KR20230173829A - Composition for preventing, alleviating or treating metabolic disease comprising Cynanchum atratum extract as an effective component - Google Patents

Abstract

The present invention relates to a composition for preventing, alleviating or treating metabolic diseases comprising a Cynanchum atratum extract as an active component. According to the present invention, a composition for preventing, alleviating or treating metabolic diseases comprises a Cynanchum atratum extract as an active component and thus can reduce blood sugar, body weight, and body fat in mice fed high-fat diet and inhibit liver damage, fatty liver production, and lipid metabolism abnormalities, thereby being useful in preventing, treating, or alleviating metabolic diseases.

Description

Composition for preventing, alleviating or treating metabolic disease comprising Cynanchum atratum extract as an effective component}

The present invention relates to a composition for preventing, improving, or treating metabolic diseases containing an extract of white rice ( Cynanchum atratum ) as an active ingredient.

Metabolic disease is a disease in which risk factors for death such as obesity, diabetes, insulin resistance, fatty liver, hyperlipidemia, arteriosclerosis or their complications exist together, and is also called 'metabolic syndrome'. The incidence of the above metabolic diseases has recently been rapidly increasing in Korea, and it is known that the incidence has increased significantly to the level of the United States and Western European countries or higher.

Obesity refers to a condition in which energy intake and consumption are not balanced due to lifestyle or genetic causes, excess energy is accumulated as fat, body fat increases abnormally, and metabolic abnormalities are caused. Obesity is common among modern people due to the improvement in living standards due to economic development, and is a major cause of increasing the risk of high blood pressure, hyperlipidemia, arteriosclerosis, heart disease, and diabetes. Obesity treatment drugs sold at home and abroad include 'Xenical', which is mainly made of orlistat, which has been approved by the U.S. FDA. There are side effects.

Diabetes mellitus is a disease in which insulin secretion is insufficient or normal function is not achieved. It is characterized by hyperglycemia, which increases the concentration of glucose in the blood. Hyperglycemia causes various symptoms and signs and causes glucose to be excreted in the urine. In addition, over a long period of time, it is a disease that causes vascular disorders and dysfunction of nerves, kidneys, and retina, and can even lead to loss of life.

Dyslipidemia refers to a condition in which the concentration of total cholesterol, triglycerides, and LDL cholesterol in the blood is higher than normal or the concentration of HDL cholesterol is lower than normal. When lipid components such as LDL cholesterol or neutral fat in the blood increase, blood flow becomes difficult and lipid components adhere to the artery walls, causing a chronic inflammatory response and narrowing the inner walls of the arteries, causing arteriosclerosis, which hardens the blood vessels. . In the long term, the blood clots generated from this block the coronary arteries or cerebral blood vessels, causing myocardial infarction, stroke, or cerebral infarction (E. Falk et al., Circulation 92, 657-671, 1995).

Fatty liver diseases (FLD) refers to a condition in which more than 5% of fat is accumulated in liver cells. Depending on the cause, it is divided into alcoholic fatty liver diseases (AFLD) and non-alcoholic fatty liver diseases (NAFLD). It is divided into The main cause of alcoholic fatty liver disease is excessive fat accumulation in the liver due to excessive alcohol consumption. Non-alcoholic fatty liver disease shows characteristic liver fat accumulation in liver imaging or biopsy without a history of significant alcohol consumption, use of drugs that can clearly induce fatty liver, or viruses. The non-alcoholic fatty liver disease (NAFLD) is a chronic metabolic disease that occurs along with obesity, type 2 diabetes, hyperlipidemia, cardiovascular disease, etc., has rapidly increased worldwide in recent decades, and is the most common chronic metabolic disease in modern society. It is reported that approximately 24% of the world's population and 16-33% of Koreans are affected by NAFLD. The severity of NAFLD varies greatly, from simple fatty liver (liver steatosis without inflammation) to nonalcoholic steatohepatitis (NASH), liver fibrosis, and even liver cirrhosis. It was reported that the number of NASH patients, which was only 6,785, increased more than four-fold to 28,368 in 5 years in 2015. In addition, the number of NASH patients in Korea increased by more than 20% per year on average over the five years from 2013 to 2017.

Meanwhile, in actual clinical practice, NAFLD occurs along with various metabolic diseases such as obesity, diabetes, and hyperlipidemia. A meta-analysis of clinical data showed that among NAFLD patients, 69.2% suffered from hyperlipidemia, 22.5% from type 2 diabetes, 42.5% from metabolic syndrome, and 39.3% from hypertension. Accordingly, some scholars have recently argued that the name NAFLD (non-alcoholic fatty liver disease) is inaccurate and that the name metabolic associated fatty liver disease (MAFLD) is more accurate. Therefore, in order to treat NAFLD, there is an urgent need for a drug that not only improves fatty liver disease but also improves metabolic diseases such as obesity, diabetes, and hyperlipidemia. Many studies related to NAFLD treatments are being conducted mainly in the Gumi region, and although the effectiveness of some treatments has been observed to date, it is difficult to use them due to unclear or various side effects that appear when taken for a long period of time. Accordingly, there is still no officially recognized treatment for improving NAFLD in the world.

Meanwhile, white rice (Cynanchum Atratum) is classified as a cleansing heat medicine in Oriental medicine, and it cools the heat of blood powder and helps to restore the body's Qi and blood in the late stages of heat-related illness or after childbirth. By consuming it, it heals the symptoms of internal fatigue and fatigue, and has been used for fever, seawater due to lung heat, diuresis, boils, sores, sore throat, and wounds caused by snake bites. In various modern pharmacological studies, white rice extract has been reported to be effective in improving various inflammations, allergies, parasites, atopic dermatitis, and anticancer. However, to date, there have been no studies related to the therapeutic effects of white rice extract on metabolic diseases such as fatty liver disease, hyperlipidemia, obesity, and diabetes.

KR 10-1727056 B1

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating metabolic diseases containing an extract of white rice ( Cynanchum atratum ) as an active ingredient.

In addition, another object of the present invention is to provide a health functional food composition for preventing or improving metabolic diseases containing white rice ( Cynanchum atratum ) extract as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating metabolic diseases containing white rice ( Cynanchum atratum ) extract as an active ingredient.

According to one embodiment of the present invention, the white rice extract may be an extract of water, alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.

According to one embodiment of the present invention, the mixed solvent may be 20 to 80% by volume of methanol, ethanol, butanol, or propanol.

According to one embodiment of the present invention, the mixed solvent may be 20 to 40% by volume of methanol or ethanol.

According to one embodiment of the present invention, the metabolic disease may be obesity, diabetes, hyperglycemia, dyslipidemia, or fatty liver.

According to one embodiment of the present invention, the dyslipidemia may be any one or more selected from the group consisting of hyperlipidemia, hyperlipidemia, hyperlipidemia, hypertriglyceridemia, and hypolipidemia.

According to one embodiment of the present invention, the fatty liver may be non-alcoholic fatty liver disease (NAFLD).

According to one embodiment of the present invention, the pharmaceutical composition may be an oral dosage form.

In addition, the present invention provides a health functional food composition for preventing or improving metabolic diseases containing white rice ( Cynanchum atratum ) extract as an active ingredient.

The present invention also provides a method for preventing or treating metabolic diseases in a subject, comprising administering to the subject an effective amount of a composition containing an extract of white rice ( Cynanchum atratum ) as an active ingredient.

The composition for preventing, improving or treating metabolic diseases according to the present invention has the effect of reducing blood sugar, body weight and body fat in mice on a high-fat diet and suppressing liver damage, fatty liver production and lipid metabolism abnormalities by containing white rice extract as an active ingredient. , can be usefully used in the prevention, treatment or improvement of metabolic diseases.

Figure 1 is a schematic diagram briefly showing the efficacy of white rice extract according to an embodiment of the present invention.
Figure 2a is a flowchart simply showing a method for producing white rice extract according to an embodiment of the present invention.
Figure 2b shows the results of high-performance liquid chromatography fingerprinting analysis of white rice extract according to an embodiment of the present invention.
Figure 2c is a pinker printing result of Paenol, an indicator component of white rice extract, according to an embodiment of the present invention.
Figure 2d is a schematic diagram simply showing the experimental process for verifying the efficacy of white rice extract according to an embodiment of the present invention.
Figure 3 is a graph showing the change in weight after administering a white rice extract according to an embodiment of the present invention to a mouse model consuming a high-fat diet and high-fructose water. Figure 3A shows body weight change over 10 weeks, Figure 3B shows final body weight, Figure 3C shows final liver weight, Figure 3D shows mesenteric fat weight, Figure 3E shows fat weight around testes, Figure 3F shows fat weight around kidney, Figure 3G shows feed intake, and Figure 3h shows water and fructose intake.
Figure 4 shows changes in fat accumulation after administering a white rice extract according to an embodiment of the present invention to a mouse model consuming a high-fat diet and high-fructose water. Figure 4a shows the results of histomorphological analysis of the liver, Figure 4b shows the results of lipid staining in the liver tissue, Figure 4c shows the alcoholic fatty liver evaluation score, Figure 4d shows the fat staining ratio, Figure 4e shows the TG (triglyceride) content in the liver tissue, Figure 4f represents the TC (total cholesterol) content in the liver tissue, Figure 4g represents the TG content in the feces, and Figure 4h represents the TC content in the feces.
Figure 5 is a graph showing changes in inflammation-related factors in liver tissue after administration of a white rice extract according to an embodiment of the present invention to a mouse model consuming a high-fat diet and high-fructose water. Figure 5a shows the NF-Kb phosphorylation immunofluorescence staining results, Figure 5b shows the Western blot results, Figure 5c shows the Western blot quantification results, Figure 5d shows the immunofluorescence staining results, Figure 5e shows the inflammatory cytokine ELISA results, and Figure 5f shows the TNF- The α/IL-10 ratio, Figure 5g shows the TLR-4 gene expression level, Figure 5h shows the endotoxin content in the feces, and Figure 5i shows the relative contents of Gram-positive and Gram-negative bacteria in the feces.
Figure 6 is a graph showing changes in lipid metabolism-related factors in liver tissue after administration of a white rice extract according to an embodiment of the present invention to a mouse model consuming a high-fat diet and high-fructose water. Figure 6a shows Western blot results for proteins related to lipid synthesis and lipid metabolism, Figures 6b and 6c show Western blot quantification results for proteins related to lipid synthesis and lipid metabolism, and Figure 6d shows realtime PCR results for genes related to fatty acid metabolism. .

Hereinafter, the present invention will be described in detail.

One aspect of the present invention relates to a pharmaceutical composition for preventing or treating metabolic diseases containing an extract of white rice ( Cynanchum atratum ) as an active ingredient.

The white rice of the present invention refers to the roots and rhizome of the white rice flower ( Cynanchum atratum Bunge ), a perennial herb belonging to the genus Cynanchum of the family Cynanchum. The white rice is also called Cynanchi atrati Radix in the Chinese Pharmacopoeia, and is used in several Asian countries to treat diseases such as fever, vasoconstrictive syncope, and lymphangitis, and is used to treat postpartum fatigue, vomiting, nephritis, urinary tract infections, edema, bronchitis, and rheumatism. It has been reported that it is also used to treat sexual back pain.

In one embodiment, the extraction solvent for the white rice extract may be water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. There is no need to specifically limit the alcohol having 1 to 4 carbon atoms, but for example, methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, or normal-butanol can be used. The mixed solvent may be 20 to 80% by volume of alcohol having 1 to 4 carbon atoms, preferably 20 to 80% by volume of methanol, ethanol, butanol, or propanol.

The extraction solvent is not particularly limited, but extracts extracted with 20 to 40% by volume of methanol or ethanol work more preferably in preventing, improving, or treating metabolic diseases of the present invention.

In one embodiment, the white rice extract is prepared by mixing white rice powder with an extraction solvent at a weight ratio of 1:0.1 to 10, preferably 1:0.1 to 5, more preferably 1:0.1 to 1, more preferably 1:0.2. Mix at a weight ratio of 0.6 to 0.6 and extract at 10 to 40 ℃, preferably 20 to 30 ℃, more preferably at room temperature for 24 to 72 hours, preferably 36 to 60 hours, more preferably 40 to 55 hours. It may be manufactured by performing reduced pressure concentration. If the weight ratio is outside the above range, the active ingredient of white rice may be extracted in a small amount.

The white rice extract may be a fraction obtained by extracting white rice with water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof and refractionating the extract with a second organic solvent. Preferably, the white rice extract may be a methanol extract or a fraction obtained by re-fractionating an ethanol extract of white rice with hexane, ethyl acetate, butanol, or water.

The term "extract" used in this specification when referring to white rice refers to not only the crude extract obtained by treatment with an extraction solvent, but also the processed product of the white rice extract, such as a concentrate obtained by additionally concentrating the white rice extract, the dried product thereof, or the powder obtained by grinding it. It can be included. For example, white rice extract can be prepared in powder form by additional processes such as reduced pressure distillation and freeze-drying, or spray drying.

In addition, the white rice extract of the present invention, in a broad sense, also includes processed products of white rice extract formulated so that white rice can be administered to animals, such as white rice extract powder. Although experiments were conducted with white rice extract in the present invention, those skilled in the art will be able to predict that the desired effect can be achieved even in the form of a processed product of white rice extract.

As used herein, the term “metabolic disease” may include obesity, diabetes, hyperglycemia, dyslipidemia, or fatty liver. The dyslipidemia may be any one or more selected from the group consisting of hyperlipidemia, hyperlipidemia, hypertriglyceridemia, and low HDL cholesterolemia. Additionally, the fatty liver may be non-alcoholic fatty liver disease (NAFLD).

In the following examples, the present inventors found that when the white rice extract was administered to an animal model of fatty liver induced by a high-fat diet, the increase in blood sugar, body weight, and body fat was reduced, and liver damage, inflammation in liver tissue, fatty liver production, and lipid metabolism abnormalities were suppressed. This was confirmed in detail. Specifically, the white rice extract of the present invention inhibits fat accumulation, reduces blood sugar, lowers the concentration of endotoxin, inflammatory cytokines, triglycerides and total cholesterol in the serum, inhibits the expression of lipid synthesis-related proteins in liver tissue, It may increase the expression of proteins related to lipid metabolism and the expression of genes related to fatty acid oxidation.

Meanwhile, the inventors of the present invention confirmed the toxicity of the white rice extract through specific experiments and confirmed that it is a substance harmless to the human body. In addition, the white rice extract of the present invention has almost no toxicity and side effects, so it can be used safely even for long-term use.

In this specification, 'containing as an active ingredient' means containing an effective amount sufficient to achieve efficacy or activity, that is, an effective amount capable of showing prevention, improvement or treatment efficacy against metabolic diseases as a pharmaceutical composition.

In one exemplary embodiment, the white rice extract may be present in the composition in any amount desired to provide effective metabolic disease treatment efficacy. According to one embodiment, the white rice extract may be 0.00001 to 99.9% by weight, 0.0001 to 30% by weight, preferably 0.0001 to 10% by weight, more preferably 0.0001 to 10% by weight, more preferably 0.0001 to 5% by weight. It may be included in weight percent. If the content of the white rice extract is less than the above range, it is difficult to expect the effect of the white rice extract, and if it exceeds the above range, not only is the increase in effect due to an increase in content not only insignificant, but the stability of the formulation is reduced. There is. In other words, by including the white rice extract of the present invention within the above range, it is possible to achieve a prevention, improvement or treatment effect on metabolic diseases, have formulation and product stability, and exhibit excellent effects at the optimal content in terms of economic efficiency.

The term “prevention” of the present invention refers to all actions that suppress the symptoms or delay the progression of metabolic disease by administering the composition of the present invention.

The term “treatment” of the present invention refers to any action that improves or beneficially modifies metabolic disease by administering the composition of the present invention.

The “pharmaceutical composition” may further include, in addition to white rice extract as an active ingredient, appropriate carriers, excipients, and diluents commonly used in the production of pharmaceutical compositions, etc.

The “carrier” is a compound that facilitates the addition of the compound into cells or tissues. The “excipient” is a compound added to give the active ingredient an appropriate form to formulate it or increase its amount to make it more convenient to use. The “diluent” is a compound diluted in water that not only stabilizes the biologically active form of the compound of interest, but also dissolves the compound.

There is no need to specifically limit the carrier, excipient, and diluent, but for example, lactose, glucose, sugar, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose. , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

The pharmaceutical composition of the present invention can be administered to mammals, including humans, by any method. For example, it 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., and oral administration is preferred.

The pharmaceutical composition of the present invention can be formulated into a preparation for oral administration or parenteral administration according to the administration route described above. When formulated, one or more buffers (e.g. saline or PBS), antioxidants, bacteriostatic agents, chelating agents (e.g. EDTA or glutathione), fillers, bulking agents, binders, adjuvants (e.g. aluminum hydroxide) side), suspending agents, thickening agents, wetting agents, disintegrants or surfactants, diluents or excipients.

Preparations for oral administration include tablets, pills, powders, granules, solutions, gels, syrups, slurries, suspensions or capsules, and such solid preparations include at least one excipient, for example, in the pharmaceutical composition of the present invention. , starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol, maltitol, cellulose It can be prepared by mixing methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, or gelatin. For example, tablets or dragees can be obtained by combining the active ingredient with solid excipients, grinding them, adding suitable auxiliaries and processing them into a granule mixture.

In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, or syrups. In addition to the commonly used simple diluents such as water or liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, or preservatives may be included. .

In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant, and anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives may be additionally included. .

When administered parenterally, the pharmaceutical composition of the present invention can be formulated with a suitable parenteral carrier in the form of injections, transdermal administration, and nasal inhalation according to methods known in the art. The above injections must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. For injections, examples of suitable carriers include, but are not limited to, solvents or dispersion media including water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), mixtures thereof, and/or vegetable oils. You can. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% dextrose. etc. can be used. In order to protect the injection from microbial contamination, it may additionally contain various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. Additionally, in most cases, the injection may additionally contain an isotonic agent such as sugar or sodium chloride.

The pharmaceutical composition of the present invention can provide desirable prevention, improvement or treatment effects on metabolic diseases when it contains an effective amount of white rice extract. In this specification, the term 'effective amount' refers to an amount that shows a greater response than the negative control, and preferably refers to an amount sufficient to improve or treat metabolic diseases. The white rice extract is used in an amount of 1 to 500 ㎍/ml, preferably 1 to 300 ㎍/ml, more preferably 1 to 200 ㎍/ml, more preferably 5 to 100 ㎍/ml, based on the total content of the pharmaceutical composition. Preferably, it may be included at a concentration of 10 to 50 μg/ml. At this time, if the content of the white rice extract is less than the lower limit, the metabolic disease prevention, improvement, or treatment effect may not be achieved to the desired extent. Conversely, if the upper limit is exceeded, the effectiveness of preventing, improving, or treating metabolic diseases may not increase as the concentration increases. Meanwhile, as a result of in vitro experiments, when the concentration of the white rice extract of the present invention was within the above range, a significant effect was observed in preventing, improving, or treating metabolic diseases, but no side effects such as cytotoxicity were observed. The effective amount of white rice extract included in the pharmaceutical composition of the present invention will vary depending on the form in which the composition is commercialized.

The total effective amount of the pharmaceutical composition of the present invention can be administered to a patient as a single dose, or may be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. . The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the severity of the disease.

The appropriate dosage of the pharmaceutical composition varies depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity, and is usually A skilled physician can easily determine and prescribe an effective dosage for desired treatment or prevention. According to a preferred embodiment, the preferred daily dosage of the white rice extract is 1 to 200 mg/kg, preferably 1 to 100 mg/kg, more preferably 2 to 50 mg/kg, even more preferably 5 to 50 mg/kg. It may be 30 mg/kg, more preferably 10 to 20 mg/kg.

The composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans through various routes. All modes of administration are contemplated, for example, oral, rectal or by intravenous, intramuscular, subcutaneous, intrathecal or intracerebroventricular injection, preferably intravenous or oral administration. It can be administered, and more preferably, it can be administered orally.

Another aspect of the present invention relates to a health functional food composition for preventing or improving metabolic diseases containing white rice extract as an active ingredient. The health functional food composition of the present invention can be usefully used to prevent or improve metabolic diseases.

Since the terms of the present invention, “white rice,” “extract,” and “metabolic disease,” have been described above, their description is omitted to avoid excessive duplication.

The term “prevention” in the present invention means anything that suppresses or delays the metabolic disease.

The term "improvement" in the present invention means at least reducing the degree of symptoms, for example, parameters related to the condition being treated by administration of a composition comprising the white rice extract of the present invention.

The health functional food composition may be provided in the form of powder, granules, tablets, capsules, syrup, or beverage, and the health functional food composition may be used with other foods or food additives in addition to the active ingredients, and may be administered appropriately according to conventional methods. It can be used effectively. The mixing amount of the active ingredient can be appropriately determined depending on its purpose of use, for example, prevention, improvement or therapeutic treatment. According to one embodiment, the white rice extract may be 0.00001 to 99.9% by weight, 0.0001 to 30% by weight, preferably 0.0001 to 10% by weight, more preferably 0.0001 to 10% by weight, more preferably 0.0001 to 5% by weight. It may be included in weight%, more preferably 0.001 to 5% by weight, and even more preferably 0.01 to 5% by weight.

The effective dose of the active ingredient contained in the health functional food composition is the type and content of the active ingredient and other ingredients contained in the composition, type of dosage form, age, weight, general health status, gender and diet of the ingestor, time of administration, It may be adjusted according to various factors including the route of administration, period of intake, and concurrently used drugs, but is not limited thereto. In addition, in the case of long-term intake for the purpose of preventing or improving metabolic diseases, the amount may be below the above range, and since the active ingredient has no safety issues, it is certain that it can be used in amounts above the above range.

Hereinafter, the present invention will be described in detail through examples, but the present invention is not limited to the following examples.

<Example>

Example: Preparation of white rice extract

The white rice was washed and ground to a 200 mesh size. 200 mL of 30% ethanol solvent was added to 500 g of the white rice powder, followed by shaking extraction and only the supernatant was recovered. After filtering with filter paper, centrifugation (3000 x g, 15 minutes) was performed to obtain the supernatant. The supernatant was concentrated under pressure to 80 mL and then freeze-dried at -80°C (Figure 2a). The yield of the powder obtained after freeze-drying (hereinafter referred to as “CAE”) was 12.25% by weight compared to the dried herbal medicine before extraction.

<Test example>

All experiments were repeated three times, and the results were expressed as mean ± standard deviation. Statistical analysis was performed using SPSS software (ver. 20.0, SPSS Inc., Chicargo, IL, USA). One-way ANOVA was performed to verify the difference between each mean value, and p < 0.05 was judged to be significant. In each data, # and ## mean significant differences at p<0.05 and p<0.01, respectively, compared to the normal group (Normal), and * and ** mean significant differences, respectively, compared to the induced group (HFHFD). It means that there is a significant difference at <0.05 and p<0.01.

Test Example 1: HPLC analysis results of white rice extract

For the white rice extract prepared in the examples, chemical analysis using HPLC was performed as follows. High-performance liquid chromatography used the Agilent 1260 HPLC system (Agilent Technologies, Wilmington, DE, USA), the column used was Agilent Eclipse XDB-C18 (5㎛, 100A, 4.6mm × 250mm), and the detector was a diode array detector. (DAD) was used.

The mobile phase used was a gradient solvent system (20-100% acetonitrile mixed solvent, time: 45 minutes, flow rate: 1 mL/min) in which the ratio of the mixed solvent of water and acetonitrile for HPLC was changed, and the standard After accurately measuring the weight of the compound Paeonol (Glentham Life Sciences, Wiltshire, UK) and the white rice extract of the example, each was dissolved in 50% methanol to prepare concentrations of 1 mg/ml and 0.1 mg/ml, and filtered through a 0.45 ㎛ filter. After that, 10 ㎕ was injected and analyzed. At this time, the UV wavelength of the diode array detector (DAD) was selected at 300 nm, where all peaks of the white rice extract are evenly distributed. As a result, as shown in Figures 2b and 2c, it was confirmed that 0.829 mg/g of Paeonol as an indicator substance was present in the white rice extract of the present invention.

Test Example 2: Effect of white rice extract on improving metabolic diseases through animal testing

2-1: Animal experiment design

laboratory animals

Thirty SPF (specific pathogen free) 6-week-old male C57BL/6J mice were purchased from Daehan Biolink, acclimatized for 1 week, and then randomly divided into five groups with 6 mice per group.

Separation of experimental groups (5 groups in total; 6 animals per group)

(1) Normal group: Untreated group

(2) Induction group (HFHFD): High-fat diet and high-fructose water intake group for 10 weeks

(3) Drug treatment group low concentration (CAE100): group administered high-fat diet and high fructose water (HFHFD) for 10 weeks and white rice extract of the example (100 mg/kg body weight)

(4) High concentration drug treatment group (CAE200): Group administered high-fat diet and high fructose water (HFHFD) for 10 weeks and white rice extract (200 mg/kg body weight) of the example.

(5) Positive control group (MET): A group that consumed a high-fat diet and high fructose water (HFHFD) and metformin (100 mg/kg body weight) for 10 weeks.

High-fat diet and high-fructose intake, and drug administration

The experimental animals were fed a high-fat diet (D12494, 60% kcal% fat, Research Diet, New Brunswick, NJ, USA) and high-fructose water (20% w/v, dissolved in distilled water, Sigma-Aldrich, Darmstadt, Germany). Metabolic disease was induced by consuming it for 10 weeks.

Then, after 4 weeks of ingesting a high-fat diet and high-fructose water, the drug-treated group was orally administered 100 and 200 mg/kg of the white rice extract of the example once daily for 6 weeks, while the normal and induced groups were orally administered only sterilized distilled water. , the positive control group was orally administered 100 mg/kg of metformin (Figure 2d).

Measure changes in body weight, liver and fat weight

The amount of food (including the amount of water) and body weight of all animals were recorded every week during the experiment, and the animals were sacrificed on the last day of the experiment and their liver and fat were weighed. Liver tissue was stored in an ultra-cold refrigerator at -70°C for subsequent analysis.

2-2: Anti-obesity effect of white rice extract

To confirm the obesity-suppressing effect or weight loss effect of white rice extract, body weight changes in mice fed a high-fat diet and high-fructose water were measured.

Specifically, while the 10-week experiment was in progress according to Test Example 2-1, the body weight of each group of mice was measured at regular times every week (FIGS. 3A and 3B).

As a result, as shown in Figures 3a and 3b, the high-fat diet and high-fructose water intake group (HFHFD) showed significantly higher body weight than the normal group, while the white rice extract administration group showed a high-fat diet and high-fructose water intake group (HFHFD). ) showed a significant obesity suppression effect or weight loss effect compared to Specifically, the white rice extract administration group (CAE100 and CAE200) showed a weight loss effect of approximately 20% compared to the high-fat diet and high-fructose diet (HFHFD) group.

2-3: Liver weight and body fat reduction effect of white rice extract

In order to confirm the effect of the white rice extract on reducing liver weight and body fat, mice from each group were sacrificed at the end of the 10-week experiment according to Test Example 2-1, and liver tissue, enterohepatic adipose tissue, fat tissue around the testicles, and fat tissue around the kidneys were sacrificed. After the fat tissues were removed, their respective weights were measured (FIGS. 3C to 3F).

As a result, as shown in Figure 3c, the high-fat diet and high-fructose water consumption group (HFHFD) showed significantly higher liver weight than the normal group, while the white rice extract administration group (CAE100 and CAE200) showed a high-fat diet and high-fructose water intake group. It was confirmed that liver weight was significantly reduced compared to the group (HFHFD).

In addition, as shown in Figures 3d to 3f, the high-fat diet and high-fructose diet group (HFHFD) had higher levels of mesenteric fat weight, epididymal fat weight, and kidney fat tissue compared to the normal group. While the weight of adipose tissue (perirenal fat weight) was significantly higher, the white rice extract administration group (CAE100 and CAE200) had mesenteric adipose tissue, peritesticular adipose tissue, and perirenal fat compared to the high-fat diet and high-fructose diet (HFHFD) group. It was confirmed that the weight of the tissue was significantly reduced.

For reference, there was no significant difference in feed intake and water/fructose intake between each animal test group except the normal group (Figures 3g and 3h).

From the above results, it can be seen that the white rice extract of the present invention can effectively suppress obesity or increase in body fat without affecting dietary intake.

2-4: Fatty liver improvement effect of white rice extract

In order to confirm the effect of the white rice extract on suppressing fatty liver formation, mice from each group at the end of the 10-week experiment were sacrificed according to Test Example 2-1, the liver was extracted, the weight of the liver was measured, and the tissue condition of the extracted liver was measured. was confirmed.

Specifically, hematoxylin and eosin and Oil Redo staining were performed to verify the effect of white rice extract on lipid accumulation in liver tissue (FIGS. 4A to 4D). As a result, as shown in Figures 4a to 4d, it was confirmed that intake of white rice extract (CAE100 and CAE200) significantly reduced the formation of vacuoles and lipid accumulation in liver tissue. In particular, looking at Figures 4c and 4d, it can be seen that the white rice extract intake group (CAE100 and CAE200) has a better fatty liver improvement effect than the positive control group (MET).

In addition, TG (triglyceride) content in liver tissue, TC (total cholesterol) content in liver tissue, TG (triglyceride) content in feces, and The TC (total cholesterol) content in feces was measured (Figures 4e to 4h).

As a result, as shown in Figures 4e and 4f, it was confirmed that the amount of neutral fat and total cholesterol in the liver tissue was significantly reduced in the white rice extract consumption group (CAE100 and CAE200) compared to the induction group (HFHFD). In addition, as shown in Figures 4g and 4h, it was confirmed that the excretion of neutral fat and total cholesterol in feces was significantly increased in the white rice extract consumption group (CAE100 and CAE200) compared to the induction group (HFHFD).

From the above results, it can be seen that the white rice extract of the present invention exhibits an effect of improving fatty liver or inhibiting fatty liver formation.

2-5: White rice extract improves liver damage and suppresses lipid metabolism abnormalities

According to Test Example 2-1, the mice in each group at the end of the 10-week experiment were anesthetized and blood was collected through the abdominal vein. Serum was separated from the blood, and liver damage, lipids, and glucose metabolism-related factors in the serum were aspartate transaminase (AST), alanine transaminase (ALT), triglycerides (TG), total cholesterol (TC), and low-density Cholesterol (LDL), high-density cholesterol (HDL), and free fatty acid (FFA) were measured using an automated biochemical analyzer (Chiron, Emeryville, Ca, USA) or a commercial diagnostic kit and are shown in Table 1 below.

division Normal HFHFD C.A.E. MET 100 200 Serum AST (IU/L) 51.0±5.1 81.4±13.4 ^## 65.3±7.4 ^* 62.4±9.7 ^* 71.0±14.6 ALT (IU/L) 17.3±3.0 48.2±17.2 ^# 31.6±16.2 20.3±4.9 ^* 44.1±31.7 TG (mg/dL) 116.9±11.4 137.4±16.9 ^# 140.7±16.8 122.7±8.4 ^* 112.5±10.8 ^** TC (mg/dL) 101.2±5.6 180.9±9.9 ^## 169.4±18.6 148.6±22.4 ^* 162.5±11.6 ^* LDL (mg/dL) 15.8±0.6 27.8±2.6 ^## 22.7±1.9 ^** 23.5±1.2 ^** 26.3±1.8 HDL (mg/dL) 114.0±14.3 72.5±3.9 ^## 114.4±9.3 ^** 124.2±13.6 ^** 113.8±6.2 ^** FFA (mEq/L) 0.89±0.12 1.03±0.01 ^# 0.92±0.04 ^** 0.85±0.16 ^* 0.97±0.23 Fasting blood glucose
(mg/dL) 88.5±7.4 163.2±40.6 ^## 106.3±10.9 ^* 119.0±18.8 ^* 130.7±16.8 ^*

Looking at Table 1, the blood AST, ALT, TG, TC, LDL, and FFA of the induced group (HFHFD) were significantly increased compared to the normal group (Normal), and the intake of white rice extract (CAE100 and CAE200) increased the blood AST. , ALT, TG, TC, LDL and FFA levels were significantly reduced. Additionally, consumption of white rice extract (CAE100 and CAE200) significantly increased blood HDL levels.

2-6: Blood sugar lowering effect of white rice extract

For all mice, after the 10-week experiment according to Test Example 2-1 was completed and before autopsy, blood was collected from the tail vein and fasting blood sugar concentration was measured using an ACCUCHEK Instant blood glucose meter (Roche Diabetes Care GmbH, Mannheim, Germany) (Table One).

Looking at Table 1 above, the fasting blood glucose concentration of the induced group (HFHFD) was significantly increased compared to the normal group (Normal), and the intake of white rice extract (CAE100 and CAE200) significantly increased the increased fasting blood glucose concentration. reduced to

2-7: Inhibitory effect of liver inflammation of white rice extract

Figure 5 is a graph showing changes in inflammation-related factors in liver tissue after administration of a white rice extract according to an embodiment of the present invention to a mouse model consuming a high-fat diet and high-fructose water.

Specifically, Figure 5a shows the results of NF-kB phosphorylation immunofluorescence staining, Figure 5b shows the Western blot results of NF-kB phosphorylated protein and IkB phosphorylated protein, and Figure 5c shows the quantification of the NF-kB phosphorylated protein by Western blot. This is the result, and Figure 5d is the immunofluorescence staining quantification result for NF-kB.

Looking at Figures 5a to 5d, the activity of NF-kB in the induced group (HFHFD) was significantly increased compared to the normal group (Normal), and the intake of white rice extract (CAE100 and CAE200) significantly increased NF-kB activity. You can see that it is decreasing significantly.

Additionally, Figure 5e shows the ELISA results of inflammatory cytokines IL-10 and TNF-α, and Figure 5f shows the TNF-α/IL-10 ratio.

Specifically, the concentrations of TNF-α and IL-10, markers related to liver tissue inflammation, were measured using enzyme-linked immunosorbent assay (ELISA kit, RBioLegend, San Diego, CA, USA for TNF-α; BD Biosciences, San Jose, CA, USA for IL- 10) was measured. Protein concentration was measured using the Pierce™ BCA kit (Thermo Fisher Scientific, Lafayette, CO, USA).

As shown in Figures 5e and 5f, serum TNF-α was significantly decreased and IL-10 was significantly increased in the white rice extract treatment group (CAE100 and CAE200) compared to the inducer group (HFHFD). The ratio of TNF-α and IL-10 was also significantly decreased in the white rice extract treatment group (CAE100 and CAE200) compared to the induced group (HFHFD).

Additionally, Figure 5g shows the TLR-4 gene expression level, Figure 5h shows the endotoxin content in the feces, and Figure 5i shows the relative contents of Gram-positive and Gram-negative bacteria in the feces.

Looking at Figures 5g and 5h, the expression level of TLR-4 gene, an endotoxin receptor, and endotoxin content in feces were significantly reduced in the white rice extract treated group (CAE100 and CAE200) compared to the induced group (HFHFD). In addition, looking at Figure 5i, it can be seen that the white rice extract treatment group (CAE100 and CAE200) significantly reduced intestinal Gram-negative bacteria and significantly increased intestinal Gram-positive bacteria compared to the induced group (HFHFD).

From the above results, it can be seen that consumption of the white rice extract of the present invention effectively suppresses inflammation in liver tissue.

2-8: Mechanism of white rice extract to inhibit hepatic lipid synthesis and promote fatty acid oxidation

The expression of proteins and genes related to hepatic lipid synthesis and fatty acid oxidation was confirmed using Western blot and real-time PCR (Real-time polymerase chain reaction) (Figures 6a to 6d).

Figure 6 is a graph showing changes in lipid metabolism-related factors in liver tissue after administration of a white rice extract according to an embodiment of the present invention to a mouse model consuming a high-fat diet and high-fructose water. Figure 6a shows Western blot results for proteins related to lipid synthesis and lipid metabolism, Figures 6b and 6c show Western blot quantification results for proteins related to lipid synthesis and lipid metabolism, and Figure 6d shows realtime PCR results for genes related to fatty acid metabolism. .

Looking at Figures 6A to 6D, it can be seen that the expression of lipid synthesis-related proteins GPAM, SEREBP1, and FAS was suppressed by ingestion of white rice extract, and the expression of lipid metabolism-related proteins pAMPK-α and PPAR-α increased. . In addition, it was confirmed that the RNA expression of SIRT1, PGC1α, CTP1, and ACSL1, genes related to fatty acid oxidation, increased due to intake of white rice extract.

From the above results, it can be seen that white rice extract inhibits liver lipid synthesis and promotes fatty acid oxidation.

Below, a formulation example of a composition containing the white rice extract of the present invention is described, but the present invention is not intended to be limited, but merely explained in detail.

<Manufacturing example>

<Preparation Example 1> Preparation of pharmaceutical preparation

1-1: Production of powder

200 mg of white rice extract of the example

lactose 100mg

Talc 10mg

The above ingredients are mixed and filled into an airtight bubble to prepare a powder.

1-2: Preparation of tablets

White Rice Extract of Examples 100mg

corn starch 100mg

lactose 100mg

Magnesium Stearate 2mg

After mixing the above ingredients, tablets are manufactured by compressing them according to a typical tablet manufacturing method.

1-3: Preparation of capsules

White Rice Extract of Examples 100mg

crystalline cellulose 3mg

lactose 14.8mg

magnesium stearate 0.2mg

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

<Manufacturing Example 2> Manufacturing of health functional food

2-1: Preparation of granules

White Rice Extract of Examples 1,000 mg

vitamin mixture Appropriate amount

Vitamin A Acetate 70 ㎍

Vitamin E 1.0mg

Vitamin B1 0.13mg

Vitamin B2 0.15mg

Vitamin B6 0.5mg

Vitamin B12 0.2 μg

Vitamin C 10mg

biotin 10 μg

Nicotinic acid amide 1.7mg

folic acid 50 μg

Calcium Pantothenate 0.5mg

mineral mixture Appropriate amount

Ferrous sulfate 1.75mg

zinc oxide 0.82mg

Magnesium Carbonate 25.3mg

Potassium Phosphate Monobasic 15mg

potassium phosphate dibasic 55mg

potassium citrate 90mg

calcium carbonate 100mg

Magnesium chloride 24.8mg

The composition ratio of the above vitamin and mineral mixture is a mixture of ingredients suitable for health functional foods in a preferred embodiment, but the mixing ratio may be modified arbitrarily, and the above ingredients are mixed according to a normal health functional food manufacturing method. Next, granules can be prepared and used to manufacture a health functional food composition according to a conventional method.

2-2: Manufacturing of health drinks

White Rice Extract of Examples 1,000 mg

citric acid 1,000 mg

oligosaccharide 100 g

plum concentrate 2g

taurine 1g

Add purified water Total 900 mL

After mixing the above ingredients according to a typical beverage production method, stirring and heating at 85° C. for about 1 hour, the resulting solution was filtered, placed in a sterilized 2 L container, sealed, sterilized, and stored in the refrigerator. Then, the solution according to the present invention was mixed. A functional beverage composition manufacturing example was used.

Although the present invention has been described in terms of the above-mentioned preferred embodiments, various modifications and variations can be made without departing from the gist and scope of the invention. Additionally, the appended claims cover such modifications or variations as fall within the subject matter of the present invention.

Claims (9)

A pharmaceutical composition for preventing or treating metabolic diseases comprising an extract of white rice ( Cynanchum atratum ) as an active ingredient. According to paragraph 1,
A pharmaceutical composition, wherein the white rice extract is an extract of water, alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. According to paragraph 2,
A pharmaceutical composition, characterized in that the mixed solvent is 20 to 80% by volume of methanol, ethanol, butanol, or propanol. According to paragraph 2,
A pharmaceutical composition, characterized in that the mixed solvent is 20 to 40% by volume of methanol or ethanol. According to paragraph 1,
A pharmaceutical composition, wherein the metabolic disease is obesity, diabetes, hyperglycemia, dyslipidemia, or fatty liver. According to clause 5,
A pharmaceutical composition, wherein the dyslipidemia is at least one selected from the group consisting of hyperlipidemia, hyperlipidemia, hypertriglyceridemia, and hypolipidemia. According to clause 5,
A pharmaceutical composition, wherein the fatty liver is non-alcoholic fatty liver disease (NAFLD). According to paragraph 1,
A pharmaceutical composition, characterized in that the pharmaceutical composition is an oral dosage form. A health functional food composition for preventing or improving metabolic diseases containing white rice ( Cynanchum atratum ) extract as an active ingredient.