KR20050082446A - Obesity treatment with tetrahydroberberine - Google Patents

Abstract

The present invention relates to a tetrahydroxyberberine alkaloid compound having the activity of inhibiting the differentiation of adipocytes (3T3-L1 and F442A cell). More specifically, the present invention relates to a tetrahydroberberine alkaloid compound, which is an active substance that inhibits the differentiation of adipocytes (3T3-L1 and F442A cells) and inhibits the accumulation of fat metabolites that cause obesity.

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

Description

Obesity prevention and treatment composition containing tetrahydro berberine as an active ingredient {Obesity treatment with tetrahydroberberine}

The present invention relates to an obesity prevention and treatment composition containing tetrahydroberberine as an active ingredient, and more particularly, tetrahydroberberine (tetrahydroberberine) represented by the following Chemical Formula 1 showing excellent activity in inhibiting the differentiation of adipocytes as an active ingredient. It relates to a composition for preventing and treating obesity.

Formula 1

The present invention relates to a tetrahydroxyberberine alkaloid compound having the activity of inhibiting the differentiation of adipocytes (3T3-L1 and F442A cells). More specifically, the present invention relates to tetrahydroberberine, an active substance that inhibits the differentiation of adipocytes (3T3-L1 and F442A cells) and inhibits the accumulation of fat metabolites that cause obesity.

The compound according to the present invention exhibits an obesity prevention and therapeutic effect by inhibiting 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 change in diet of the modern man is showing a tendency to increase the fast obesity 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, resulting in various diseases such as hypertension, diabetes, hyperlipidemia and coronary artery disease, as well as breast cancer, uterine cancer and colon cancer. 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 serotonin (5HT) nervous system according to each mechanism, 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 advent of differentiation cell has been searched for less obesity and 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, adipocytes not only increase in number but also form a large amount of triglycerides by differentiation of excessive adipocytes, which are accompanied by morphological changes including the increase of adipocyte size 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 adipocytes is generally controlled by diet, but the process of differentiating new adipocytes 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-β (C / EBPβ) and peroxisome proliferator-activated receptor An increase in transcription factors such as (PPARr) is observed. These transcription factors, together with adipocyte regulators, promote the differentiation of adipocytes and increase the expression levels of enzymes such as Glycerol-3-phosphate dehydrogenase (GPDH) and fatty acid synthase.

Therefore, based on the idea of regulating the number of adipocytes produced by inhibiting the differentiation of adipocytes and controlling the excess energy accumulated accordingly, researches are actively conducted to search for substances that inhibit adipocyte differentiation. .

Therefore, the present inventors treated the isoquinoline-based tetrahydroxyberberine alkaloid compound in DIO mice, a model animal causing obesity due to the High Fat Diet, and observed the effects of obesity prevention and treatment including weight loss. Therefore, it was confirmed that the tetrahydroberberine alkaloid compound has an obesity prevention and therapeutic effect and completed the present invention.

Still another object of the present invention is to inhibit the differentiation of 3T3-L1 and F442A cells which are tetrahydroberberine alkaloid compounds.

The present invention relates to a composition for the prevention and treatment of obesity comprising tetrahydroberberine represented by the following formula (1) and a pharmaceutically acceptable carrier.

Formula 1

The present invention will be described in more detail as follows.

The present invention relates to a tetrahydroberberine alkaloid showing an obesity prevention and treatment effect in DIO mice, which is a model animal causing obesity due to an inhibitor of differentiation of fat cells and a high fat diet.

Tetrahydroberberine (tetrahydroberberine) used in the present invention is a reduction reaction by a general hydrogenation (hydrogenation) or hydrogen anion (hydride) using berberine and its derivatives or dihydroberberine and its derivatives extracted or synthesized in nature as a raw material It was prepared through. This is described as a chemical formula.

That is, the tetrahydroberberine alkaloid compound inhibits adipocyte differentiation and observes for the first time that it has an anti-obesity and therapeutic effect in DIO mice, a model animal causing obesity due to a high fat diet, and contains the composition as an active ingredient. It has been developed an active composition having the effect of preventing and treating obesity.

In addition to the active compositions according to the invention, pharmaceutically acceptable carriers or excipients may be used in the form of unit or multiple dosage forms for tablets, powders, granules, capsules, ointments, suspensions, emulsions or parenteral administration. Can be.

The effective dose of the active ingredient represented by the active substance can be varied depending on the age, physical condition, weight, etc. of the patient, but in general, 0.1 to 1,000 mg / kg (weight) / day range, preferably 0.1 to 50 It is administered within mg / kg (weight) / day range. In addition, within a daily effective dosage range is divided into once a day or several times a day.

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

Example 1 Tetrahydroberberine (tetrahydroberberine)

3.72 g of berberine chloride was dissolved in 50 ml of methanol, and 540 mg of NaBH ₄ was added dropwise while stirring vigorously, followed by further stirring at room temperature for 1 hour. Dilute hydrochloric acid is added little by little to the reaction solution to completely decompose the remaining NaBH ₄ without reaction, and then neutralize it by treating with saturated aqueous NaHCO ₃ solution. The reaction solution thus treated was distilled under reduced pressure, concentrated thoroughly, and then chromatographed on silica gel to obtain 3.63 g of tetrahydroberberine.

NMR (CDCl ₃ ): 6.87 (d, 1H), 6.79 (d, 1H), 6.74 (s, 1H), 6.60 (s, 1H), 5.93 (s. 2H), 4.24 (d, 1H), 3.86 ( s, 3H), 3.85 (s, 3H), 3.54 (d, 2H), 3.12-3.26 (m, 3H), 2.82 (m, 1H), 2.64 (m, 2H)

Example 2 Physicochemical Properties of Tetrahydroberberine

Appearance: Yellow powder

Molecular Formula: C20H21NO4

Molecular weight: 339.3947

Composition: C70.78% H6.24% N4.13% O18.86%

Soluble: Methanol, DMSO

Insoluble: Water

Example 3 Measurement of Adipocyte Differentiation Inhibitory Activity

    Adipose precursor cells 3T3-L1 and F442A cells were cultured in DMEM containing 10% bovine calf serum. When the fat precursor cells are about 90% densified in cell culture, 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, they 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. . Determination of adipocyte differentiation inhibitory activity is compared with the control by treating the tetrahydroberberine alkaloid compound at a concentration of 5μg / 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 the experimental group was treated until the same time as the control group to observe its efficacy and by photographing micrographs.

Figure 1 shows the comparison of adipocyte differentiation ability between the control and tetrahydroberberine alkaloid compounds according to the induction period of adipocyte differentiation. In the control group, it took about 11 days for 80-90% of F442A cells to differentiate into adipocytes, whereas berberine, a tetrahydroberberine alkaloid compound, was treated at the same time when treated at a concentration of 5 ug / ml from the early stage of differentiation. Only 5-10% or less cells differentiated into adipocytes.

Example 4 Cytotoxicity Measurement

Adipocytes 3T3-L1 and F442A cells were cultured in DMEM containing 10% bovine calf serum. When the cell density is about 85-90%, add the solution for measuring cytotoxicity to measure the degree of cell activity. As shown in FIG. 3, tetrahydroberberine was treated with the adipocyte line, and no cytotoxicity was observed.

Example 5 Obesity Prevention and Therapeutic Effect Assay in DIO Mice

DIO mice were fed a diet diet D12451 45% kcal from C57BL / 6 male animals at 4 weeks of age. As a result, fat accumulates excessively in the body, which causes the body to maintain more than 31 to 32 g, which is twice the weight of a normal mouse at about 3 months after birth. Therefore, in order to investigate the effect of preventing and treating obesity, 16 three-month-old DIO mice weighing 31-32 g or more were included. Eight experimental groups were diluted tetrahydroberberine alkaloid compound in DW and administered orally at a constant time (11:00 am) for 4 weeks at a concentration of 50 mg / kg once a day for 4 weeks, the same amount in the eight control groups Only DW was administered. After 4 weeks of administration, the body weight of the test group and the control group was measured and analyzed, and compared to the control body weight (33.2 ± 1.7 g), the weight of the experimental group to which the sample was administered was significantly lower (p <0.01) (p <0.01). 28.6 ± 0.9 g) was observed to prevent and treat obesity.

Example 6: Preparation of Tablets

600 g of tetrahydroberberine

Arginine 240 g

140 g of crystalline cellulose

10 g of magnesium stearate

10 g of hydroxypropyl methyl cellulose

Example 7 Preparation of Hard Capsules

10 g of active ingredients

50 g of corn starch

40 g of carboxy cellulose

Total amount 100 g

A powder was prepared by crushing and mixing the ingredients listed above. 100 mg of powder was put into 7 hard capsules to prepare a capsule.

As described above, the tetrahydro-berberine alkaloid compound according to the present invention can be used as an inhibitor containing fat cell differentiation inhibition, obesity prevention and therapeutic effect because it is excellent.

1 is a structure of a tetrahydroberberine alkaloid compound.

Figure 2 is a photograph showing the difference that the differentiation of adipocytes is inhibited by treating the tetrahydroberberine-based alkaloid compound to 3T3-L1 cell line which is adipocytes.

3 is a graph showing that there is no cytotoxicity by treating a tetrahydroberberine-based alkaloid compound to a 3T3-L1 cell line which is an adipocyte.

Figure 4 is a graph comparing the difference between the control group and the weight loss (obesity prevention and treatment) after administration of tetrahydroberberine-based alkaloid compounds to obese model animals for 4 weeks to mature DIO (Diet Induced Obesity) mice.

Claims (10)

A composition for preventing or treating obesity, characterized in that it contains a tetrahydroxyberberine compound of Formula 1 as an active ingredient Formula 1) The composition of claim 1, wherein the composition has adipocyte differentiation inhibitory activity. The composition of claim 2, wherein the adipocytes are 3T3-L1 and F442A cells. The composition of claim 1, wherein the active ingredient Tetrahydroxyberberine is derived from a herbal medicine. The composition of any one of claims 1 to 4, further comprising a pharmaceutically acceptable carrier or excipient. The method for preventing or treating obesity according to any one of claims 1 to 4, wherein the composition further comprises tetrahydroxyberberine as an active ingredient and optionally further comprises one or more pharmaceutically acceptable carriers or excipients. Formulation. 7. The method for preventing or treating obesity according to claim 6, wherein the preparation is a tablet, powder, hard or soft capsule, suspending agent, injectable preparation, emulsion, unit dosage form or parenteral dosage form for parenteral administration. Formulation. 8. The composition for preventing or treating obesity according to claim 7, wherein the content of the active ingredient is administered in the range of 0.1 to 1000 mg / day per 1 kg of adult body weight. 6. The composition for preventing or treating obesity according to claim 5, comprising a pharmaceutically acceptable excipient in the form of an edible drink or food. Tetrahydroberberine comprising a step of purifying or purifying a natural berberine, a derivative or dihydroberberine, or a derivative thereof, by a hydrogenation reaction or a hydrogen anion according to Scheme 1 below. Manufacturing method Scheme 1)