KR20030088528A - Method for producing inhibitor against pancreatic lipase from eggplant extracts and compositions containing same for inhibiting lipid uptake - Google Patents

Abstract

PURPOSE: Provided are a method for producing inhibitor against pancreatic lipase from an eggplant extract and a composition containing the same to inhibit lipid uptake. The eggplant extract directly inhibits the decomposition of lipid in the small intestine, and is thus useful for the prevention and treatment of obesity. CONSTITUTION: A method for producing inhibitor against pancreatic lipase comprises the steps of: extracting active ingredients from an egg plant with hot water or an organic solvent; inhibiting pancreatic lipase using the extract; and fractioning the extract to give active ingredients having the highest inhibition effect on lipase. A pharmaceutical composition for the inhibition of lipid uptake contains the egg pant extract as an active ingredient.

Description

METHOD FOR PRODUCING INHIBITOR AGAINST PANCREATIC LIPASE FROM EGGPLANT EXTRACTS AND COMPOSITIONS CONTAINING SAME FOR INHIBITING LIPID UPTAKE}

The present invention relates to a method for producing an inhibitor for pancreatic lipase using eggplant and a composition for inhibiting absorption of fat in the body using the same, specifically, extracting an active ingredient from eggplant using hot water or an organic solvent; Inhibiting pancreatic lipase which is an lipase using the extract; The method for inhibiting pancreatic lipase using eggplant extract, comprising the step of fractionating an active ingredient having a high inhibitory effect on lipase in the extract, and using the extract active ingredient obtained by the method, the activity of lipase which is a lipolytic enzyme in vivo The present invention relates to a composition for inhibiting absorption of fat in the body.

In vivo digestion of ingested food usually results in the breakdown of some proteins in the stomach, including the initial breakdown of carbohydrates in the oral cavity. However, full-fledged digestion and absorption occurs in the duodenum to small intestine. In general, the large intestine is known to reabsorb water and salts and to absorb free fatty acids. Therefore, the most important part of digestive absorption occurs in the small intestine, where the proteolytic enzymes such as trypsinogen, chymotrypsinogen, and carboxy peptidase, lipolytic enzymes such as pancreatic lipase and stipin, pancreatic lipase, amylosin Masses are broken down by various digestive enzymes such as lipolytic enzymes such as, maltase, and various peptidase, nucleic acid, alginase, phosphatase, and lactose, various glycolytic enzymes present in the small intestine, and villi on the small intestine Absorbed by the nutrients absorbed through the dark ducts and blood vessels will be moved into the body.

Lipase is the most important role in the digestion and absorption of fat in the small intestine. Lipase is an enzyme that plays an important role in the body's absorption of fat as it breaks down fats from foods into fatty acids and glycerol, which can be absorbed through the small intestine.

Therefore, considering the fact that the intake of fat is increasing following the dietary pattern of Western countries, it is possible to locally inhibit the absorption of fat in the small intestine without inhibiting the absorption of amino acids and other essential inorganic ions and proteins. Development of the method is required. Lipase inhibitors such as Xenical (pharmaceutical name Olistat), developed in line with these trends, are being used for the treatment of severe obesity patients, and are approved by the US Food and Drug Administration (FDA). If you have health risk factors such as diabetes, hypertension, hyperlipidemia, apply to more than 25 if used for 3-6 months with diet and exercise therapy.

Reducing fat intake also lowers blood cholesterol levels and helps improve high blood pressure and diabetes. Xenical, which only affects the stomach, is known to have the least adverse side effects of any obesity drug ever. In 1996, he had a sensational diet sensation prior to Xenical, but it was much safer than Redux, which had become obsolete after a year due to the discovery of heart valve abnormalities in some users. However, all drugs carry the risk of side effects. In the case of Xenical, about 6% of people discontinue treatment due to side effects. It can range from mild symptoms such as fatty stools, abdominal bloating, farts, frequent bowel movements, and abdominal pain, to more or less dangerous symptoms such as gallstones. Zenical also interferes with the intake of fat-soluble vitamins A, D, E, and K. The US Food and Drug Administration (FDA) recommends taking multivitamins about two hours before and after Zenical to prevent this. The more high-fat foods you eat, the more severe the side effects.

In addition, Xenkal and the like must be followed by a doctor's prescription and has the disadvantage that it can not be used as a raw material of functional food.

On the other hand, in addition to these drugs for the treatment of obesity, various studies for the prevention and improvement of obesity are being actively conducted, and various herbal extracts, such as green tea, have been used to help the decomposition of fat accumulated in the body.

In order to develop a substance capable of inhibiting pancreatic lipase, an enzyme that plays an important role in the digestion and absorption of fat, the present inventors extracted natural substances from eggplants, which are foodstuffs having proven safety, and used the extracted and separated substances as described above. By directly inhibiting the digestive absorption of fat in vivo, the present invention was completed by revealing that it can be usefully used as a composition for improving and treating obesity and hyperlipidemia.

It is an object of the present invention to provide a method for producing a substance that inhibits enzymatic activity against pancreatic lipase, a lipolytic enzyme, from eggplant.

Another object of the present invention to provide a composition for inhibiting fat absorption containing the branch extract.

1 is a result of separating pancreatic lipase from pigs using DEAE-Sepharose and the like,

Figure 2 shows the change in reaction rate of the pancreatic lipase isolated in Figure 1 with temperature and pH,

Figure 3 shows that the activity of porcine pancreatic lipase is directly inhibited by the eggplant extract prepared according to the present invention.

In order to achieve the above object, the present invention provides a method for producing a lipase inhibitor material using hot water and organic solvent extraction method from eggplant.

In addition, the present invention provides a pharmaceutical composition for inhibiting fat absorption containing the eggplant extract produced by the method as an active ingredient.

In addition, the present invention provides a food and beverage composition containing the eggplant extract produced by the method as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for producing eggplant extracts that inhibit pancreatic lipase.

The production method

1) producing an extract using hot water or an organic solvent using eggplant; And

2) separating the substance that inhibits lipase from the extract.

The above steps will be described in more detail below.

As a preferred embodiment of the present invention, pancreatic lipase is purified from pigs using anion exchange column chromatography (see FIG. 1 ). In order to measure the enzyme activity of the purified pancreatic lipase, the optimum reaction temperature and pH of each enzyme activity were examined, and the highest activity was found at 50 ° C. and pH 5 (see FIG. 2 ).

The pancreatic lipase used as a lipase in the present invention can be isolated from pigs as well as rats, mice, guinea pigs, rabbits or humans, and can be used separately from those expressed in recombinant microorganisms containing genes encoding these enzyme proteins. And chemically synthesized ones may be used.

The inhibitory effect of eggplant extract can be measured using the isolated pancreatic lipase as lipase.

Step 1) is a step of producing an extract using hot water or an organic solvent using eggplant. In order to facilitate the extraction of the extract with a lipase inhibitor, pure separation with high efficiency is required, and in particular, it should not be harmful to a living body. For this purpose, it is most preferable to extract with hot water from the eggplant, and when extracting with an organic solvent, it is important to select a solvent that is harmless to the human body, and it is important to completely remove the remaining organic solvent after extraction.

The isolated and purified eggplant extract effectively inhibited pancreatic lipase activity (see FIG. 3 ), and effectively inhibited fat absorption by inhibiting the enzyme activity when applied in vivo, thereby reducing subcutaneous fat accumulation ( FIG. 4 ).

In another aspect, the present invention provides a pharmaceutical composition for inhibiting fat absorption containing the eggplant extract produced by the method as an active ingredient.

Natural extracts inhibiting pancreatic lipase produced according to the present invention directly inhibits the breakdown of fat in vivo to reduce the digestive absorption of fat in the body, so that the pharmaceutical composition using the eggplant extract as an active ingredient is excessive without restriction of food intake. It can be usefully used for the prevention and treatment of diseases due to absorption of fat, for example, obesity, hyperlipidemia and the like.

The pharmaceutical formulations can be prepared according to conventional methods using the compositions of the present invention. The formulation may be in the form of tablets, pills, powders, assays, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft and hard gelatin capsules, sterile injectable solutions, sterile packaged powders and the like.

Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, poly Vinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The formulation may further comprise fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like. Compositions of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

The pharmaceutical compositions of the invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular. For humans, the usual daily dosage of eggplant extract may range from 5 to 30 mg / kg body weight, preferably 10 to 20 mg / kg body weight, and may be administered once or in several doses.

However, it should be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors such as the disease to be treated, the route of administration chosen, the age, sex, weight of the patient and the severity of the patient's symptoms, and thus the dosage may be The method does not limit the scope of the present invention.

In addition, the present invention provides a food and beverage composition containing the eggplant extract produced by the method as an active ingredient.

Since the food and beverage composition of the present invention inhibits the breakdown of fat in the body, it is possible to maintain and improve health through inhibition of obesity, control of blood lipid level, and the like.

The eggplant extract of the present invention may also be used as a dietary ingredient for improving obesity, or as an additive or food supplement to foods or beverages for the purpose of exhibiting prophylactic and therapeutic effects such as diseases caused by excessive fat absorption, such as hyperlipidemia and hypertension. Can be incorporated. As the food or beverage, meat; Vegetable juices (eg, carrot juice and tomato juice) and fruit juices (eg, orange juice, grape juice, pineapple juice, apple juice and banana juice); Chocolate; Snacks; confectionery; Pizza; Food products made of grain powder such as bread, cakes, crackers, cookies, biscuits, noodle, etc .; Gums; Dairy products such as milk, cheese, yogurt and ice cream; soup; Juicy; Pastes, ketchups and sauces; car; Alcoholic beverages; Carbonated beverages; Vitamin complexes; And various health foods. In this case, the content of the eggplant extract in food or beverage may be in the range of 0.05 to 0.5% by weight, preferably 0.1 to 0.2% by weight.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Isolation and Activity of Pancreatic Lipase

(Step 1) Isolation of Pancreatic Lipase from Pigs

For isolation of pancreatic lipase involved in digestive enzymes, fresh pig small intestine is washed with phosphate buffer containing sodium phosphate (abbreviated as PBS) and recovered. The protein was adsorbed onto a DEAE-Sepharose column (Amersham Pharmacia Biotec Co.) equilibrated with -HCl buffer (pH 8), and the proteins were selectively fractionated with the same buffer containing 0-1.0 M sodium chloride. Pancreatic lipase was isolated ( FIG. 1 ).

(Step 2) Determination of Enzyme Activity of Isolated Pancreatic Lipase

In order to investigate the activity of the enzyme isolated in step 1, an enzyme reaction was performed by adding a fat emulsion as a substrate. The reaction solution contains a substrate 1%, 50 mM phosphate buffer, enzyme (0.2munit unit), distilled water and the reaction capacity was reacted for 20 minutes at 37 ℃ with 0.1ml. At this time, the temperature was reacted in the temperature range of 30 ℃ to 70 ℃ for the investigation of the optimum reaction temperature and pH of the enzyme and the pH was reacted in the pH range of 3 to 9. After the reaction, the enzyme activity was stopped at 100 ° C and the resulting fatty acid concentration was quantified using a fatty acid quantification kit.

As a result, it showed the highest activity at 40 ℃ and pH 6.5 ( Fig. 2 ).

Example 2 Confirmation of Inhibition of Pancreatic Lipase Activity of Eggplant Extract

Extracts using 10 liters of hot water or organic solvent (ethanol, ethyl acetate, chloroform, etc.) per 1 kg of eggplant. After extraction, the residue was removed by centrifugation and filtration, and then the liquid component was dried under lyophilization or reduced pressure to form a powder, which was then tested for inhibition of activity of pancreatic lipase. In order to confirm whether the extracted eggplant component can actually inhibit the activity of pancreatic lipase, the enzymatic activity of pancreatic lipase was measured in the same manner as in step 2 of Example 1 above. During the enzymatic reaction, eggplant extract was added at a concentration of 100-500 µg / ml to determine the degree of activity inhibition. To compare the inhibitory activity of eggplant extracts against pancreatic lipase, a control group without enzymes, eggplant extract-free group, and pancreatic lipase-only group were used.

As a result, as shown in Figure 3 , it was confirmed that about 40% of the activity of the pancreatic lipase is inhibited by the eggplant extract prepared according to the present invention.

(Step 2) Inhibition of Pancreatic Lipase Activity Using Eggplant Extract

Example 3 Inhibitory Activity of Fats in Vivo

In order to investigate whether the eggplant extract prepared according to the present invention exhibits the inhibitory activity of fat absorption in vivo, lipid emulsions (6 mL of cron oil; 80 mg of cholic acid; cholesteryl oleic acid) using male C57BL / 6J mice Eight 2 g; saline 6 mL) was orally administered once at 20 mg / mouse to ingest lipids, and then eggplant hot water extract was orally administered at 10-100 mg / mouse. After ingestion of lipid and eggplant extract, blood was collected by heart bleeding after 0.2 hours. Neutral lipid and total cholesterol content were analyzed using an analysis kit (Asan Pharmaceutical). (Table 1).

From this, it was confirmed that the eggplant extract prepared according to the present invention effectively inhibits the absorption of glucose in vivo in a concentration-dependent manner.

Table 1. Changes in Serum Total Cholesterol and Triglycerides in Egg Extracts and Control Groups after Oral Fat Administration in C57BL / 6J Mice

Time (hr) 0 2 4 Total cholesterol Control 114.63 ± 3.52 120.34 ± 3.25 115.69 ± 13.52 Eggplant extract 113.77 ± 5.27 112.8 ± 2.17 * 114.48 ± 2.11 Total triglycerides Control 108.86 ± 3.16 115.69 ± 13.63 120.34 ± 3.52 Eggplant extract 107.39 ± 10.38 113.59 ± 8.22 117.37 ± 13.27

Preparation Example 1 Preparation of Pharmaceutical Formulation

Capsules were prepared by mixing the following ingredients to fill hard gelatine capsules:

Volume (mg / capsule)

Active Ingredient (Eggplant Extract of Example 2) 20

Dry starch 160

Magnesium Stearate 20

Total 200 mg

Preparation Example 2 Preparation of Functional Food Containing Eggplant Extract

Functional foods containing the eggplant extract of the present invention were prepared as follows.

(1) Manufacture of dairy products

Eggplant extract was added to the milk in an amount of 5% by weight, and the milk was used to prepare various dairy products such as butter and ice cream.

However, eggplant extract was added to the coagulated milk protein in cheese production and to the coagulated milk protein obtained after fermentation in yogurt production.

(2) Preparation of Mixed Drinks

1 g of eggplant extract was added to 1,000 ml of tomato, carrot, apple or grape juice to obtain a mixed beverage for health promotion.

As described above, the enzyme activity inhibitor for pancreatic lipase extracted from the eggplant according to the present invention can be produced safely and in large quantities using natural materials, which is very economical and does not inhibit absorption of other essential inorganic ions and proteins. By effectively inhibiting only the absorption of fat, it can be usefully used as a composition for improving and treating obesity and hyperlipidemia diseases.

Claims (7)

1) extracting an activity inhibitory substance against pancreatic lipase from eggplant; And 2) using the extract to inhibit activity against pancreatic lipase. The method of claim 1, wherein the pancreatic lipase protein is isolated from porcine small intestine, rat, mouse, guinea pig, rabbit or human. The method of claim 1 wherein the pancreatic lipase protein is expressed by a recombinant microorganism or is chemically synthesized. A pharmaceutical composition for inhibiting fat absorption containing eggplant extract produced by the method of claim 1 as an active ingredient. The pharmaceutical composition according to claim 4, which is used for the prevention and treatment of obesity or hyperlipidemia related diseases. Food and beverage compositions comprising the eggplant extract produced by the method of claim 1 as an active ingredient. 7. A food and beverage composition according to claim 6 for use in preventing or improving obesity.