KR100390766B1 - Active fraction having inhibitory effects on fatty acid synthase isolated from piper longum - Google Patents

Abstract

The present invention relates to an active fraction composition for inhibiting fatty acid biosynthetic enzymes obtained from essential hairs. More specifically, the present invention relates to fats that cause obesity by inhibiting fatty acid biosynthetic enzymes (FAS) from piper longum . The present invention relates to an active fraction composition capable of inhibiting the accumulation of the same, a method for efficiently extracting and purifying the same, and an obesity prevention and treatment containing the extract as an active ingredient, a herbal medicine for inhibiting blood sugar and fatty liver.

Description

Active fraction having inhibitory effects on fatty acid synthase isolated from piper longum}

The present invention relates to an active fraction composition for inhibiting fatty acid biosynthetic enzymes obtained from essential hairs. More specifically, the present invention relates to fats that cause obesity by inhibiting fatty acid biosynthetic enzymes (FAS) from piper longum . The present invention relates to an active fraction composition capable of inhibiting the accumulation of the same, a method for efficiently extracting and purifying the same, and an obesity prevention and treatment containing the extract as an active ingredient, a herbal medicine for inhibiting blood sugar and fatty liver.

Recently, due to the improvement of living standards according to economic development, the hygiene environment is improved and the intake of calories is rapidly increased due to the improvement of diet. However, while calorie intake increases with food, the amount of calories consumed due to lack of exercise, etc. tends to increase obesity. Obesity not only harms young people's skin-like beauty, but also keeps obesity alive and can lead to a variety of diseases such as hypertension, diabetes, hyperlipidemia and coronary artery disease, as well as breast cancer, uterine cancer and colon cancer. It is reported to cause it and is now treated as a fatal disease [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, orlistat, which is approved as an obesity drug by inhibiting pancreatic lipase and reducing fat absorption by acting on the gastrointestinal tract and inhibiting obesity, is used as a representative drug. However, drugs such as fenfluramine, which have been used previously, have been banned because of side effects such as primary pulmonary hypertension or heart valve lesions. Other drugs also have problems such as blood pressure reduction or lactic acidosis, which lead to heart failure and kidney disease. There is a problem that the patient can not use.

Therefore, in search of improved methods for the treatment of obesity, we searched for fatty acid biosynthetic enzyme inhibitors, new drugs that are closely related to the fat biosynthesis process and do not act on the nervous system.

Fatty acid biosynthetic enzymes are one of the enzymes initially expressed in the differentiation of adipocytes [J. Biol. Chem., 255, 4745-4750 (1980)] Palmitate is produced from acetyl-CoA and malonyl-CoA. The calories that are not consumed due to excessive food intake are stored in cells in the form of triglycerides, which are storage lipids, by the action of fatty acid biosynthetic enzymes, causing obesity. On the other hand, in the case of type 2 diabetes in which obesity progresses to diabetes, it is known that palmitate, a triglyceride, gradually progresses to diabetes by destroying β cells of the pancreas [Proc. Natl. Acad. Sci. 95, 2498 to 2502 (1998), reports that triglyceride accumulation is involved in the progression of fatty liver [J. Clin. Invest., 98, 1575-1584 (1996).

Cerulenin derivatives, recently known as selective fatty acid biosynthetic inhibitors,Lepr ^ob / Lepr ^ob Specific weight loss effects have been observed in mice and reported to have no side effects [Science, 288, 2379-2381 (2000)]. There are active studies to develop such new inhibitors [New Engl. J. Med., 343, 1888-1889, (2000).

In general, among the various methods for the development of drugs with new ingredients, there is a high possibility of finding new active ingredients from natural medicines used in traditional medicine rather than the effort by experimental modification of existing drugs. There is less concern about toxicity due to the drugs developed.

Therefore, the present inventors have explored fatty acid biosynthetic enzyme inhibitory actions against drugs that have been used in traditional medicine of Korea including obesity and obesity in Lepr ^db / Lepr ^db mice, which are model animals causing obesity above leptin receptor. As a result of observing prevention and treatment, hypoglycemic effect and fatty liver inhibitory effect, the present invention was completed by obtaining an active fraction composition showing specific inhibitory activity from Piper longum , which is a pepper plant.

Accordingly, the present invention provides a method of extracting the extract of Piper longum , a drug having a pronounced effect on fatty acid biosynthesis enzyme inhibition, obesity prevention and treatment, hypoglycemia and fatty liver inhibition, and the extract as an active ingredient. The purpose is to provide a herbal medicine containing.

1 is an obese model animal extracted and separated from the active extractLepr ^db / Lepr ^db This is a photograph comparing the apparent difference between the experimental group in which the weight was administered to the mouse for 4 weeks and the control group in the obese state not receiving the active substance.

FIG. 2 is a bar graph comparing the difference between the experimental group and the control group that inhibited weight gain (prevention of obesity) after administering the active fraction extracted from the essential extract to four-week-old immature Lepr ^db / Lepr ^db mice, which are obese model animals. to be.

FIG. 3 is a bar graph comparing the difference between the experimental group and the control group of which weight was reduced after administration of the active fraction extracted from the shot for 4 weeks to an obese model animal, 3 months old mature Lepr ^db / Lepr ^db mice.

FIG. 4 is a bar graph comparing the difference between the experimental group and the control group in which the blood glucose level was lowered after administration of the active fraction extracted from the shot for 4 weeks to the obese model animal, 3 months old mature Lepr ^db / Lepr ^db mice.

Figure 5 is a micrograph comparing the liver tissue of the experimental group and the control group, which recovered almost normally after the administration of the active fraction extracted from the pilates for 4 weeks to the obese model animal, 3 months old mature Lepr ^db / Lepr ^db mice. to be.

The present invention is characterized by an active fraction composition for inhibiting fatty acid biosynthetic enzymes obtained from the essential.

In the present invention, in the preparation of herbal medicines by extraction with alcoholic aqueous solution,

(1) pulverizing the piper longum and extracting the solvent three times by adding 3-10 times the aqueous alcohol solution to the content of the hairbrush;

(2) After the filtrate obtained in the extraction step was concentrated by evaporation, the concentrated solution was suspended in distilled water of 50 to 100-fold volume and passed through Diaion HP-20 resin to be adsorbed. Elution removal using;

(3) eluting the adsorbed active fraction using 10 to 20 times the amount of acetone, and then concentrating to obtain an extract;

(4) suspending the extract with distilled water 10 to 100 times the total amount of the extract, extracting and concentrating the active substance with an organic solvent such as chloroform and ethyl acetate to obtain an extract;

(5) a method for separating and purifying the active fraction composition for inhibiting fatty acid biosynthetic enzymes from the infiltration comprising the step of adding the distilled water 1 to 2 times the total amount of the extract to homogeneously suspend and then freeze drying to separate the active fraction. It is another feature.

In addition, the present invention includes the use of the isolated extract according to the extraction and purification method as an active ingredient as an anti-obesity agent, therapeutic agent, hypoglycemic agent and fatty liver inhibitor.

Referring to the present invention in more detail as follows.

The present invention relates to an essential active fraction composition showing obesity prevention and treatment, hypoglycemia and fatty liver inhibition in Lepr ^db / Lepr ^db mice, which are model animals that cause obesity by fatty acid biosynthetic enzyme inhibition and leptin receptor abnormalities. .

In other words, the active fraction composition obtained by fractionation from Piper longum inhibits fatty acid biosynthesis enzymes and prevents obesity and lowers blood sugar levels in Lepr ^db / Lepr ^db mice, a model animal that causes obesity above leptin receptor. And it was observed for the first time to have a fatty liver inhibitory effect, to develop an active composition having the effect of preventing and treating obesity, hypoglycemic and fatty liver containing the composition as an active ingredient.

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 100 mg / kg (weight) per day. 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 Examples.

Example 1 Preparation of Active Fractions from Primary

The pulverized piper longum was extracted under reflux for 5 hours so that the active substance was eluted using 10 times the amount of alcoholic solvent, and the alcoholic solvent extract was concentrated under reduced pressure. The active concentrate was then suspended in 50-fold distilled water and passed through Diion HP-20 resin to adsorb the active material onto the resin, followed by elution of the inactive fraction using 30% methanol as the developing solvent. In order to elute the active substance, 10 times the amount of acetone was passed through the diion HP-20 as a developing solvent, and the eluate was concentrated under reduced pressure. Thereafter, the concentrated active fraction was suspended in 50 times of distilled water and solvent fractionated three times with the same amount of an organic solvent such as ethyl acetate or chloroform to obtain a crude extract containing the active substance. In order to remove the trace amount of the solvent which was not removed in the solvent used in the crude extract process obtained above, distilled water of 1 times the total amount of the crude extract was added to the mixture, suspended and lyophilized to separate and purify the powdered active fraction.

Example 2 Measurement of Fatty Acid Biosynthesis Enzyme Inhibitory Activity

Fatty acid biosynthetic enzymes were isolated from the liver using a method of maximizing expression of fatty acid biosynthetic enzymes in male Sprague-Dewley rats weighing 250 g.

1. Spectroscopically measuring the activity of fatty acid biosynthetic enzymes

To 1 ml of 75 mM phosphate buffer containing 3 mM acetyl-CoA, 0.2 M DTT, 2.8 mM NADPH and the assay sample, 50 μl of fatty acid biosynthetic enzyme fraction was added and allowed to stand at room temperature for 30 minutes. . Then, 3 mM malonyl-coA (malonyl-CoA) was added to the reaction solution and gently shaken. After leaving for 30 minutes, the absorbance was measured at 340 nm. Enzyme inhibition rate was calculated by the following equation (1), IC ₅₀ value was determined by the concentration of the inhibitor to the inhibition rate of the enzyme activity 50%.

A: absorbance after the reaction without the inhibitor

B: absorbance after reaction of the sample

C: absorbance after the reaction with the inhibitor

The obtained active fraction was found to be 5 μg / ml as a result of measuring a concentration (IC ₅₀ ) which inhibits the activity of the fatty acid biosynthetic enzyme by 50%. On the other hand, serel runin known as an optional fatty acid biosynthesis inhibitors was found to be 5 μ M in these experiments.

2. Methods for Measuring the Activity of Fatty Acid Biosynthesis with Radioisotopes

Spectroscopically searching for fatty acid biosynthetic enzyme inhibitory active substances, some of the tested samples may have absorbance at 340 nm, which may lead to false results. Thus, an isotope method was used for more accurate measurements. To 1 ml of 75 mM phosphate buffer containing 3 mM acetyl-CoA, 0.2 M DTT, 2.8 mM NADPH and the assay sample, 50 μl of fatty acid biosynthetic enzyme fraction was added and allowed to stand at room temperature for 30 minutes. . Again, 3 mM malonyl coei ([malonyl-2-14C] -CoA) labeled with carbon isotopically labeled with position 2 was added to the reaction solution, shaked lightly, and reacted for 30 minutes. In order to terminate the reaction, 0.5 ml of 0.5 N NaOH was added thereto, followed by saponification in boiling water for 15 minutes. Then, 0.5 ml of 1N HCl aqueous solution was added to neutralize the reaction solution, and the same amount of nucleic acid was added twice to extract and centrifuged. After centrifugation, the supernatant nucleic acid layer was taken and radioactivity was measured. Enzyme inhibition rate was calculated by the following equation (2), IC ₅₀ value was determined by the concentration of the inhibitor to the inhibition rate of the enzyme activity 50%.

A-C

% Inhibition = -------- # 12316; × 100

A-B

A: Radioactivity after reaction of the one without inhibitor

B: Radioactivity after reaction of enzyme-free solution

C: radioactivity after reaction of inhibitor

The obtained active fraction was found to be 7 [mu] g / ml as a result of measuring a concentration (IC ₅₀ ) that inhibits the activity of the fatty acid biosynthetic enzyme by 50%. On the other hand, serel runin known as an optional fatty acid biosynthesis inhibitors was found to be 5 μ M in these experiments.

Example 3 obese model animal Lepr ^db / Lepr ^db Obesity Prevention Test in Mice

Lepr ^db / Lepr ^db mice are deficient in leptin receptors, which result in an inadequate appetite, which results in excessive food intake. 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, 16 Lepr ^db / Lepr ^db mice, 4 weeks of age (weight: 22-24 g), whose body weight began to increase rapidly, were examined for the purpose of preventing obesity. Eight experimental groups were diluted with DMSO (dimethyl sulfoxide) in active fractions and administered orally and intraperitoneally at a constant time (11:00 am) for 4 weeks at a concentration of 10 mg / kg once daily for 4 weeks. In the case of horses, only the same amount of DMSO was administered. Four weeks after the start of the administration, the body weight of the test group and the control group was measured and analyzed. As compared with the body weight of the control group (34.2 ± 2.7 g), the body weight of the experimental group that received the active fraction extracted from the shot was about 28% significantly (p <0.01) It was observed that obesity was prevented by showing a low level (24.7 ± 2.2 g) [Fig. 2].

Example 4 Obese Model Animals Lepr ^db / Lepr ^db Test of Obesity Treatment in Mice

As mentioned above, Lepr ^db / Lepr ^db mice maintain about 50 g at about three months after birth, which is twice the normal mouse weight. Therefore, 16 rats of 3 months of age (weight: 47-50 g) of Lepr ^db / Lepr ^db mice whose weight was nearly increased were examined in order to investigate the effect of obesity. In eight experimental groups, the active fractions extracted from the inocula were diluted in dimethyl sulfoxide (DMSO) and administered orally and intraperitoneally at a constant time (11:00 am) for 4 weeks at a concentration of 10 mg / kg once daily for 4 weeks. In the case of horses, only the same amount of DMSO was administered. As a result of measuring body weight after 4 weeks of administration, the body weight (39.3 ± 3.3 g) of the experimental group that received the active fraction extracted from the shot compared to the body weight of the control group (49.8 ± 3.9 g) was significantly (p. 20%) (p) <0.01) it was observed that obesity was treated [FIGS. 1 and 3].

Example 5 Obese Model Animals Lepr ^db / Lepr ^db Measurement of hypoglycemic effect in mice

Lepr ^db / Lepr ^db mice have very high hyperglycemia and consequent diabetes with obesity. Therefore, in order to investigate the hypoglycemic effect of the active fractions extracted from the essentials, experiments were conducted on 16 adult 3 month old Lepr ^db / Lepr ^db mice. In eight experimental groups, the active fraction extracted from the inoculation was diluted in DMSO (dimethyl sulfoxide) and administered orally and intraperitoneally once a day at a concentration of 10 mg / kg. In the eight control groups, only the same amount of DMSO was administered. Four weeks after administration, blood was collected from the retro-orbital sinus using a heparin-treated capillary tube, centrifuged to separate plasma, and then hemochemical analyzer [CIBA Corning 550 Express, USA] blood glucose was measured to analyze the difference between the control group and the experimental group. As a result, the blood sugar level was lowered (397 ± 112 mg / ㎗) about 35% compared to the blood sugar level of the control group (611 ± 106 mg / ㎗) [Fig. 4].

Example 6 Obese Model Animals Lepr ^db / Lepr ^db Fatty liver inhibitory effect in mice

Lepr ^db / Lepr ^db mice have fatty liver in the liver cells due to excessive nutrient inflow due to continuous food intake, causing fatty liver. Therefore, to investigate the effect of the active fraction extracted from the essential liver on fatty liver, fourteen weeks of treatment with the active substance extracted from the essential hair of four adult Lepr ^db / Lepr ^db mice was performed. The animals were sacrificed and the liver was excised and placed in 10% neutral formalin for 24 hours fixation. Then, wash thoroughly with running water, dehydrate with 70%, 80%, 90%, and 100% alcohol, embedding with paraffin, and remove the tissue slice to a thickness of about 4 μm with a tissue slicer [LEICA, RM2045, Germany]. Produced. Then, hematoxylin and eosin were stained and liver tissues were observed by light microscopy. As a result, in the control group, hepatic cells containing abnormally excessive fat granules in the cytoplasm of hepatic tissue and fatty denaturation observed in fear form of various sizes were frequently observed. However, the experimental group was not able to observe such fatty degeneration findings as well as most of the recovery to normal form was found to be suppressed fatty liver [Fig. 5].

Example 7: Preparation of Tablets

10 g of active ingredients

70 g of lactose

15 g of crystalline cellulose

5 g of magnesium stearate

Total amount 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 8 Preparation of Powder

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 added to the 6 times hard capsule to prepare a capsule.

Example 9 Toxicity Test

Acute Toxicity

This study was conducted to investigate the toxicity of the active fraction extracted from the short-term ingestion in a short time period (within 24 hours) and to determine the mortality.

50 ICR mouse strains, which are general mice, were assigned to 10 control groups and 20 experimental groups. In the control group, only DMSO was administered, and the experimental group contained the active fraction extracted from the essential concentrations of 2,000 times (20 g / kg) and 5,000 times (100 g / kg) of 10 mg / kg, which is the dose used in the above-described example. Each was administered orally. After 24 hours of administration, the mortality rate of each group survived in the control group and the experimental group administered with the 20 g / kg concentration extract. However, 12 mortalities were observed in the experimental group administered the 100 g / kg concentration extract.

The experimental results show that the extract is a very safe substance with almost no toxicity, with one animal lethal dose of about 100 g / kg (100,000 mg / kg) and about 10,000 times the concentration that shows a general efficacy (10 mg / kg). there was.

2. Organ and tissue toxicity

The active fractions extracted from the essential extracts were administered at a concentration of 10 mg / kg for 4 weeks to study Lepr ^db / Lepr ^db mice used in observation tests for obesity prevention and treatment, hypoglycemia and fatty liver inhibition. In order to investigate the effect on each organ (tissue) of animals, blood was collected after 4 weeks from animals of experimental group and DMSO-only control group, and GOT (glutamate-oxalate-transferase) and GPT (glutamate). Blood concentrations of pyruvate-transferase, creatine kinase and creatinine were measured. As a result, in the case of GOT and GPT known to be related to hepatotoxicity, the experimental group had a lower value (GOT: 157 ± 15 IU) compared to the control group (GOT: 233 ± 47 IU / L, GPT: 116 ± 31 IU / L). / L, GPT: 78 ± 21 IU / L), hepatoprotective activity was observed, and creatinine, an indicator of kidney toxicity, and creatine kinase, an indicator of muscle toxicity, were compared to the control group. There was no difference.

In addition, the heart, lung, pancreas, adrenal gland, liver, kidney, and muscles were removed from each animal, and histological observations were performed by optical microscopy through a conventional tissue fabrication process. As a result, in the liver tissues, the control group showed severe fatty degeneration, while the experimental group showed almost normal condition, indicating that there was a protective effect on the liver along with low GOT and GPT levels in the blood. No abnormality was observed in the histological observations of other organs, so no toxicity was found for the organ organs of the extract.

As described above, the active fraction composition obtained from the invention according to the present invention is excellent in inhibiting fatty acid biosynthetic enzyme inhibition, obesity prevention and treatment, hypoglycemia and fatty liver can be used as a herbal medicine containing it as an active ingredient.

Claims (4)

Active fraction composition for inhibiting fatty acid biosynthetic enzymes obtained from Piper longum . In the preparation of herbal medicines by extraction from aqueous solution with alcoholic aqueous solution, (1) pulverizing the piper longum , and then extracting the solvent three times by adding 3-10 times the aqueous alcohol solution to the content of the hairbrush; (2) After evaporating and concentrating the filtrate obtained in the extraction step, the concentrate was suspended in distilled water of 50 to 100 times volume and passed through DIION HP-20 resin to be adsorbed, and then the inactive fraction using 30% methanol. Eluting and removing; (3) eluting the adsorbed active fraction using 10 to 20 times the amount of acetone, and then concentrating to obtain an extract; (4) suspending the extract with distilled water 10 to 100 times the total amount of the extract, extracting and concentrating the active substance with an organic solvent such as chloroform and ethyl acetate to obtain an extract; And (5) separating the extract by adding 1 to 2 times the total amount of the distilled water to homogeneously suspend the extract, and then freeze-drying to separate the active substance. A method for separating and purifying an active fraction composition for inhibiting fatty acid biosynthetic enzymes obtained from a seed comprising a. Fatty acid biosynthetic enzyme inhibitor pharmaceutical composition, characterized in that the active fraction composition obtained from the essential ingredient is contained as an active ingredient. 4. The fatty acid biosynthetic enzyme inhibitor pharmaceutical composition according to claim 3, wherein the pharmaceutical composition is effective in preventing and treating obesity, lowering blood sugar and inhibiting fatty liver.