from China since the Three Kingdoms period and there are many native species native to each region. Radish contains about 93% of moisture, 1% of crude protein, 3% of sugar mainly glucose, and 19~39 mg% of vitamin C. The tissue of radish contains fiber, pectin, various minerals and amylase, a digestive enzyme, and has unique aromatic components such as methyl mercaptane and mustard oil.

Radish greens (Raphanus sativus Leaves or Radish greens) correspond to the upper part of the root of radish, that is, the stem and leaf part, and the dried radish greens (Siraegi and/or Radish greens; hereinafter referred to as radish greens) are ours. Although it is a favorite food of most of the people, it is not fulfilling its role as a commodity like other traditional foods because the harvest season is limited and it is not commercialized so that it can be eaten throughout the year.

In the present invention, 'obesity' refers to a state in which body fat is excessively accumulated, rather than simply being overweight. Even if you appear to be of normal weight on the outside, it means that you can say that you are obese if your body fat percentage is high. The body mass index (BMI) is usually used to determine obesity. A score of 23 to 24.9 is considered overweight, a score of 25 to 29.9 is considered mildly obese, a score of 30 to 34.9 is moderately obese, and a score of 35 or more is considered severely obese. are doing Obesity is caused by a combination of multiple causes rather than a single cause. Wrong eating habits, including westernized eating habits, reduced activity, emotional factors, and genetic factors, etc. increases the risk of disease.

In the present invention, 'metabolic disease' means a condition or disease closely related to or caused by obesity, specifically, any one selected from the group consisting of dyslipidemia, non-alcoholic fatty liver, insulin resistance syndrome, and diabetes. There can be more than one. The dyslipidemia may be any one or more selected from the group consisting of hyperlipidemia, hypercholesterolemia, and hypertriglyceridemia. The fatty liver refers to a condition or disease in which fat is accumulated in hepatocytes in an excessive amount due to a fat metabolism disorder of the liver, and may specifically be non-alcoholic fatty liver.

In the present invention, hyperlipidemia refers to a condition or disease in which the concentration of fat components, particularly cholesterol or triglyceride, in the blood is higher than normal levels, and includes all conditions in which lowering the blood lipid concentration is required. is used in a broad sense.

In the present invention, hypercholesterolemia means a condition or disease in which cholesterol in the blood is excessively high, and hypertriglyceridemia means a condition or disease in which the concentration of triglyceride in the blood is excessively high.

Specifically, the composition for improving, preventing or treating obesity or metabolic diseases may also be referred to as a composition for improving, preventing, alleviating, suppressing, or treating an increase in body weight or body fat due to the various causes. More preferably, it has an effect of improving, preventing, or treating obesity or metabolic diseases caused by a high-fat diet.

According to the experimental results of the present invention, when a high-fat diet was administered for a short or long period, body weight and body fat increased significantly. Over time, this increases body weight, body fat, and white epididymal fat, and increases blood triglyceride and LTB4 concentrations as well as serum endotoxin concentrations, leading to obesity or metabolic diseases.

At this time, it was confirmed that the administration of the polysaccharide fraction derived from nocheong reduces the increase in body weight, body fat and white epididymal fat weight, decreases the increase in blood triglyceride and LTB4 concentration, and suppresses the increase in endotoxin concentration in serum to lower it to normal levels. can Therefore, it can be seen that the composition containing the polysaccharide fraction derived from radish greens as an active ingredient according to the present invention has an effect of improving, preventing or treating obesity or metabolic diseases.

In the polysaccharide fraction derived from nocheong of the present invention, neutral polysaccharide is 65 to 75% by weight, uronic acid is 20 to 30% by weight, KDO analog (2-keto-3-deoxy- D-manno-octulosonic acid) may include 1 to 10% by weight.

In addition, the polysaccharide fraction derived from grass green of the present invention may further include 0.5 to 5% by weight of protein.

In addition, the green-derived polysaccharide fraction of the present invention may further include 0.5 to 10% by weight of polyphenol.

The neutral polysaccharide may include rhamnose, fucose, arabinose, xylose, mannose, galactose, glucose, glucuronic acid and galacturonic acid, and more specifically, The neutral polysaccharide contains 1 to 15 mol% rhamnose, 1 to 15 mol% fucose, 10 to 30 mol% arabinose, 0.1 to 5 mol% xylose, 1 to 10 mol% mannose, 30 to 30 mol% 50 mol% of galactose, 1 to 5 mol% of glucose, 1 to 5 mol% of glucuronic acid and 5 to 20 mol% of galacturonic acid.

The polysaccharide fraction derived from green-radish of the present invention comprises the steps of (a) first extracting at 80 to 110 ° C. by mixing an extraction solvent with green-radish powder; (b) recovering fractions by precipitating the firstly extracted extract with C1 to C4 alcohol; and (c) recovering a fraction having a molecular weight of 10 to 20 kDa or more from the fraction.

First, (a) primary extraction is performed at 80 to 110 ° C. by mixing an extraction solvent with radish green powder. The extraction solvent may be water, alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof, preferably water.

In the present invention, the radish leaves can be cut into a certain size, dried in the shade or using a dryer, and then crushed or used as is. The drying period is not particularly limited as long as the moisture content in the radish is less than 20%.

Preferably, the green-free powder may be used after being suspended in distilled water at an amount of 5 to 20 times (w/v).

After the step (a), a step of removing the residue may be additionally performed. A method for removing the residue may be used without particular limitation as long as it is a known method such as centrifugation or filtering, and preferably centrifugation.

Next, (b) precipitate the firstly extracted extract with C1 to C4 alcohol to recover fractions. Alcohol precipitation may be performed by a known ethanol precipitation method, and the ethanol used for the ethanol precipitation is preferably ethanol having a concentration of 70% to 95% (v/v) by mixing with water.

Finally (c) a fraction having a molecular weight of 10 to 20 kDa or more is recovered from the above fractions. A method for obtaining the fraction is not particularly limited as long as it is purified based on molecular weight, but a method by dialysis is preferred. The dialysis may be performed for 0.5 to 5 days, preferably for 1 to 3 days.

As a result of analyzing the constituent sugars of the obtained polysaccharide fraction derived from noeng by HPAEC-PAD, neutral polysaccharide was 70.8% by weight, uronic acid was 22.3% by weight, KDO analog (2- keto-3-deoxy-D-manno-octulosonic acid) was 1.6% by weight and polyphenol was 4.5% by weight, and as a result of analyzing the components of the neutral polysaccharide, 5.4 mole% of rhamnose and 5.0 mole of fucose compared to the total neutral polysaccharide. %, arabinose 22.9 mole%, xylose 0.4 mole%, mannose 2.0 mole%, galactose 40.7 mole%, glucose 2.1 mole%, glucuronic acid 1.9 mole%, and galacturonic acid 12.7 mole% were confirmed (Table 1). Reference).

A process of lyophilizing the final polysaccharide fraction may be further performed.

Specifically, in the present invention, a polysaccharide fraction derived from noen with a molecular weight of 10 kDa or more was obtained by using the same method as above for the fraction obtained by extracting the noen with an extraction solvent and then precipitating with an alcohol having 1 to 4 carbon atoms.

In this specification, the term 'fraction' used while referring to nocheong includes not only a fraction obtained by precipitating an alcohol having 1 to 4 carbon atoms in an extract obtained by treating an extraction solvent, but also a processed product of a polysaccharide fraction derived from nocheong. For example, the green-derived polysaccharide fraction may be prepared in a powder state by additional processes such as distillation under reduced pressure and freeze drying or spray drying.

In a broad sense, the green-free polysaccharide fraction of the present invention also includes a processed product obtained by drying the no-green polysaccharide fraction, which is formulated to be administered to animals. Although the experiment was conducted with the polysaccharide fraction derived from noen, it will be expected by those skilled in the art that the desired effect can be achieved even in the same form as the processed noen-noisy polysaccharide.

In the present invention, 'contained as an active ingredient' or 'contained as an active ingredient' means containing an amount sufficient to achieve the efficacy or activity of the polysaccharide fraction derived from radish greens of the present invention, specifically improving obesity or metabolic diseases. , means containing an amount sufficient to prevent or treat. For example, the polysaccharide fraction derived from radish greens may be used at a concentration of 10 to 15,000 μg/ml, preferably 100 to 10,000 μg/ml. Since the polysaccharide fraction derived from noen is a natural product and does not cause side effects on the human body even when administered in excess, the upper and lower limits of the quantity of the polysaccharide fraction derived from noen contained in the composition of the present invention can be selected and implemented by those skilled in the art within appropriate ranges.

The composition of the present invention is a food composition, and provides a food composition for improving or preventing obesity or metabolic diseases containing the polysaccharide fraction derived from radish greens as an active ingredient. The food composition according to the present invention may be formulated in the same way as the pharmaceutical composition described later and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, alcoholic beverages, confectionery, diet bars, dairy products, meat, chocolate, pizza, ramen, other noodles, chewing gum, ice cream, vitamin complexes, and health supplements. etc.

The food composition of the present invention may include, as an active ingredient, a polysaccharide fraction derived from radish greens, as well as ingredients commonly added during food manufacturing, and include, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. do. Examples of the aforementioned carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose, oligosaccharides and the like; and polysaccharides such as conventional sugars such as dextrins and cyclodextrins and sugar alcohols such as xylitol, sorbitol and erythritol. As flavoring agents, natural flavoring agents [thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is prepared as a drink or beverage, citric acid, high fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be further included in addition to the radish extract of the present invention.

The present invention provides a health functional food comprising a food composition for improving, suppressing or preventing obesity or metabolic diseases containing the polysaccharide fraction derived from radish greens as an active ingredient. Health functional food refers to a food made by adding green-free polysaccharide fraction to food materials such as beverages, teas, spices, chewing gum, and confectionery, or by encapsulating, powdering, or suspensioning. It means to come, but unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time by using food as a raw material. The health functional food of the present invention obtained in this way is very useful because it can be consumed on a daily basis. The amount of added polysaccharide fraction derived from noh in such health functional foods varies depending on the type of target health functional food and cannot be uniformly defined, but can be added within a range that does not impair the original taste of the food. It is usually in the range of 0.01 to 50% by weight, preferably 0.1 to 20% by weight. In addition, in the case of health functional foods in the form of pills, granules, tablets or capsules, it is usually added in the range of 0.1 to 100% by weight, preferably 0.5 to 80% by weight. In one embodiment, the health functional food of the present invention may be in the form of pills, tablets, capsules or beverages.

The present invention can be used as a pharmaceutical composition for preventing or treating obesity or metabolic diseases containing a polysaccharide fraction derived from radish greens as an active ingredient. In addition to the active ingredient, the green-derived polysaccharide fraction, it can be prepared using pharmaceutically suitable and physiologically acceptable adjuvants, such as excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, glidants, or Flavoring agents etc. can 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 pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc., preferably oral administration.

The suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, medical condition, food, administration time, administration route, excretion rate and reaction sensitivity, usually This allows the skilled physician to readily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.0001-10 g/kg, more preferably 0.001 g/kg to 10 g/kg.

The pharmaceutical composition of the present invention may be prepared in a unit dose form by formulation using a pharmaceutically acceptable carrier and/or excipient, or prepared by putting it into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally contain a dispersing agent or stabilizer.

In addition, the present invention provides a use of a polysaccharide fraction derived from radish greens for the manufacture of medicines or foods for the improvement, prevention or treatment of obesity or metabolic diseases. As described above, the polysaccharide fraction derived from radish greens can be used for the improvement, prevention, or treatment of obesity or metabolic diseases.

In addition, the present invention provides a method for improving, preventing or treating obesity or metabolic disease, which includes administering an effective amount of a polysaccharide fraction derived from radish greens 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 "effective amount" means 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 is contains an amount that induces relief of The effective amount and frequency of administration of the active ingredient of the present invention can be varied depending on the desired effect. Therefore, the optimal dose to be administered can be easily determined by a person skilled in the art, and the degree of obesity, body weight, body fat, concentration of triglyceride and endotoxin in serum, active ingredient and other ingredients contained in the composition, type of formulation, and patient It can be adjusted according to various factors, including age, weight, general health condition, sex and diet, administration time, administration route, treatment period, and concurrently used drugs. In the prevention, treatment, or improvement method of the present invention, in the case of adults, it is preferable to administer the polysaccharide fraction derived from nohyeon at a dose of 0.0001 g/kg to 10 g/kg when administered once or several times a day, more preferably. Preferably, it may be administered at a dose of 0.001 g/kg to 10 g/kg.

In the treatment method of the present invention, the composition containing the polysaccharide fraction derived from green tea as an active ingredient can be administered through oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular or intradermal routes. It can be administered in a manner, preferably intraoral.

Hereinafter, preferred embodiments are presented to aid understanding of the present invention, but the following examples are merely illustrative of the present invention, and various changes and modifications are possible within the scope and spirit of the present invention. It is obvious to those skilled in the art, It goes without saying that these variations and modifications fall within the scope of the appended claims.

Example 1. Radish-derived crude polysaccharide fraction (PRG)

Domestic radish (radish leaf) produced by Wanju Nonghyup, Jeonbuk was purchased and used. Radish green (radish leaves) was dried in a drying oven at 50 ° C. for 24 hours, and then pulverized to a size of 1.0 mm or less with a grinder to prepare a much green powder.

A hot water extract was obtained by adding 20 times the volume of distilled water to 100 g of the green-radish powder and performing reflux extraction at 80° C. for 3 hours. The hot water extract was filtered, and then concentrated under reduced pressure using a rotary vacuum evaporator to make 1/10 of the extract, and 80% alcohol corresponding to 4 times the volume (v/v) of the hot water extract concentrated under reduced pressure. was added to the hot water extract to precipitate for 24 hours. The precipitate was obtained by centrifugation (6000 rpm, 635 × g, 30 minutes), and a small amount of distilled water was added thereto to re-dissolve the precipitate, and the dialysis membrane (MW cut off 12,000, Sigma-Aldrich Co., St. Louis, MO, USA) for 2-3 days to remove low-molecular substances, and lyophilized for 72 hours to obtain polysaccharides from radish greens (PRG).

Comparative Example 1. Radish hot water extract

Domestic radish (radish leaf) produced by Wanju Nonghyup, Jeonbuk was purchased and used. Radish green leaf powder was prepared by drying radish green in a drying oven at 50 ° C. for 24 hours and then pulverizing to a size of 1.0 mm or less with a grinder.

Distilled water corresponding to 20 times the volume was added to 100 g of radish green leaf powder, and reflux extraction was performed at 80 ° C. for 3 hours to obtain a hot water extract. After filtering the hot water extract, it was concentrated under reduced pressure using a rotary vacuum evaporator and freeze-dried for 72 hours. The hot water extract of radish greens obtained by the above method was described as WERG (Water-soluble extract from radish greens).

<Test Example>

Test Example 1. Yield, sugar and protein content analysis of the crude polysaccharide fraction derived from radish greens of Example 1

For the analysis of constituent sugars, the method of Albersheim et al. was partially modified, and each constituent sugar was derivatized with alditol acetate and aldonolactone after hydrolysis, and GC (Gas Chromatography ACME-6100, Young-Lin Co. Ltd. Anyang, Korea ) was analyzed using. The sample was hydrolyzed in 2 M TFA (trifluroacetic acid) at 121° C. for 1.5 hr, then dissolved in 1 ml of 1 M NH ₄ OH (ammonia solution) and reduced with 10 mg of NaBH ₄ for 4 hours. Acetic acid was added in an appropriate amount to remove residual NaBH ₄ , and then methanol was added and dried repeatedly to remove excess acetic acid, which was converted into alditol. In order to know the composition of the neutral sugar, 1 ml of acetic anhydride was added to the alditol and reacted at 121 ° C. for 30 minutes to convert it into alditol acetate, which was converted into alditol acetate and chloroform / H ₂ O 2 The phase solvent was separated and extracted, and the extract was dried and dissolved in a small amount of acetone to be used as a sample for analysis.

Protein quantification was measured according to the Bradford method, and BSA (Bovine Serum Albumin, sigma aldrich) was used as a standard and diluted by a constant multiple with a maximum concentration of 1 mg/ml. 980 μl of Bradford reagent was reacted by adding 20 μl of each standard and sample, and the absorbance was measured at 595 nm using a microplate reader. Protein quantification was converted to the amount for BSA.

Total sugar was measured according to the phenol-sulfuric acid method. After adding the same amount of 5% phenol solution to 0.5 mL of the sample solution and stirring, 2.5 mL of concentrated sulfuric acid (98%, v/v) was added and stirred at room temperature for 20 minutes. After the reaction, absorbance was measured at 470 nm using a microplate reader. Quantification of total sugar was converted into a calibration curve using a glucose standard (sigma aldrich).

Total polyphenol content was analyzed according to the Folin Denis method using gallic acid (sigma aldrich).

In the case of constituent sugars, HPAEC-PAD (high-performance anion-exchange chromatography-pulsed amperometric detector) was used after hydrolysis of sugar chains in the sample into monosaccharides, and data were analyzed using Chromelon 6.80 software (Dionex). Monosaccharides (fucose, rhamnose, arabinose, galactose, glucose, xylose) were used as standard solutions.

Physicochemical properties, such as content of major components, of the crude polysaccharide fraction derived from Example 1 and the hot water extract of Radish Green of Comparative Example 1 were analyzed, and the results are shown in Table 1 below.

Comparative Example 1 (WERG) Example 1 (PRG) Extraction yield (%) 34.3 7.9 Chemical composition (%) Protein 4.5 ± 0.2 0.7 ± 0.1 Neutral sugar 72.4 ± 1.2 70.8 ± 0.7 Uronic acid 10.9 ± 0.1 22.3 ± 0.4 KDO-like material - 1.6 ± 0.1 polyphenol 12.2 ± 0.2 4.5 ± 0.3 Component sugar (PMP) (mole %) Rhamnose 6.6 ± 0.1 5.4 ± 0.3 Fucose 0.6 ± 0.0 5.0 ± 0.7 Arabinose 5.7 ± 0.1 22.9 ± 2.3 Xylose 0.4 ± 0.1 0.4 ± 0.1 Mannose 1.7 ± 0.1 2.0 ± 0.2 Galactose 8.7 ± 0.1 40.7 ± 3.6 Glucose 55.8 ± 0.6 2.1 ± 0.1 Glucuronic acid - 1.9 ± 0.1 Galacturonic acid 3.8 ± 0.3 12.7±1.9

* The content ratio (%) of the chemical component represents the weight % in the dry sample.

* The above mole% is calculated from the total amount of neutral sugars detected.

As shown in Table 1, the yield of the green-radish hot water extract prepared in Comparative Example 1 was 34.3%, and the protein content was 4.5% by weight and the neutral sugar content was 72.4% by weight. In addition, it was confirmed that most of the neutral polysaccharide in the green-radish hot water extract prepared in Comparative Example 1 was glucose (55.8 mole%), and the rest contained a small amount, and glucuronic acid and KDO analogues did not exist.

On the other hand, although the extraction yield of the crude polysaccharide fraction derived from grape seed extract prepared in Example 1 was only 7.9%, the protein content was 0.7% by weight and the neutral sugar content was 70.8% by weight, confirming that the sugar content was high. In the crude polysaccharide fraction derived from the green tea extract of Example 1, galactose (40.7 mole%) was the highest in the neutral polysaccharide, followed by arabinose and galacturonic acid at 22.9 and 12.7 mole%, respectively, unlike the hot water extract. , It was confirmed that trace amounts of fucose, glucose, mannose, glucuronic acid and xylose (5.4, 5.0, 2.1, 2.0, 1.9, 0.4 Mole%, respectively) were present.

Test Example 2. Animal testing

laboratory animal

Six-week-old male C57BL/6J mice raised in an aseptic environment were purchased from Central Laboratory Animals Co., Ltd. and used, and the mice were acclimatized for 2 weeks before the experiment. Mice were bred with free food in a breeding room maintained at a temperature of 22 ± 2 ° C and a humidity of 40-60% during the experiment, and the light and dark cycles were adjusted at 12-hour intervals. All animal experiments were performed in accordance with the Korea Food Research Institute's Animal Experiment Operation Regulations.

Administration and sampling of samples

For the administration of the sample, the mice were randomly divided into five groups of nine mice each. The NC (normal group) was a negative control group fed a normal diet, and the HFD (high fat diet group) and sample administration group were fed a high-fat diet (Modified AIN 76A purified rodent diet (60 cal%) in which calories were increased by 60 cal% by adding lard. % fat)) was voluntarily provided. Sample PRG4 was orally administered 4 mg/kg of the crude polysaccharide fraction derived from the radish extract of Example 1, sample WERG4 was orally administered 4 mg/kg of the hot water extract of the radish greens of Comparative Example 1, and sample WERG50 was orally administered the radish extract of Comparative Example 1 50 mg/kg of the hot water extract was orally administered every day, and the same amount of distilled water was orally administered to the NC and HF groups for the same period of time. Body weight was measured every week at 10 am for a total of 8 weeks.

For the statistical analysis of the experiment, one-way ANOVA (Dunnett's test vs. HFD) was used to confirm the significant difference in mean values between groups. In case of p-value lower than 0.05, *, in case of p-value lower than 0.01, **, Significant difference was marked with *** when the p value was lower than 0.001.

5 groups of animal models

Group 1 (NC, negative control group): normal diet, oral administration of distilled water

Group 2 (HFD): intake of high-fat diet, oral administration of distilled water

Group 3 (PRG4): Intake of high-fat diet, oral administration of 4 mg/kg of the crude polysaccharide fraction derived from no-hung in Example 1

Group 4 (WERG4): intake of high-fat diet, oral administration of 4 mg/kg of the radish green hot water extract of Comparative Example 1

Group 5 (WERG50): intake of high-fat diet, oral administration of 50 mg/kg of the radish green hot water extract of Comparative Example 1

Test Example 2-1. Measurement of body composition and body fat in animal models

1 is a picture of the body shape of an animal model divided into groups 1 to 5 measured using a body composition analyzer, and FIG. 2 shows group 1 (NC, negative control group), group 2 (HFD) measured using a body composition analyzer, It is a graph showing the change in body fat mass (%) of animal models classified into group 3 (PRG4) and group 5 (WERG50). At this time, the body composition analyzer was measured according to the protocol using dual-energy X-ray absorptiometry (DXA).

As shown in FIGS. 1 and 2, in the experimental group (group 3) orally administered the crude polysaccharide fraction (PRG4) derived from noen (PRG4) prepared according to Example 1 of the present invention daily for 8 weeks, significant body weight and It was confirmed that there is an effect of reducing body fat.

Compared to the HFD group that ate only a high-fat diet, the NC group ate a normal diet had significantly higher body fat. When the green-free crude polysaccharide fraction (PRG4) prepared in Example 1 was administered, the body fat content was significantly higher than that of the HFD group by 17.6%. It was confirmed that there was a negative decrease.

In addition, neither of the experimental groups administered with 4 mg/kg or 50 mg/kg of the radish green hot water extract (WERG4) prepared according to Comparative Example 1 could not confirm a significant decrease in body fat.

Therefore, it can be seen that the crude polysaccharide fraction derived from radish greens of the present invention has an effect on reducing body fat as well as reducing body weight.

The group administered with 4 mg/kg of the radish green hot water extract (WERG4) prepared according to Comparative Example 1 was judged to have no effect on obesity or metabolic diseases and was not used in further experiments.

Test Example 2-2. Weight measurement of animal models

Figure 3 is a graph showing the measured weight gain of animal models divided into group 1 (NC, negative control group), group 2 (HFD), group 3 (PRG4) and group 5 (WERG50).

As shown in Figure 3, the body weight of the NC group increased by 4.9 g over 8 weeks, while the body weight of the HFD group increased by 11.9 g. When the crude polysaccharide fraction (PRG4) derived from radish greens prepared in Example 1 of the present invention was administered, the weight gain decreased by 16.9% compared to the HFD group.

In addition, no significant weight loss was observed in any of the experimental groups administered with 50 mg/kg of the radish green hot water extract (WERG4) prepared according to Comparative Example 1.

Test Example 2-3. Measurement of white epididymal fat in animal models

4 is a white epididymal adipose tissue extracted from animal models classified into group 1 (NC, negative control group), group 2 (HFD), group 3 (PRG4) and group 5 (WERG50), and the weight of the white epididymal adipose tissue This is a graph showing the measured results.

As shown in Figure 4, the weight of white epididymal adipose tissue in the NC group was significantly increased compared to the HFD group. The weight of the white epididymal adipose tissue in the group (PRG4) administered with 4 mg/kg of the crude polysaccharide fraction derived from grass green in Example 1 was significantly reduced compared to the HFD group. On the other hand, in the case of the group administered with the hot water extract of Comparative Example 1 (WERG50), the weight of white epididymal adipose tissue increased significantly compared to the NC group, and did not show a significant difference compared to the HFD group.

Test Example 2-4. Liver Weighing

After 8 weeks, the animal models divided into group 1 (NC, negative control group), group 2 (HFD), group 3 (PRG4), and group 5 (WERG50) were fasted for 12 hours, anesthetized using Ipran solution, and the abdominal and After removing the liver by incising the thoracic cavity, the weight was measured, and the results are shown in Table 2 below.

division Liver weight (g) NC 0.83 ± 0.09g HFD 1.52 ± 0.11g PRG4 1.00 ± 0.1 g WERG50 1.47 ± 0.08g

As shown in Table 2, it can be seen that the liver weight significantly increased in the HFD group consuming a high-fat diet compared to the NC group consuming a normal diet, and 4 mg/kg of crude polysaccharide fraction derived from radish in Example 1 It can be confirmed that this increase is significantly reduced upon administration. No significant difference was observed when 50 mg/kg of the radish green hot water extract of Comparative Example 1 was administered.

Test Example 2-5. Measurement of blood lipids and LTB4

After 8 weeks, the animal models divided into group 1 (NC, negative control group), group 2 (HFD), group 3 (PRG4), and group 5 (WERG50) were fasted for 12 hours, anesthetized using Ipran solution, and the abdominal and Blood was collected from the hepatic portal vein by incision in the thoracic cavity, coagulated, and serum was separated and used to analyze blood fat and LTB4 concentrations.

For triglyceride (TG) analysis from the serum, it was performed according to the manufacturer's protocol using a Triglyceride Colorimetric Assay Kit (Cayman). For the analysis of LTB4 (leukotriene B4) from the serum, it was performed according to the manufacturer's protocol using the LTB4 Parameter Assay Kit (R&D systems).

For the statistical analysis of the experiment, one-way ANOVA (Dunnett's test vs. HFD) was used to confirm the significant difference in mean values between groups. In case of p-value lower than 0.05, *, in case of p-value lower than 0.01, **, Significant difference was marked with *** when the p value was lower than 0.001.

5 is a graph showing blood triglyceride concentrations of animal models classified into group 1 (NC, negative control group), group 2 (HFD), group 3 (PRG4), and group 5 (WERG50). Figure 6 is a graph showing the concentration of LTB4 in animal models divided into group 1 (NC, negative control group), group 2 (HFD), group 3 (PRG4) and group 5 (WERG50).

As shown in Figures 5 and 6, it can be seen that the HFD group ate a high-fat diet compared to the NC group consuming a normal diet significantly increased both blood triglyceride and LTB4, It can be confirmed that this increase is significantly reduced when the crude polysaccharide fraction is administered at 4 mg/kg.

Meanwhile, in the group administered with 50 mg/kg of the radish green hot water extract of Comparative Example 1, neither blood triglyceride nor LTB4 showed a significant decrease even though the dose was increased 12.5 times compared to PRG4. As a result of this experiment, it was confirmed that the crude polysaccharide fraction derived from radish greens lowers normal blood triglycerides, suppresses inflammation caused by obesity, and is particularly effective in treating excessive high fat intake in a short period of time.

Test Example 2-6. Blood endotoxin measurement

After 8 weeks, the animal models divided into group 1 (NC, negative control group), group 2 (HFD), group 3 (PRG4), and group 5 (WERG50) were fasted for 12 hours, anesthetized using Ipran solution, and the abdominal and Blood was collected from the hepatic portal vein by incision in the thoracic cavity, coagulated, and serum was separated and used to analyze endotoxin concentration. In order to analyze the endotoxin concentration from the serum, it was performed according to the manufacturer's protocol using Pierce™ LAL Chromogenic Endotoxin Quantitation Kit (Thermo scientific).

For the statistical analysis of the experiment, one-way ANOVA (Dunnett's test vs. HFD) was used to confirm the significant difference in mean values between groups. In case of p-value lower than 0.05, *, in case of p-value lower than 0.01, **, Significant difference was marked with *** when the p value was lower than 0.001.

7 is a graph showing endotoxin concentrations in the blood of animal models classified into group 1 (NC, negative control group), group 2 (HFD), group 3 (PRG4), and group 5 (WERG50).

As shown in Figure 7, it was confirmed that endotoxin significantly increased in the HFD group fed a high-fat diet compared to the NC group. In the case of the group (PRG4) administered with the crude polysaccharide fraction derived from grass green in Example 1, it was significantly decreased compared to the HFD group, and it was confirmed that the endotoxin concentration in the blood was lowered to the same level as the NC group. Meanwhile, despite the 12.5-fold higher dose than PRG4, the group (WERG50) administered with the hot water extract of Comparative Example 1 showed no significant difference compared to the HFD group.

Considering the above results, the crude polysaccharide fraction derived from radish greens of Example 1, unlike the hot water extract of radish greens, shows an effect of improving, preventing or treating obesity or metabolic diseases when used at 1 mg/kg or more, preferably 4 mg/kg or more. confirmed that Although this is 12.5 times lower than the Radish Green hot water extract, it achieves a remarkable and unpredictable effect compared to the Radish Green hot water extract.

Hereinafter, formulation examples of the composition containing the powder of the present invention will be described, but the present invention is not intended to limit them, but only to be specifically described.

Formulation Example 1. Preparation of powder

Crude polysaccharide fraction derived from radish greens obtained in Example 1 500 mg

Lactose 100 mg

Talc 10 mg

A powder is prepared by mixing the above ingredients and filling them in an airtight bag.

Formulation Example 2. Preparation of tablets

Crude polysaccharide fraction derived from radish greens obtained in Example 1 300 mg

corn starch 100 mg

lactose 100 mg

Magnesium stearate 2 mg

After mixing the above ingredients, tablets are prepared by tableting according to a conventional tablet manufacturing method.

Formulation Example 3. Preparation of capsule formulation

200 mg of crude polysaccharide fraction derived from radish greens obtained in Example 1

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium stearate 0.2 mg

Capsules are prepared by mixing the above ingredients and filling them into gelatin capsules according to a conventional capsule preparation method.

Formulation Example 4. Preparation of Injections

Crude polysaccharide fraction derived from radish greens obtained in Example 1 600 mg

Mannitol 180mg

Sterile Distilled Water for Injection 2974 mg

Na ₂ HPO _4, 12H ₂ O 26 mg

It is prepared with the above component content per 1 ampoule according to the conventional method for preparing injections.

Formulation Example 5. Preparation of liquid formulation

Crude polysaccharide fraction derived from radish greens obtained in Example 1 4g

Isomerized sugar 10 g

5 g mannitol

Appropriate amount of purified water

According to the conventional liquid formulation manufacturing method, each component is dissolved in purified water, lemon flavor is added in an appropriate amount, the above components are mixed, and then purified water is added to adjust the total amount to 100g, and then filled into a brown bottle to be sterilized. to prepare a liquid.

Formulation Example 6. Preparation of granules

Crude polysaccharide fraction derived from radish greens obtained in Example 1 1,000 mg

Appropriate amount of vitamin mixture

Vitamin A Acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 μg

Vitamin C 10 mg

10 μg of biotin

Nicotinamide 1.7 mg

Folic acid 50 μg

Calcium Pantothenate 0.5 mg

Appropriate amount of mineral mixture

Ferrous sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium Carbonate 25.3 mg

Potassium Phosphate Monobasic 15 mg

Dibasic Calcium Phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium Chloride 24.8 mg

Although the composition ratio of the above vitamin and mineral mixture was prepared by mixing ingredients suitable for granules in a preferred embodiment, the mixing ratio may be arbitrarily modified, and after mixing the above ingredients according to a conventional granule manufacturing method, It can be prepared and used for preparing a health functional food composition according to a conventional method.

Formulation Example 7. Manufacturing of functional beverages

Crude polysaccharide fraction derived from radish greens obtained in Example 1 1,000 mg

Citric Acid 1,000 mg

100 g of oligosaccharides

2 g plum concentrate

1 g of taurine

Add purified water to make a total of 900 ml

After mixing the above ingredients according to the normal health drink manufacturing method, stirring and heating at 85 ° C. for about 1 hour, the resulting solution is filtered and collected in a sterilized 2 L container, sealed and sterilized, and then refrigerated. It is used for preparing the functional beverage composition of the present invention.

Although the composition ratio is a mixture of ingredients suitable for a relatively favorite beverage in a preferred embodiment, the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as the class of demand, the country of demand, and the purpose of use.

Claims (15)

Food composition for improving or preventing obesity or metabolic disease containing a polysaccharide fraction derived from radish greens as an active ingredient According to claim 1,
The noon-derived polysaccharide fraction contains 65 to 75% by weight of neutral sugar, 20 to 30% by weight of uronic acid, and a KDO analog (2-keto-3-deoxy-D- manno-octulosonic acid) is a food composition for improving or preventing obesity or metabolic diseases, characterized in that it contains 1 to 10% by weight. According to claim 2,
The radish-derived polysaccharide fraction is a food composition for improving or preventing obesity or metabolic diseases, characterized in that it further comprises 0.5 to 5% by weight of protein. According to claim 2,
The polysaccharide fraction derived from green tea is a food composition for improving or preventing obesity or metabolic diseases, characterized in that it further comprises 0.5 to 10% by weight of polyphenols. According to claim 2,
Obesity or metabolism, characterized in that the neutral polysaccharide includes rhamnose, fucose, arabinose, xylose, mannose, galactose, glucose, glucuronic acid and galacturonic acid A food composition for improving or preventing disease. According to claim 2,
The neutral polysaccharide is 1 to 15 mol% of rhamnose, 1 to 15 mol% of fucose, 10 to 30 mol% of arabinose, 0.1 to 5 mol% of xylose, 1 to 10 mol% of mannose, 30 to 50 mol% of galactose, 1 to 5 mol% of glucose, 1 to 5 mol% of glucuronic acid and 5 to 20 mol% of galacturonic acid. A food composition for improving or preventing obesity or metabolic disease. According to claim 1,
The polysaccharide fraction derived from noen (a) firstly extracted at 80 to 110 ° C. by mixing an extraction solvent with noen green powder; (b) recovering fractions by precipitating the firstly extracted extract with C1 to C4 alcohol; And (c) recovering a fraction having a molecular weight of 10 to 20 kDa or more from the fraction. According to claim 1,
The metabolic disease is a food composition for improving or preventing obesity or metabolic disease, characterized in that at least one selected from the group consisting of dyslipidemia, non-alcoholic fatty liver, insulin resistance syndrome and diabetes. A pharmaceutical composition for preventing or treating obesity or metabolic disease, comprising a polysaccharide fraction derived from radish greens as an active ingredient. According to claim 9,
The noon-derived polysaccharide fraction contains 65 to 75% by weight of neutral sugar, 20 to 30% by weight of uronic acid, and a KDO analog (2-keto-3-deoxy-D- manno-octulosonic acid) is a pharmaceutical composition for preventing or treating obesity or metabolic diseases, characterized in that it comprises 1 to 10% by weight. According to claim 10,
The radish-derived polysaccharide fraction is a pharmaceutical composition for preventing or treating obesity or metabolic disease, characterized in that it further comprises 0.5 to 5% by weight of protein. According to claim 10,
The polysaccharide fraction derived from green tea is a pharmaceutical composition for preventing or treating obesity or metabolic diseases, characterized in that it further comprises 0.5 to 10% by weight of polyphenols. According to claim 10,
Obesity or metabolism, characterized in that the neutral polysaccharide includes rhamnose, fucose, arabinose, xylose, mannose, galactose, glucose, glucuronic acid and galacturonic acid A pharmaceutical composition for preventing or treating a disease. According to claim 10,
The neutral polysaccharide is 1 to 15 mol% of rhamnose, 1 to 15 mol% of fucose, 10 to 30 mol% of arabinose, 0.1 to 5 mol% of xylose, 1 to 10 mol% of mannose, 30 to 50 mol% of galactose, 1 to 5 mol% of glucose, 1 to 5 mol% of glucuronic acid and 5 to 20 mol% of galacturonic acid. A pharmaceutical composition for preventing or treating obesity or metabolic disease. According to claim 9,
The metabolic disease is a pharmaceutical composition for preventing or treating obesity or metabolic disease, characterized in that any one selected from the group consisting of dyslipidemia, non-alcoholic fatty liver, insulin resistance syndrome and diabetes.

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