JPH05339168A - Pancreatic lipase inhibitor and cholesterol esterase inhibitor - Google Patents

Abstract

PURPOSE:To provide the subject pancreatic lipase inhibitor inhibiting absorption of fats or cholesterols in a food through the intestine. CONSTITUTION:A pancreatic lipase inhibitor containing protamine as the active component. This inhibitor can retard absorption of fats or cholesterols in a food through the intestine and it, therefore, can prevent degenerative diseases, especially hyperlipidemia. The inhibitor can, in its turn, be used for prevention or improvement of arteriosclerosis and there is no risk of a bad influence due to side effects.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel lipase activity inhibitor or cholesterol esterase activity inhibitor in pancreatic juice, or a food additive substance that suppresses absorption of fat and cholesterol.

More specifically, the present invention delays the absorption of fat and cholesterol in foods from the intestinal tract,
A lipase activity inhibitor or a cholesterol esterase activity inhibitor in pancreatic juice that can be used for the prevention and treatment of adult diseases, particularly hyperlipidemia and arteriosclerosis, by suppressing an increase in the concentration of fat and cholesterol in blood, or The present invention relates to a food additive substance that suppresses absorption of fat and cholesterol.

[0003]

2. Description of the Related Art Generally, hyperlipidemia is a pathological condition in which the concentration of fat and cholesterol in blood increases, and it is said to cause arteriosclerosis. Further, arteriosclerosis is a disease in which cholesterol ester is mainly accumulated in the intima of the arterial wall, and not only cholesterol in blood but also fat is important in this accumulation. That is, fat and cholesterol in food are absorbed through the intestine,
It is taken into the lipoprotein called chylomicrons, enters the blood from the intestine through the lymphatics, and is transported to each tissue. Subsequently, at the stage where fat and cholesterol taken into the lipoprotein are transferred to each tissue, the transfer order is strictly present. That is, fat is first removed from chylomicrons and then cholesterol is transferred to the tissues, and vice versa cannot occur. Removal of fat from chylomicrons is carried out by the action of lipoprotein lipase present on the wall of blood vessels.

By the way, when a food containing the same amount of fat and cholesterol is ingested, the rate of absorption from the intestinal tract greatly affects the concentration of fat and cholesterol appearing in the blood. For example, when fat and cholesterol are absorbed within 1 hour and when they are absorbed over 3 hours, the concentrations of fat and cholesterol that appear in blood are extremely low when absorbed over 3 hours. It becomes a value and can be an effective means of reducing the risk of hyperlipidemia and arteriosclerosis. That is, delaying the absorption of fat and cholesterol from the intestinal tract as much as possible leads to the improvement of hyperlipidemia and the prevention of arteriosclerosis.

By the way, fat in food is not absorbed in the intestinal tract as it is. First, small oil droplets (micelles) are formed with bile acids and phospholipids in bile, and pancreatic lipase, which is a digestive enzyme, acts on these oil droplets, and fatty acids and β
-Monoglyceride or glycerol is first taken up by chylomicron and absorbed from the intestinal tract, then fatty acids and β-monoglyceride are mainly transported into the blood through lymphatic vessels, and glycerol is transferred to the portal vein. Therefore, by suppressing the process of fat decomposition by pancreatic lipase, the decomposition of fat can be delayed and the absorption from the intestinal tract can be delayed.

On the other hand, cholesterol in foods exists in the form of free cholesterol and cholesterol ester. For example, about 60% of cholesterol in butter and milk exists in the form of cholesterol ester. Are known. Absorption of such cholesterol from the intestine is carried out with free cholesterol, and cholesterol ester is first absorbed from the intestine after being hydrolyzed to free cholesterol and fatty acid by cholesterol esterase which is a digestive enzyme.
Therefore, by suppressing the cholesterol esterase activity, absorption of cholesterol ester in food from the intestine can be delayed.

However, the activity of the pancreatic lipase or cholesterol esterase can be suppressed in the process of decomposing fat or cholesterol by such a digestive enzyme, pancreatic lipase or cholesterol esterase, and the pancreatic lipase or cholesterol esterase can be prevented by the inhibitory effect. However, the present situation is that no suitable functional substance free of side effects has been found.

[0008] On the other hand, protamine is a strong basic simple protein with a small molecular weight (about 4000 to several thousand) which is usually present in the sperm nucleus of many vertebrates, and its constituent amino acids are almost basic amino acids. It contains a large amount of arginine (40 to 88% by weight), and the protamine molecule of fish has a typical linear molecular form, and DNA is present in the sperm cell nucleus.
It is known that the expression of genetic information is blocked and protected by binding to and forming a complex by forming a complex, and the anticoagulant action of heparin is neutralized.

[0009] More recently, a part of protamine separated and purified from fish algae such as herring has been used as a medicinal substance by binding with insulin, and it has been reported that it has a function of maintaining the medicinal effect of insulin. In addition, protamine sulfate is produced as a purified mixture from fish and is used as a heparin antagonist used for treatment of bleeding associated with an increase in circulating blood heparin-like substances and heparin overdose. In addition to drugs, it is also partially used as an antibacterial agent.

However, to date, no report has been made on a lipase activity or cholesterol esterase activity inhibitor in pancreatic juice or a food additive substance that suppresses absorption of fat and cholesterol in food from the intestinal tract using protamine, Conventionally, its usefulness as a functional substance in food has not been recognized at all.

[0011]

SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a protamine preparation which suppresses the action of pancreatic lipase which hydrolyzes fat in foods and promotes absorption from the intestinal tract.

Another object of the present invention is to provide a protamine preparation which suppresses the action of cholesterol esterase capable of decomposing cholesterol in foods, particularly cholesterol ester, into free cholesterol which can be absorbed from the intestinal tract.

A further object of the present invention is to provide a food additive containing protamine which inhibits absorption of fat and cholesterol in food from the intestinal tract.

[0014]

[Means for Solving the Problems] The present inventor has conducted extensive studies on various substances isolated from marine product resources that can exert various functions related to promotion of human health and prevention of adult diseases. As a result, it was found that protamine, which is one of the proteins isolated and purified from fish such as herring sardine, inhibits absorption of fat and cholesterol in food from the intestinal tract, and the protamine is used as a drug or a food additive. It was found that the above-mentioned various objects can be achieved by doing so, and the present invention has been completed based on this finding.

That is, the object of the present invention is achieved by a lipase activity inhibitor in pancreatic juice containing protamine as an active ingredient.

The object of the present invention is also achieved by a cholesterol esterase activity inhibitor containing protamine as an active ingredient.

The object of the present invention is also achieved by a food additive substance comprising protamine.

[0018]

The inhibitor of lipase activity and cholesterol esterase activity in the pancreatic juice of the present invention and protamine contained in the food additive are effective for administration of human or animal to hyperlipemia, Is an origin of arteriosclerosis, the activity of lipase and cholesterol esterase, which are digestive enzymes that promote the absorption of fat and cholesterol from the intestinal tract into the body (in the blood), against an increase in blood fat and cholesterol concentrations. By suppressing or inhibiting the change, the absorption of the fat and cholesterol into the blood in the intestinal tract can be delayed, and the fat and cholesterol concentrations in the blood can be stabilized.

Therefore, the protamine used in the present invention is present in the sperm nucleus of many vertebrates, and is not particularly limited as long as it can be extracted and purified from fish, mammals and the like. For example, sperm nuclei of fish such as herring, salmon, cod, mackerel, sturgeon or carp (hereinafter, also referred to as albino), and those extracted and purified from sperm nuclei of mammals such as chicken or mouse are preferable, but are preferable. Is an alga that is extracted and purified from fish. In this case, it can be expected that it can be stably obtained as a natural marine product resource, and that the cabbage that was also extracted with viscera can be effectively used at the time of food processing and can be obtained at low cost.

Since such protamine is a strong basic simple protein having a small molecular weight, it is easily dissolved in water and easily mixed with various foods, and also when it is prescribed as a drug in various states. It can be contained.

Therefore, the lipase activity inhibitor or cholesterol esterase activity inhibitor in pancreatic juice used in the present invention is mainly orally administered in the form of tablets, capsules, powders, granules, tablets, fluids, gel preparations and the like. Is done in. Similarly, the food additive substance comprising protamine of the present invention can be added to various food products such as solid foods (including powders and granules), semi-solid foods (including gels), fluid foods (including beverages) and the like. The addition method is arbitrary, such as the case where it is added together with the food material at the stage of food processing or the case where it is added later in the finished food. Furthermore, it may be pre-coated with a retarder or the like so that it can act appropriately in the body (intestinal tract) for a long time.

Further, tablets and capsules for oral administration of lipase activity inhibitor or cholesterol esterase activity inhibitor in pancreatic juice used in the present invention are:
Those which carry a single dose are preferred and contain conventional excipients such as syrup, acacia or sorbitol as a binder drug, eg fillers such as lactose, sugar or corn-starch. To do.

Oral liquid preparations are those which are in the form of suspensions, solutions, emulsions, syrups or elixirs containing water or oil, and are formulated with water or other suitable form before use. It may be a dried product which can be added with an agent and returned to a liquid state. Such oral liquid formulations include suspensions such as sorbitol, syrup or gelatin,
Emulsifiers such as acacia and lecithin, coconut oil, non-aqueous excipients such as oily esters (propylene glycol, etc.), other preservatives and additives used as usual chemicals, and if necessary seasonings and colorants Alternatively, vitamin supplements are used.

Further, the amount (or dose) of the protamine added in the lipase activity inhibitor or cholesterol esterase activity inhibitor in the food additive substance or pancreatic juice of the present invention
Is different depending on the intake of fat or cholesterol, the degree of hyperlipidemia and arteriosclerosis, weight, age, sex, etc., and it is preferable to determine an appropriate amount according to the patient in use. But weight 70
For adult male patients with kg hyperlipidemia, usually 500-
The 700 mg / day range is a rough guideline for the treatment.

[0025]

EXAMPLES Next, the present invention will be described in more detail with reference to examples.

Example 1 Method for extracting protamine and the method
Evaluation of Protamine The protamine used in the examples of the present invention was obtained using the extraction method detailed below.

First, 1000 ml of 5% sulfuric acid was added to 100 g of the herring homogenate of herring as a raw material, and the mixture was stirred at 20 ° C. for 2 hours and then centrifuged to obtain a supernatant 950 of the solution.
ml was separated and collected. Next, a 2N sodium hydroxide solution was added to the supernatant to adjust the pH to 6.5, and then the solution was centrifuged again to separate and collect the supernatant of the solution. Subsequently, the supernatant was allowed to stand at 0 ° C. for 15 hours in a cold place, and then subjected to centrifugal separation to precipitate the solute, which was separated and collected. The resulting precipitate was dissolved in deionized water and subjected to ion exchange chromatography (Amberlito 402
"[OH ^-]. Filtered using), after concentration of the effluent was collected solution, we were able to extract the desired protamine powder 2g by lyophilization.

The composition of amino acids of protamine obtained by the above extraction method is shown in Table 1 below.

[0029]

[Table 1]

The protamine used in the following examples was obtained by using the protamine powder obtained by the above-mentioned extraction method (1) unless otherwise specified.

Example 2 Prota for pancreatic lipase activity
Confirmation of the inhibitory action of min fats in the test food fat droplets formed with bile acids and phospholipids (micelles) because pancreatic lipase is a digestive enzyme acts on the substrate solution corresponding to the oil droplet component, A desired substrate solution was prepared by ultrasonically treating a solution containing triolein, which is one of the neutral fats, as a main component and having the composition shown in Table 2 below.

[0032]

[Table 2]

The enzyme solution 5 containing 100 μl of the obtained substrate solution and 0.1 μg of pancreatic lipase which is a digestive enzyme
0 μl and 50 μl of a protein solution appropriately containing protamine were mixed and reacted at 37 ° C. for 30 minutes. At this time, the pancreatic lipase activity when the protamine content was changed was measured. The obtained results are shown in FIG.

Comparative Example 1 Other parameters for pancreatic lipase activity
Comparative test of protein inhibitory activity Bovine serum albumin (a type of soluble simple protein with a large molecular weight of 69000, instead of protamine, is usually a plasma protein, fatty acid in blood, bile pigment,
The reaction was carried out in the same manner as in Example 2 except that a poorly soluble substance such as a drug was bound to carry these substances. Furthermore, the pancreatic lipase activity when the bovine serum albumin content was changed was also measured by the same method as in Example 2, and the obtained results are shown in FIG.

FIG. 1 shows that when the concentration of protamine exceeds about 5 μg / ml in the sample solution, the decomposition of fat (triolein) in the sample solution by pancreatic lipase activity is almost completely inhibited. On the other hand, it was confirmed that when the albumin concentration exceeds about 5 mg / ml in the sample solution, it inhibits the decomposition of fat (triolein) in the sample solution by pancreatic lipase activity. .. Therefore, it can be said that the inhibitory action of protamine on the pancreatic lipase activity is not the action of general proteins, but the specific action of protamine.

Comparative Example 2 Other parameters for pancreatic lipase activity
Comparative test of protein inhibitory effect Further, the reaction was carried out in the same manner as in Example 2 except that a substrate solution in which triolein (a fat component) was emulsified with gum arabic was used instead of the substrate solution. On this occasion,
Pancreatic lipase activity when the protamine content was changed was also measured using the same method as in Example 2 (not shown), but no inhibitory action was shown at any protamine concentration. Therefore, in this case, protamine does not act on the pancreatic lipase directly, but acts on the substrate (fat) side of the pancreatic lipase by binding to the substrate (fat) side, especially taurocholic acid (bile acid) which has a surfactant action. Is indirectly expressed.

This means that when protamine directly acts (bonds or adsorbs) on pancreatic lipase (serine enzyme) which is a digestive enzyme, trypsin (digestive enzyme for food proteins) and chymotrypsin (which are other serine enzymes) Mainly one of the major digestive enzymes that cleaves the peptide bond on the C-terminal side of aromatic amino acid residues) will be inhibited, and not only the absorption of fat but also the absorption of protein will be inhibited. However, when the protamine of the present invention is used, it is a substrate (fat) side that forms a micelle with bile acid, while it is not suitable as a food additive or an inhibitor such that a side effect rather than a medicinal effect appears. By acting on, the inhibitory effect is localized and there is no fear of adverse effects due to side effects.

Example 3 Cholesterol esterase activity
Confirmation of inhibitory effect of protamine on sex Since cholesterol esterase, which is a digestive enzyme, acts on oil droplets (micelles) formed by cholesterol ester in foods together with bile acids and phospholipids, a substrate corresponding to the oil droplet component As a solution, a desired substrate solution was prepared by sonicating a solution containing cholesterol ester as a main component and having the composition shown in Table 3 below.

[0039]

[Table 3]

30 μl of the obtained substrate solution, 20 μl of the enzyme solution containing 20 μg of cholesterol esterase as a digestive enzyme, 20 μl of an inhibitor solution containing appropriately protamine and a buffer solution (1.43% BS)
A, 80 mM KCl, 20 mM NaCl and 45
The one prepared at pH 6.8 consisting of mM BES was used), and the mixture was reacted at 37 ° C. for 1 hour. On this occasion,
Cholesterol esterase activity was measured when the protamine content was changed. The obtained results are shown in FIG.

From FIG. 2, it was confirmed that when the concentration of protamine exceeds about 100 μg / ml in the sample solution, the degradation of cholesterol ester in the sample solution by cholesterol esterase activity is inhibited by about 60%.

[0042]

INDUSTRIAL APPLICABILITY The lipase activity inhibitor and cholesterol esterase activity inhibitor in pancreatic juice containing protamine of the present invention, and the food additive containing protamine can prevent absorption of fat and cholesterol in food from the intestinal tract. By delaying it, it is possible to prevent the appearance of adult diseases, particularly hyperlipidemia, and thus be used for the prevention and treatment of arteriosclerosis.

Further, when protamine directly acts (binds or adsorbs) on digestive enzymes (pancreatic lipase (serine enzyme) and cholesterol esterase), trypsin (digestive enzyme for food proteins) and chymotrypsin which are other serine enzymes. (Mainly one of the major digestive enzymes that cleaves the peptide bond on the C-terminal side of aromatic amino acid residues) is also inhibited, and not only the absorption of fat but also the absorption of protein is also inhibited. As such, while not suitable as a food additive or an inhibitor such as the appearance of adverse effects rather than medicinal effects, the lipase activity inhibitor and cholesterol esterase activity inhibitor in the pancreatic juice containing the protamine of the present invention, When using food additive substances consisting of and protamine, along with bile acids and phospholipids By acting on form oil droplets (micelles), the inhibitory effect is localized, in which fear of adverse effects of side effects is eliminated.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between pancreatic lipase activity and protamine concentration.

FIG. 2 is a graph showing the relationship between cholesterol esterase activity and protamine concentration.

Claims (3)

[Claims] 1. A lipase activity inhibitor in pancreatic juice containing protamine as an active ingredient. 2. A cholesterol esterase activity inhibitor containing protamine as an active ingredient. 3. A food additive substance comprising protamine.