KR20040100406A - Active fraction having enhancive effects on adipocytes (NIH3T3-L1 cell) isolated from medicinal plants - Google Patents

Abstract

PURPOSE: Provided is an active fraction having enhancive effects on adipocytes(NIH3T3-L1 cell) isolated from medicinal plants. The active fraction has reducing effect on the body weight and thus inhibits obesity. CONSTITUTION: The composition for prevention and treatment of obesity in the Leprdb/Leprdb mouse is characterized by containing a prophylactically or therapeutically effective amount of the active fraction of Carpesil fructus as a main ingredient. It characteristically inhibits the differentiation of adipocytes, and further contains pharmaceutically acceptable carrier or excipient.

Description

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

The present invention relates to an active fraction composition for promoting the differentiation of adipocytes obtained from herbal extract fractions, and more particularly, to promote the differentiation of NIH3T3-L1 cells, which are adipocytes, from the extracts of Hakseul traditionally used for therapeutic purposes in the Orient. The present invention relates to an active fraction composition capable of inhibiting fat accumulation, a method for efficiently extracting and purifying the same, and an herbal medicine for preventing and treating obesity containing the extract as an active ingredient.

The compound according to the present invention exhibits an obesity prevention and therapeutic effect by promoting adipocyte differentiation.

Recently, due to the improvement of living standard according to the economic development, the hygiene environment is improved, and the frequent food intake and the meat-based eating habits cause excessive calorie intake. However, the changes in the diet of the modern man is showing a tendency of rapid increase in the obese population because the calorie consumption is low due to lack of exercise lacking. Obesity is now fatal as it is reported to cause not only cosmetic problems but also obesity, causing various diseases such as hypertension, diabetes, hyperlipidemia and coronary artery disease, as well as adult diseases such as breast cancer, uterine cancer and colon cancer. Is treated as one of the diseases [ J. Biol. Chem ., 273, 32487-32490 (1998); Nature, 404, 652-660 (2000).

Current treatments for treating obesity can be divided into drugs that affect the central nervous system and affect appetite and drugs that inhibit absorption by acting on the gastrointestinal tract. Drugs that act on the central nervous system include drugs such as fenfluramine and dexfenfluramine that inhibit the serotonin (5HT) nervous system according to their respective mechanisms, drugs such as ephedrine and caffeine through the noradrenaline nervous system, and recently serotonin and noradrenaline nervous system. Drugs such as sibutramine that act and inhibit obesity 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 ( PPAR) Gamma and other transcription factors are observed to increase. 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).

In the case of Hak-Seul, which is used in the past, it is used in the composition of caries prevention material having excellent caries prevention effect, and also in the composition of the hair growth promoting composition, and it is also used in the composition of whitening cosmetics containing the extract of Hak-Seul. It is also known to be used in the manufacture of herbal extract fertilizers for plant disease control.

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.

Therefore, the present inventors explored the action of adipocyte differentiation of adipocytes, 3T3-L1 and F442A, to the drugs that have been used in traditional Korean medicine, including synonymous gamma, and model animals causing obesity above leptin receptor. As a result of observing obesity prevention and treatment effect in Leprdb / Leprdb mice, the present invention was completed by obtaining an active fraction composition showing both specific adipocyte-promoting activity and weight loss effect in animal experiments.

Accordingly, an object of the present invention is to provide a method for extracting Hakseul extract, which is a drug having a pronounced effect in promoting fat cell differentiation and preventing obesity and treatment, and a herbal medicine containing the extract as an active ingredient. .

Figure 1 is a photograph showing the difference that the differentiation of adipocytes is promoted by treating the active fraction extracted from the carcass ( Carpesil fructus ) to the NIH3T3-L1 cell line as adipose cells.

FIG. 2 is a precipitate obtained by concentrating an extract extracted from Carpesil fructus with 70% ethanol and then suspending it in distilled water and adjusting the pH to 10, for 15 days (oral 10 days) in an obese model animal Leprdb / Leprdb mice. , 5 days to the abdominal cavity is a graph comparing the difference between the experimental group and the control group inhibited weight gain (obesity prevention and treatment effect).

Figure 3 is an active fraction obtained by adsorbing HP-20 extract extracted with 70% ethanol from Hakseul ( Carpesil fructus ) was administered to the abdominal cavity model Leprdb / Leprdb mice for 15 days intraperitoneal weight inhibition (prevention and treatment of obesity) Effect) It is a graph comparing the difference between the experimental group and the control group.

Figure 4 is an extract of 70% ethanol from Carpesil fructus adsorbed to HP-20, and the active fraction separated orally administered to Leprdb / Leprdb mice, obese model animals for 30 days to inhibit weight gain (obesity prevention and treatment effect ) Is a graph comparing the difference between the experimental group and the control group.

The present invention is characterized by an active fraction composition having both anti-obesity and therapeutic effects in Leprdb / Leprdb mice, which are model animals for promoting obesity and differentiation of adipocytes obtained from the crane.

In the present invention, at least room temperature is extracted for at least 24 hours in a state in which reflux is carried out by putting the organic solvent and water of 5 to 50 times the weight at room temperature after drying the crane. 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 is adsorbed to the filler, and then recovered using an organic solvent, and then 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 extract 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 medicines were extracted with a water bath with purified water containing 5-50 times 3% magnesium oxide by weight and then the precipitates were 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 freeze-dried. 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 by fractionation from the school chain has the effect of preventing and treating obesity in the Leprdb / Leprdb mouse, a model animal that causes obesity above the leptin receptor, and has an effect of promoting the differentiation of fat cells, one of its important mechanisms. By observing for the first time to have an active composition, an active composition having an obesity prevention 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 a daily effective dosage range is divided into once a day or several times a day. In addition, as a result of the toxicity test for 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, it was confirmed that there is no toxicity because no abnormal symptoms can be found.

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

Example 1 crane Carpesil fructus Of active fractions from

5 to 50 times the amount of methanol was dried and pulverized, and the mixture was extracted under reflux for more than 24 hours to elute the active substance, and the methanol extract was concentrated under reduced pressure at a temperature 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 to 10 times the weight of the sample) 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: crane Carpesil fructus From active fractions

The pulverized hazelnut is refluxed for 24 hours to allow the active substance to elute using 5-50 times the amount of 70% ethanol, and then purified water is added to the 70% ethanol extract and diluted with 50% ethanol.

HP-20 resin (5 to 10 times the weight of the extract sample) is sufficiently wetted with ethanol and filled in the next column. After ethanol is poured into the packed column to wash the resin, 50% ethanol (column volume 5 times or more) is sufficiently poured to replace the solvent in the column with 50% ethanol. After injecting the extract to the column 50% ethanol is sufficiently flowed. The active fraction is then eluted with 100% ethanol. After concentration of the active fraction obtained under reduced pressure, a small amount of distilled water was added to the active fraction content in order to remove a trace amount of solvent which was not removed in the solvent used during the extraction and concentration process. Got.

Example 3: crane Carpesil fructus From active fractions

The pulverized crane is extracted at reflux for 24 hours using 5 to 50 times the amount of 70% ethanol so that the active substance can be eluted. The 70% ethanol extract was concentrated under reduced pressure with water. Adjust the pH to 10 using 1M NaOH in the extract suspension suspended in purified water. The active substance which is not dissolved in aqueous pH 10 solution is recovered by centrifugation.

After concentration of the active fraction obtained under reduced pressure, a small amount of distilled water was added to the active fraction content in order to remove a trace amount of solvent which was not removed in the solvent used during the extraction and concentration process. Got

Example 4: Obesity animal model of obesity prevention and treatment in Leprdb / Leprdb Mouse Black

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, in order to prevent obesity and to treat treatment, 10 adult Leprdb / Leprdb mice weighing about 50 g were included in each group. 10 aliquots per experimental group were diluted in 1% DMSO (dimethyl sulfoxide), and the active fractions extracted from Hakseol were sampled in Example 3 at a constant time for 15 days at a concentration of 50 mg / kg each for 15 days. After 5 days of intraperitoneal administration, the sample of Example 2 was administered intraperitoneally 15 days, orally 30 days, and in the case of 10 control groups, only the same amount of 1% DMSO was administered. After 15 days of administration, the weights of the test group and the control group were analyzed, and in the case of the intraperitoneal administration test, the weight of the control group was increased by 5.5% (49.6 ± 1.3 → 52.3 ± 1.3) than at the beginning of the experiment, but the school chain was 4.5% ( 49.4 ± 0.1 → 47.2 ± 1.3), and in the oral and post-abdominal test, the weight of the control group was increased by 10.9% (47 ± 1.0 → 52.1 ± 0.8) than at the beginning of the experiment, but the agglomeration was 7.9% (56.5 ± 0.4 → 52 ± 0.3). In the oral administration test, the body weight of the control group was increased by 7.2% (55.4 ± 0.8 → 59.4 ± 0.5) than at the beginning of the experiment, but the school group was 4.3% (57.8 ± 0.3 → 55.3 ± 0.5). p <0.01) the weight was lowered to show that obesity is prevented and treated [Fig. 2-4].

Example 5 Measurement of Adipocyte Differentiation Promoting 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. In the measurement of adipocyte differentiation promoting activity, the active fractions extracted and separated from the Hakse were treated at a concentration of 10 ㎍ / ml from the initial stage of induction of adipocyte differentiation and compared with the control group. 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 by microscopic photographing.

Table 1. Adipocyte differentiation promoting activity (10 days) when the active fraction of Hakseul was treated at a concentration of 10 ug / ml.

Sample Name Differentiation promotion rate (%) School 58.3

* Measurement of differentiation degree at 10 days after active fraction treatment

In the control group, it took about 11 days for 60% of the F442A cells to differentiate into adipocytes, whereas the active fractions isolated from the cranes were treated at the same time when treated with a concentration of 10 ug / ml from the initial stage of differentiation. Differentiated into adipocytes by%.

Example 6: 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 100 mg, of which the active ingredient content is 10 mg.

Example 7 Preparation of Powder

10 g of active ingredient

Corn Starch 50g

Carboxy Cellulose 40g

100 g

A powder was prepared by crushing and mixing the ingredients listed above. In addition, 100 mg of powder was added to the 6 times hard capsule to prepare a capsule.

As described above, the active fraction composition obtained from the crane according to the present invention is not known the exact mechanism of action, but obesity in Leprdb / Leprdb mice, a model animal that promotes differentiation of adipocytes and at the same time causes obesity above leptin receptor Because of its excellent preventive and therapeutic effects, it can be used as a herbal medicine containing it as an active ingredient.

Claims (18)

Composition for the prevention or treatment of obesity, containing the active fraction of Carpesil fructus as a main ingredient in a therapeutically or prophylactically effective amount According to claim 1, wherein the composition is a composition for preventing or treating obesity, characterized in that it has a fat cell differentiation inhibitory activity The composition of claim 1, wherein the composition comprises one or more pharmaceutically acceptable carriers or excipients. The composition for preventing or treating obesity according to claim 3, 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. Preparation for the prevention and treatment of obesity comprising the composition of claim 1 The formulation of claim 5, wherein the formulation comprises one or more pharmaceutically acceptable carriers or excipients. The preparation according to claim 5 or 6, wherein the preparation is a tablet, powder, hard or soft capsule, suspension, injectable preparation, emulsion, unit dosage form for parenteral administration or multiple dosage form. The composition for preventing or treating obesity according to claim 1, wherein the composition further comprises a pharmaceutically acceptable excipient in the form of an edible beverage or food. A) extracting water or organic solvent from Carpesil fructus to obtain a crude extract, b) filtering the crude extract and then (decompressing) and c) optionally, removing the solvent. Or a method of preparing the therapeutic composition 10. The method of claim 9, 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. 10. The method according to claim 9, wherein the solvent used for extraction is purified water, alcohols having 1 to 5 carbon atoms, or organic solvents. The method of claim 11, wherein the alcoholic solvent is ethanol, methanol, propanol, butanol, an ethanol aqueous solution, or an aqueous methanol solution. 10. The method according to claim 9, wherein the herbal medicine is a dry medicine or herbal medicine The method of claim 11, 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 the silica gel adsorption column and / or HP-20 column is used. Composition for the prevention or treatment of obesity, characterized in that it contains the Hakseul ( Carpesil fructus ) extract prepared by claim 9 as an active ingredient 17. The composition according to claim 16, wherein the extract is a fraction adsorbed or not adsorbed on a silica gel adsorption column and / or an HP-20 column. An obesity prophylactic or therapeutically active fraction prepared by the method of any one of claims 9 to 15 and derived from Carpesil fructus .