KR20040100404A - Active fraction having inhibitory effects on adipocytes (NIH3T3-L1 cell) isolated from medicinal plants - Google Patents

Abstract

PURPOSE: Provided is an active fraction having inhibitory effects on adipocytes(NIH3T3-L1 cell) isolated from medicinal plants. The active fraction inhibits fat accumulation and prevent and treat obesity. CONSTITUTION: The composition for prevention and treatment of obesity is characterized by containing a prophylactically or therapeutically effective amount of at least one active fraction selected from the group consisting of Euphoria longana and Calystefia Japonica (Thunb.) Choisy.

Description

Active fraction having inhibitory effects on adipocytes (NIH3T3-L1 cell) isolated from medicinal plants}

The present invention relates to an active fraction composition for inhibiting adipocyte differentiation, which is isolated from herbal medicine resources, and more specifically, to the differentiation of NIH3T3-L1 cells, which are adipocytes, from the extracts of longan meat and buckwheat, which have traditionally been used for therapeutic purposes in the East. Active fraction composition that inhibits the accumulation of fat, which causes obesity, and inhibits weight gain of Leprdb / Leprdb mice, which are obese laboratory animals, and methods for efficiently extracting and purifying the same. It relates to herbal medicine for the prevention and treatment of obesity.

The compound according to the present invention can be identified as an obese experimental animal by showing the effect of preventing and treating obesity by inhibiting adipocyte differentiation.

Recently, due to the improvement of living standards due to economic development, people live in a rich and complex life, and various factors such as overeating, lack of physical activity, excessive drinking, irregular eating patterns, etc. are combined to consume less calories than consumed. The increase in body weight, ie, obesity, is caused by the abnormal increase in fat cells, not by weight gain. The increase in body weight by 20% increases the incidence of hypertension (more than 160/95) by six times, hyperlipidemia by two times, and diabetes by four times. In addition, the risk of coronary heart disease, arthritis, gout, gallstones, respiratory dysfunction, and breast cancer increases. Just as the obese population grows sharply, so does the disease population. Obesity has tended to look healthy in the past, but in recent years, obesity has persisted, leading to a number of diseases such as hypertension, diabetes, heart disease, gout, arteriosclerosis, cerebral hemorrhage, liver dysfunction, hyperlipidemia, and coronary artery disease. In addition, it has been reported to cause breast cancer, uterine cancer and colorectal cancer, and is now treated as one of the fatal diseases [J. Biol. Chem., 273, 32487-32490 (1998); Nature, 404, 652-660 (2000).

Current treatments for obesity can be divided into drugs that act on the central nervous system and affect appetite, and drugs that inhibit absorption by the gastrointestinal tract. Serotonin depends on each mechanism. (5HT) Drugs such as fenfluramine and dexfenfluramine that inhibit the nervous system, drugs such as ephedrine and caffeine through the noradrenaline nervous system, and drugs such as sibutramine that simultaneously inhibit obesity by simultaneously acting on the serotonin and noradrenaline nervous system, are commercially available. . In addition, drugs that inhibit obesity by acting on the gastrointestinal tract typically include orlistat, which has recently been approved as an obesity treatment agent by inhibiting lipase produced in the pancreas to reduce fat absorption. However, fenfluramine has been banned in recent years because of side effects such as primary pulmonary hypertension or heart valve lesions, and other drugs have been banned due to problems such as decreased blood pressure and lactic acidosis. The patient has a problem that cannot be used.

Therefore, the side effects are small, and the search for adipocyte differentiation inhibitors for better obesity treatment and prevention. In other words, they searched for and identified new drugs that are closely related to the formation of fat cells but are unlikely to act on the nervous system.

Fat stored in fat cells is used as an important energy source in the body. However, as obesity progresses, not only does the adipocytes increase in number, but the synthesis of a large amount of triglycerides by the differentiation of excessive adipocytes is accompanied by morphological changes including the size of the adipocytes and various gene expression changes. Increasing the size of fat cells is induced by synthesizing and storing surplus energy in the form of triglycerides. On the other hand, with the storage of fat, the size of fat cells can be increased by about 20 times its diameter, and as a result, the cell volume is known to increase by several thousand times. The size of the adipocytes is generally controlled by diet, but the process of differentiating new progenitor cells into adipocytes is not effective as dietary control. It is important to adjust. Adipocyte differentiation is promoted by stimulation of insulin, insulin like growth factor-1, growth hormone, and the like, and in the process, CCAAT enhancer-binding protein (C / EBP) family, peroxisome proliferator-activated receptor ( An increase in transcription factors such as PPAR) gamma is observed. These transcription factors, along with adipocyte regulators, promote the differentiation of adipocytes and increase the expression levels of enzymes such as fatty acid binding proteins aP2 and fatty acid synthase. On the other hand, in the case of type 2 diabetes in which obesity progresses and diabetes mellitus, triglycerides, which are triglycerides, gradually induce insulin resistance, and ultimately destroy pancreatic beta cells to induce diabetes. Proc. Natl. Acad. Sci. 95, 2498-2502 (1998). Meanwhile, it has been reported that excessive triglyceride accumulation is involved in the progression of fatty liver [J. Clin. Invest., 98, 1575-1584 (1996).

Recently, based on the idea that if it can inhibit the differentiation of adipocytes, the number of produced adipocytes can be controlled and the extra energy accumulated can be released. Is actively underway.

In general, among the various methods for the development of drugs with new ingredients, the experimental modification of existing drugs or the synthesis and function screening of new materials requires very much time and investment. On the other hand, natural medicines used in traditional medicine have been used for a long time, so there is little concern about the toxicity of the drug to be developed, and there is a possibility of discovering a new active ingredient based on the confirmed drug efficacy. Very high.

Searching for substances that inhibit the differentiation of fat cells, which are the cause of obesity, from the herbal resources, and using the Leprdb / Leprdb mouse, an obesity model animal, to verify the effectiveness and prevention of obesity. It was confirmed to have an effect.

Longan flesh is the flesh of the longan belonging to the fig tree family, also known as Wonan, and it is called Yongan because it looks like the eyes of a dragon. Insomnia, emotional anxiety, nervous breakdown, stress is widely used in the medicine.

The name of the flower is called Guguang or Daffodil, and it is a grass commonly used in fields such as Micho, Donjangcho, Kohwa-hwa, Geuneungi, Geunjiji, Koji-yeon, Cheongeumcho, Guaseok, and Mee. Messiah is a perennial herb in the family Narcissus, which is a vine, with white shoots spreading in all directions, where new shoots emerge and entangle. Stems, leaves, flowers, roots and whole grasses are widely used as diuretics. It is used for edible and medicinal purposes. In oriental medicine and folk medicine, flowers and roots are used as medicinal herbs for stroke, asthma and diuretic cold. Daffodils are used for women's insensitivity, cystitis, energy loss, diabetes, and diuretics. In herbal medicine, diuretics, stimulants, stimulants, and wounds are used.

In this regard, the present inventors have studied the effects of obesity prevention and treatment and leukocyte proliferation in the Leprdb / Leprdb mouse, a model animal that causes obesity over leptin receptor for drugs that have been used in traditional medicine in Korea, including consent. As a result of observing the action of differentiation of L1 and F442A for adipocyte differentiation, the present invention was completed by acquiring from the active fractions, yongan meat and buckwheat, which showed specific inhibitory activity.

Accordingly, an object of the present invention is to provide a method for extracting longan meat, buckwheat extract, which is a drug having a distinct effect on obesity prevention and treatment, and a herbal medicine containing the extract as an active ingredient.

Figure 1 is a photograph showing the difference in the differentiation of adipocytes was inhibited by treatment of the active fraction extracted from Longanae arillus extracted to the NIH3T3-L1 cell line of fat cells.

Figure 2 is a photograph showing the difference that the differentiation of adipocytes was inhibited by treating the active fraction extracted from Calystegia japonica to the NIH3T3-L1 cell line as adipose cells.

3 is a graph comparing the difference between the experimental group and the control group for weight gain inhibition (prevention and treatment of obesity) after administration of the active fraction extracted from Longanae arillus for 15 days to the Leprdb / Leprdb mouse, an obese model animal. to be.

Figure 4 is a graph comparing the difference between the experimental group and the control group for weight gain inhibition (prevention and treatment of obesity) after administration of the active fraction extracted from Calystegia japonica for 15 days in the obese model animal Leprdb / Leprdb mice to be.

The present invention is characterized in that the active fraction composition for inhibiting adipocyte differentiation obtained from longan meat and buckwheat flower.

In the present invention, after the dry grinding and crushing of yongan meat, buckwheat, at least at room temperature to extract the organic solvent and water of 5 to 50 times the weight at reflux state for at least 24 hours. The organic solvent refers to methanol, ethanol, butanol, ethyl acetate, chloroform, nucleic acid, and the like. The extracted solution is concentrated under reduced pressure at a temperature of 40 degrees or less. The concentrated extract is completely dissolved using an organic solvent and water. A part of the completely dissolved extract is dried, the total weight is converted, and then, the silica gel is added to 1.5 to 5 times the weight and concentrated under reduced pressure at a temperature of 40 degrees or less to adsorb the extract onto the silica gel. After filling the column with activated silica (more than 1.5 times the amount of adsorbed silica gel) as a developing solvent, the column is sufficiently flowed into the developing solvent. The silica gel adsorbed on the packed silica gel column is filled, and the active substance is eluted with the developing solvent. The eluted active substance is concentrated under reduced pressure at a temperature below 40 degrees, suspended with water to remove residual organic solvent, and then lyophilized. Drying method is selected from the general hot air, vacuum drying, freeze drying, spray drying. Depending on the formulation or use desired, more effective and additional separation methods may be employed. For example, the column operation of the extract may be a filler other than silica gel. Other fillers refer to activated carbon, Amberlite XAD-4, and the like. The extract was concentrated under reduced pressure, suspended in purified water, adjusted to pH 10 and lowered to pH 3.5. The precipitate was recovered by centrifugation, suspended in purified water and freeze-dried. In addition, HP-20 column equilibrated to pH 10 in the state adjusted to pH 10, adsorb | sucked to a filler, it collect | recovered using an organic solvent, and it concentrated under reduced pressure, and freeze-dried.

In addition, the mixture is extracted at least 24 hours under reflux with an alcoholic solvent and water of 5 to 50 times its weight at room temperature. Alcoholic solvent refers to methanol, ethanol, butanol, isopropyl alcohol and the like. Purified water is added to the extracted solution to produce a certain concentration of alcohol. The constant concentration is the concentration that the active part is eluted in the HP-20 column without melting according to the sample, currently 30% ethanol, 50% ethanol, 70% ethanol. The HP-20 resin (5 to 10 times the weight of the sample) is sufficiently stabilized with alcoholic solvent and then filled into the column. Rinse the resin by pouring an alcoholic solvent into the packed column, and then stabilize it by flowing a sufficient amount of the developing solvent (column volume 5 times or more). The diluted extract is injected into a column to receive an active fraction. The eluted active substance is concentrated under reduced pressure at a temperature below 40 degrees, suspended with water to remove residual organic solvent, and then lyophilized.

In addition, the dried herbal medicine is extracted with a bath with purified water containing 5 to 50 times 3% magnesium oxide by weight and then the precipitate is removed by centrifugation and filtration. The precipitate is removed, and the active portion is loaded on the HP-20 column and the active portion is eluted through an alcoholic solvent. The eluted portion is concentrated under reduced pressure and lyophilized. For detailed purification, the active substance may be separated using size exclusion chromatography (LH-20) and a reversed phase column filler.

In other words, the active fraction composition obtained from fractions of longan and meat flowers has the effect of preventing and treating obesity in the Leprdb / Leprdb mouse, a model animal that causes obesity above leptin receptor, and inhibits the differentiation of fat cells, one of its important mechanisms. By observing the effect for the first time, an active composition having an anti-obesity and therapeutic effect containing the composition as an active ingredient was developed.

In addition to the active compositions according to the invention, pharmaceutically acceptable carriers or excipients may be used in the form of tablets, powders, granules, capsules, suspensions, emulsions, or parenteral or multi-dose formulations for parenteral administration. .

The effective dose of the active ingredient represented by the active fraction may vary depending on the age, physical condition, weight, etc. of the patient, but is generally administered within the range of 1 to 500 mg / kg (weight) per day. In addition, within the effective daily dosage range, it is injected once a day or divided several times a day. In addition, as a result of toxicity experiments on the active ingredient, healthy rats and mice were administered to the active ingredient up to 500mg / weight Kg and observed for more than a week, no abnormality was found no toxicity was confirmed.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the Examples.

1 to conduct: Dragon meat ( Longanae arillus) , Buckthorn ( Calystegia japonica) Of active fractions from

The dried and pulverized longan meat and buckwheat were added with 5 to 50 times the amount of methanol, and the mixture was extracted under reflux for more than 24 hours so that the active substance could be eluted, and the methanol extract was concentrated under a reduced pressure of 40 degrees or less. The concentrated extract is completely dissolved in an organic solvent, and then part of the total weight is converted. Silica resin (1.5-10 times the sample weight) was added to the dissolved sample solution and concentrated under reduced pressure. At least 1.5 times of the silica gel compared to the amount used for adsorption is sufficiently wetted with methanol and then filled in a column. After filling, allow sufficient methanol to flow. On the packed column is filled with silica gel adsorbed so that no bubbles enter. Thereafter, more than five times as much methanol as the column volume is flowed out so that the active substance is eluted. Methanol was used as the developing solvent primarily to see the compatibility of the active material with silica columns, but in the next step, the active material appeared after removing impurities in the condition that the active material was not eluted with a lower polarity organic solvent. It can be eluted with a solvent of. Polar low organic solvents include nucleic acids, chloroform, methyl chloride, ethyl acetate, butanol, acetone, tetrahydrofuran, benzene, ethanol, acetonitrile, isopropyl alcohol and the like.

The active fraction obtained above was concentrated under reduced pressure at a temperature of 40 ° C. or lower, and then, in order to remove a small amount of solvent that was not removed from the solvent used in the extraction and concentration process, a small amount of distilled water was added to the active fraction content, and then suspended homogeneously, followed by freeze drying. To obtain an active fraction in powder form.

Example 2 obese model animal Leprdb / Leprdb Obesity Prevention and Treatment Test in Mice

Leprdb / Leprdb mice have a dietary deficiency due to lack of leptin receptors, resulting in a continual overdose of food. As a result, the fat is excessively accumulated in the body, which causes about 3 months after birth to maintain about 50g, which is twice the weight of a typical mouse. Therefore, we investigated 10 adult Leprdb / Leprdb mice weighing 50 g in each group for the purpose of prevention and treatment of obesity. 10 aliquots per experimental group were diluted with 1% DMSO (dimethyl sulfoxide) in active fractions extracted from longan meat and buckwheat, and then intraperitoneally for 10 days orally at a constant time for 15 days at 50 mg / kg concentration. Five days of administration, the control group was administered the same amount of only 1% DMSO. In the oral administration test, the body weight of the control group was increased by 5.5% (49.6 ± 1.3 → 52.3 ± 1.3) than the beginning of the experiment. ± 0.9 → 43.3 ± 0.6), and 16.2% (43.4 ± 0.4 → 36.4 ± 2.3) of the flowers, all of the experimental groups showed a significantly lower weight (p <0.01), indicating that obesity was prevented and treated [ 4 to 6].

Example 3 Measurement of Adipocyte Differentiation Inhibitory Activity

Adipocytes 3T3-L1 and F442A cells were cultured in DMEM containing 10% bovine calf serum. If the cell density is about 90% when culturing the adipose progenitor cells, 3T3-L1 induces adipocyte differentiation by treating Dexamethasone, IBMX, insulin, etc. for 48-55 hours, and then fetal calf every two days. Replace with a medium containing serum and insulin. In the case of F442A cells, when the progenitor cells have a density of about 90% in cell culture, the cells are replaced with a culture solution containing 10% fetal calf serum and insulin, and the cell culture fluid is changed every two days to induce differentiation of fat cells. do. Determination of adipocyte differentiation inhibitory activity is compared to the control by treating the active fractions extracted and isolated from yongan meat and broccoli at a concentration of 10 ㎍ / ml from the initial stage of induction of adipocyte differentiation. It takes about 12 to 15 days for more than 90% of the cells to differentiate into adipocytes, and the activity of each fraction was treated until the same time as the control group to observe its efficacy and to take a photomicrograph (Figs. 1 to 3). ).

Table 1. Adipocyte differentiation inhibitory activity when 10 ug / ml concentrations of active fractions of long meat and broccoli were treated (10 days).

Sample Name Differentiation inhibition rate (%) Dragon meat 50.0 convolvulus 54.5

* Differentiation degree measured at 10 days after active fraction treatment

In the control group, it took about 11 days for 80-90% of the F442A cells to differentiate into adipocytes, whereas the active fractions isolated from longan meat and buckwheat were treated with 10 ug / ml concentration from the early stage of differentiation. At the same time, only 45-50% or less of the cells differentiated into adipocytes.

Example 4: Preparation of Tablets

10 g of active ingredient

Lactose 70g

15 g of crystalline cellulose

Magnesium Stearate 5g

100 g

The tablets were prepared by direct tableting method after mixing the ingredients listed above finely. The total amount of each tablet is 500 mg, of which the active ingredient content is 50 mg.

Example 5 Preparation of Powder

10 g of active ingredient

50 g of corn starch

Carboxy Cellulose 40g

100 g

A powder was prepared by crushing and mixing the ingredients listed above. In addition, 500 mg of powder was put in a 5 times hard capsule to prepare a capsule.

As described above, the active fraction composition obtained from the longan meat, buckwheat flower according to the present invention can be used as a herbal medicine containing it as an active ingredient because it is excellent in the prevention and treatment of obesity.

Claims (21)

Composition for the prevention or treatment of obesity, containing therapeutically or prophylactically effective amount, based on one or more active fractions selected from the group consisting of long-term meat and broccoli The composition of claim 1, wherein the composition comprises at least two active fractions. According to claim 2, wherein the composition is characterized in that it comprises all the active fractions of longan meat, buckwheat flower The composition for preventing or treating obesity according to any one of claims 1 to 3, wherein the composition has an adipocyte inhibitory activity. 4. A composition according to any one of claims 1 to 3, wherein said composition comprises one or more pharmaceutically acceptable carriers or excipients. The composition for preventing or treating obesity according to claim 5, wherein the content of the active ingredient is administered in the range of 1 to 500 mg / day per 1 Kg of adult body weight. Formulation for the prevention and treatment of obesity comprising the composition of any one of claims 1 to 3. 8. A formulation according to claim 7, wherein the formulation comprises one or more pharmaceutically acceptable carriers or excipients. The preparation according to claim 7 or 8, wherein the preparation is a tablet, powder, hard or soft capsule, suspension, injectable preparation, emulsion, unit dosage form or parenteral administration for parenteral administration. The composition for preventing or treating obesity according to any one of claims 1 to 3, wherein the composition further comprises a pharmaceutically acceptable excipient in the form of an edible beverage or food. A) extracting water or an organic solvent from any one selected from the group consisting of long-term meat and a broccoli to obtain a crude extract, b) filtering the crude extract and then concentrating (decompression) and optionally removing the solvent. Method for producing a composition for preventing or treating obesity comprising the step of 12. The method of claim 11, wherein the step of obtaining the crude extract by extracting water or organic solvent is performed by reflux extraction in a temperature range of 25 to 100 degrees. The method according to claim 11, wherein the solvent used for extraction is purified water, alcohols having 1 to 5 carbon atoms, or an organic solvent. The method of claim 13, wherein the alcoholic solvent is ethanol, methanol, propanol, butanol, an ethanol aqueous solution, or an aqueous methanol solution. The method of claim 11, wherein the herbal medicine is a manufacturing method characterized in that the dry medicine or herbal medicine The method of claim 13, wherein the organic solvent is ethyl acetate, chloroform, or hexane. The method of claim 11, wherein the step of obtaining the crude extract is characterized in that using a silica gel adsorption column. The composition for preventing or treating obesity, characterized in that it comprises a long-term meat, buckwheat extract or a mixture thereof prepared as claimed in claim 11 as an active ingredient. 19. The composition according to claim 18, wherein the extract is a fraction adsorbed or not adsorbed on the silica gel adsorption column. An active fraction prepared by the process according to any one of claims 11 to 17. 21. The herbal fraction according to claim 20, wherein the herbal fraction is derived from any one selected from longan meat and buckwheat.