JPH04300839A - Lipolytic enzyme inhibitor - Google Patents

Abstract

PURPOSE:To suppress or inhibit lipolysis, prevent ingested lipids from being rapidly absorbed in bodies, suppress the amount of the total absorbed fats and prevent hyperlipemia and obesity by inhibiting activity of a lipolytic enzyme. CONSTITUTION:A lipolytic enzyme inhibitor containing a basic protein derived from wheat embryo buds as an active ingredient. Furthermore, a basic protein, derived from wheat embryo buds and having amino acids ranging from the 1st to the 40th residues counted from the N-terminal as follows. Gly-Lys-Val-His- GlyS-er-Leu-Ala-Arg-Ala-Gly-Lys-Val-Arg-Gly-Gln-Thr-Pro-Lys-Val-Ala-Ly s-Gln- Asp-Lys-Lys-Lys-Gln-Pro-Arg-Gly-Arg-Ala-H-is-Lys-Arg-Ile-Gin-Tyr-Asn. The number of total bound amino acids is within the range of about 60-100.

Description

[Detailed description of the invention]

0001

TECHNICAL FIELD The present invention relates to a lipolytic enzyme inhibitor containing a basic protein lipid derived from wheat germ as an active ingredient, a method for preparing the basic protein, and a specific basic protein.

0002

[Prior Art] Various reports have been made to date that proteins such as serum albumin, β-lactoglobulin, and certain soybean proteins inhibit the function of lipase and suppress or prevent the decomposition of emulsified lipids. Jour
al of Lipid Research Vol.
25, 1984p. 1214-1221 etc.). However, these proteins lose their lipase inhibitory effect in the presence of bile acids and the like, and therefore do not function as lipase inhibitors in vivo.

0003

[Contents of the Invention] The present inventors have continued to study the lipase inhibitory effect of proteins. As a result, basic proteins can be obtained by extracting wheat germ with an acidic solution.
They also discovered that this basic protein has the effect of inhibiting or suppressing the function of lipolytic enzymes even in the presence of bile acids. In addition, when we isolated a specific basic protein from the wheat germ-derived basic proteins mentioned above and determined its structure, we found that the amino acids from the 1st to the 40th from the N-terminus are shown in SEQ ID NO: 1. It was discovered that this basic protein also has a lipolytic enzyme inhibitory effect.

[0004] Therefore, the present invention is a lipolytic enzyme inhibitor containing a basic protein derived from wheat germ as an active ingredient. In the present invention, 40 from the first one counting from the N-terminus
It is a basic protein derived from wheat germ and has the sequence shown in SEQ ID NO: 1. Furthermore, the present invention includes a method for obtaining the basic protein from wheat germ.

The basic protein derived from wheat germ used in the present invention has an isoelectric point on the alkaline side and exhibits basicity. The term "lipid degrading enzyme inhibitor" as used in the present invention refers to inhibiting or suppressing the action of lipolytic enzymes such as lipase, thereby inhibiting or suppressing the decomposition of lipids and inhibiting the absorption of lipids from the intestinal tract. An agent that has the function of inhibiting or suppressing. The lipolytic enzyme inhibitor of the present invention is effective in a system where lipids are emulsified in the presence of bile acids, etc., and therefore works effectively in vivo.

[0006] The above basic protein can be obtained by extracting wheat germ with an acidic solution and, if necessary, further purifying the extract. The basic protein obtained by extracting wheat germ with an acidic solution is a mixture of multiple basic proteins, and the molecular weights of these multiple basic proteins range from about 5,000 to 42,000. In the present invention,
The mixture of the plurality of basic proteins described above can be used as it is as a lipolytic enzyme inhibitor. Alternatively, a specific basic protein may be isolated from the mixture and used as a lipolytic enzyme inhibitor.

The lipolytic enzyme inhibitor of the present invention can be administered to humans and various animals (for example, livestock such as cows, horses, pigs, and chickens, poultry, and pets such as dogs and cats). . The effective dosage of the lipolytic enzyme inhibitor of the present invention varies depending on the type, age, physical condition, etc. of the subject to whom it is administered, and is preferably administered in an amount suitable for each subject. Furthermore, the lipolytic enzyme inhibitor of the present invention is preferably prepared and administered as an oral preparation. Furthermore, the lipolytic enzyme inhibitor of the present invention may be administered alone, together with a carrier commonly used in the pharmaceutical industry, or in combination with other drugs. Furthermore, the lipolytic enzyme inhibitor of the present invention can be used in any dosage form such as tablets, granules, capsules, and powders. Furthermore, the lipolytic enzyme inhibitor of the present invention can also be administered by being added to food or feed.

[0008] The basic protein derived from wheat germ can be obtained by extracting wheat germ with an acidic solution as described above, and a specific example of its preparation method is as follows. Example of preparation of basic protein derived from wheat germ (i) Removing the water-soluble fraction from defatted wheat germ or undefatted wheat germ by washing with water or other suitable washing method,
(ii) Wheat germ solids from which the water-soluble fraction has been removed have a pH of approximately 1.
(iii) neutralize the acidic solution containing the acid-soluble fraction and add a buffer solution; , (iv) pass the neutralized and buffered solution through a column filled with ion exchange resin or adsorbent that adsorbs basic proteins, (v) elute the basic proteins adsorbed on the column by an appropriate method, (vi) desalting the eluate; and (vii) drying by lyophilization or other suitable methods to obtain a dry product consisting of a mixture of basic proteins.

The dried product obtained above can be used as it is as a lipolytic enzyme inhibitor. Also,
The above-mentioned products may be isolated and recovered into each basic protein by SDS electrophoresis, membrane separation, etc. In the above-mentioned method for preparing basic proteins, the buffer used in step (iii) includes Tris buffer (pH 8.0), phosphate buffer (pH 7.5), etc., and the ion exchange in step (iv). Examples of the resin and adsorbent include CM-Toyopearl (manufactured by Tosoh Corporation), CM-Sephadex (manufactured by Pharmacia Corporation), and the like. Furthermore, the desalting treatment in step (vi) is carried out using a microacylizer [manufactured by Asahi Kasei Corporation].
, a permeable membrane, etc. However, the method for preparing the basic protein derived from wheat germ is not limited to the above method, and any basic protein obtained from wheat germ can be used in the lipolytic enzyme inhibitor of the present invention.

Furthermore, the present inventors have developed a product consisting of a mixture of a plurality of basic proteins obtained by the above-mentioned preparation method, by CM
- An experiment was conducted to isolate each basic protein using Toyopearl and reverse phase HPLC. When the separated basic protein was recovered and its structure determined, it was found that this basic protein consists of the
The amino acids up to position 0 have the sequence shown in SEQ ID NO: 1, and the total number of amino acid bonds is approximately 60 to 100.
The protein was found to be in the range of and,
This basic protein also had a lipolytic enzyme inhibitory effect like the basic protein mixture described above.

[0011] The present invention will be specifically explained below with reference to Examples, but the present invention is not limited thereto. Example 1 [Preparation of basic protein derived from wheat germ] Defatted wheat germ 5
After adding 5 times the amount of water to 0 g and stirring at room temperature for 2 hours,
Centrifugation was performed at 000 rpm for 20 minutes. The supernatant liquid was removed and the precipitate was collected. Five times the amount of water was added to this precipitate and centrifuged in the same manner as above, and this operation was repeated three times to remove the water-soluble fraction in the wheat germ. After adding 5 times the amount of water to the precipitate obtained above and stirring, 6N hydrochloric acid was added to adjust the pH of the liquid to 2.0. After stirring at room temperature for 2 hours, 5
After centrifugation at 000 rpm for 20 minutes, the supernatant containing the acid-soluble fraction was collected. Furthermore, the above-mentioned acid extraction treatment was repeated on the precipitate, and the supernatant liquid containing the acid-soluble fraction was collected and combined with the supernatant liquid containing the acid-soluble fraction collected above.

[0012] Sodium hydroxide was added to the acid-soluble fraction-containing solution obtained above to adjust the pH to 7.0. Next, sodium chloride and Tris buffer (pH 8.0) were added to this solution.
(hereinafter referred to as "Tris buffer"), sodium chloride concentration 200mM and Tris buffer concentration 2
The solution was adjusted to 0 mM and further centrifuged to obtain a supernatant (adjusted supernatant). CM-Toyopearl column (
A column (with an inner diameter of 1.6 cm and a length of 20 cm) was prepared, and this column was equilibrated in advance with a solution having a sodium chloride concentration of 200 mM and a Tris buffer concentration of 20 mM. The above-mentioned adjusted supernatant liquid was passed through the column which had been equilibrated, and then thoroughly washed with the same liquid used for equilibration of the column. Next, the sodium chloride concentration is 1M
A solution having a Tris buffer concentration of 20 mM was passed through the column to elute the components adsorbed on CM-Toyopearl.

[0013] After desalting the eluted solution using a microacylizer, the obtained solution was freeze-dried and
0 mg of powdered product was obtained. The powdered product obtained above was analyzed by the Pico-Tag method to determine the content ratio of each amino acid, and the amino acid analysis values were as shown in Table 1.

[0014]

[Table 1]

When this powdered product was subjected to SDS electrophoresis, it was separated into about 20 types of basic proteins each having a molecular weight between about 5,000 and 42,000. This revealed that the powdered product obtained above was a mixture of multiple basic proteins.

[Preparation of olive oil emulsion] To 250 mg of olive oil, 60 mg of phospholipid, 26.9 mg of sodium taurocholate, which is a component of bile acid, and 2
5ml of 0mM Tris buffer was added. This liquid was treated with ultrasound to prepare an olive oil emulsion.

[Preparation of basic protein-containing solution] Add 200 mM Tris buffer to the wheat germ-derived basic protein fraction obtained above to dissolve it, until the basic protein concentration is 25%.
Two types of solutions were prepared: 0 μg/ml and 500 μg/ml.

[Preparation of lipolytic enzyme solution] 200 mM Tris buffer was added to porcine pancreatic lipase (L0382, manufactured by Sigma) to prepare a lipolytic enzyme solution containing 100 units of porcine pancreatic lipase/ml.

[Measurement of lipolytic enzyme inhibitory properties of basic protein] Add 100 μl of the olive oil emulsion prepared above.
I prepared three sets of each. 50 μl of Tris buffer was added to the first set of emulsions, and 50 μl of the basic protein-containing solution prepared above was added to each of the second and third sets of emulsions.
In addition, after shaking for 5 minutes, add the lipolytic enzyme solution 5 prepared above.
0 μl was added and shaken at 37° C. for 1 hour. Next, extraction solvent (chloroform: methanol: heptane = 49:
After adding 3 ml of 1:49) and shaking for 5 minutes,
Centrifugation was performed at 000 rpm for 5 minutes. After removing the upper layer liquid with an aspirator, a copper reagent (0.
45M triethanolamine, 0.05N acetic acid, 3.4
% copper sulfate pentahydrate and 20% sodium chloride) 1 ml
was added, and after shaking for 5 minutes, centrifugation was performed at a rotation speed of 3000 rpm for 5 minutes.

Next, 0.5 ml of the upper layer liquid was collected, and a coloring agent (0.1% bathocuproine, 0.0
．． 0.5 ml of 0.05% butylated hydroxyanisole dissolved therein was added, and absorbance at a wavelength of 480 nm (Abs480) was measured to determine the amount of free fatty acids produced, that is, the activity of porcine pancreatic lipase. The above results were as shown in Table 2 below. In addition, the activity in Table 2 is Abs480 when an emulsion of olive oil added with Tris buffer is decomposed with porcine pancreatic lipase.
is expressed as a percentage of 100.

[0021]

[Table 2]

From the results in Table 2 above, it can be seen that when the inhibitor of the present invention consisting of a basic protein derived from wheat germ is added, lipase activity is suppressed in the presence of bile acids.

Example 2 Two groups of SD male rats (average weight: 290 g/animal) were prepared, with 5 rats in each group. Separately, mix 10 g of olive oil and 1.2 g of egg yolk lecithin, add distilled water to this and bring the total amount to 20 g.
ml, and then treated with ultrasound to prepare an emulsion, which was divided into 2 ml portions. For each of the rats in the first section, 2 ml of the above aliquoted emulsion was added per rat.
A sample to which 100 mg of the basic protein obtained in Example 1 was added was orally administered using a gastric tube, and blood was collected from the tail vein of the rat over time and the neutral fat concentration in the blood was measured using the Kyowa Metics Enzyme Kit TG. The average value per animal (m
g/dl) was determined (example of the present invention). In addition, to each of the rats in the second group, a sample prepared by adding 100 mg of soybean protein hydrolyzate to 2 ml of the above-mentioned aliquoted emulsion was orally administered to each rat, and the blood concentration was increased in the same manner as above. Sexual fat concentration was measured over time and the average value was determined (comparative example). The above results are shown in Table 3 below.

[0024]

[Table 3]

From the results in Table 3 above, in the case of the present invention example in which the sample containing wheat germ-derived basic protein was administered,
Compared to the comparative example in which a sample containing soybean protein hydrolyzate, which is not a basic protein, was administered, the inhibitory effect on lipid absorption was greater, and the concentration of neutral fat in the blood was suppressed to a lower level. ”

Example 3 The powdered product consisting of a mixture of a plurality of basic proteins obtained in Example 1 was subjected to SDS polyacrylamide electrophoresis using a Fast system manufactured by Pharmacia. As a result, approximately 20 separate bands consisting of each basic protein appeared on the gel. The molecular weight of each of these basic proteins was between about 5,000 and 42,000.

[0027] Furthermore, when the amino acid sequence of one of the basic proteins isolated using reversed-phase HPLC was investigated, the amino acids from the 1st to the 40th amino acids counting from the N-terminus were shown in SEQ ID NO: 1. It has been revealed that the sequence is similar to the following. Furthermore, the total number of amino acid bonds in this basic protein is approximately 60 to 60, calculated from its molecular weight.
It was estimated that 100.

[0028]

[Effect of the invention] When the lipolytic enzyme inhibitor of the present invention is administered to humans or animals, the action of lipolytic enzymes is inhibited and lipid decomposition is suppressed or inhibited, so that ingested lipids are rapidly absorbed in the body. Absorption can be prevented, and the total amount of absorbed fat can also be kept low, resulting in various effects such as prevention of hyperlipidemia and obesity. The wheat germ-derived basic protein used in the lipolytic enzyme inhibitor of the present invention can be easily obtained by extracting wheat germ with an acidic solution, and is highly safe because it is derived from wheat germ. .

[Sequence list] Sequence number: 1 Array length: 40 Sequence type: amino acid Topology: linear Sequence type: protein array

Claims (4)

[Claims] Claim 1: A lipolytic enzyme inhibitor containing a basic protein derived from wheat germ as an active ingredient. 2. A method for preparing basic protein, which comprises extracting wheat germ with an acidic solution. 3. A basic protein derived from wheat germ, in which amino acids 1 to 40 counting from the N-terminus have the sequence shown in SEQ ID NO: 1. 4. The basic protein according to claim 3, which is isolated from a mixture of basic proteins obtained by extracting wheat germ with an acidic solution.