JP2753686B2 - Blood coagulation inhibitors and functional foods - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a perilla hot water extract having a fibrin aggregation inhibitory activity, a blood coagulation inhibitor having a fibrin aggregation inhibitory activity obtained from a perilla hot water extract,
Purified perilla hot water extract, method for producing the same , blood coagulation
Functional foods with added inhibitor and purified perilla hot water
The blood coagulation according to the present invention relates to a functional food to which an extract is added.
Inhibitors and functional foods are useful for treating and preventing thrombosis.

[0002]

2. Description of the Related Art Blood vessels are lined with vascular endothelial cells. When the endothelial cells are damaged, collagen is exposed. Platelets adhere and aggregate to the exposed collagen, and blood coagulation occurs. The system is activated. In this process, a fibrin network is formed, and the formed fibrin undergoes intermolecular cross-linking and also cross-linking of fibronectin, whereby the fibrin is stabilized, fixed to the tissue, and a strong thrombus is formed.

However, disseminated intravascular coagulation (DIC)
Is a syndrome characterized by activation of blood coagulation in the bloodstream due to various underlying diseases, which is characterized by organ symptoms due to microvascular thrombus and bleeding symptoms due to consumable coagulopathy. In the treatment, anticoagulant therapy is effective in addition to the treatment of the underlying disease. Currently, for example, fibrin aggregation inhibitors such as heparin, various synthetic antithrombin drugs which are thrombin inhibitors, and plasminogen activator Thrombolytic agents such as beta have been used.

[0004]

Since thrombosis therapeutic agents are frequently used in many cases, a safe therapeutic agent having few side effects is first required. Therefore, the present invention finds a blood coagulation inhibitory substance in foods taken daily , provides the substance or a purified extract, and
Use foods with these additives as functional foods.
aimed to.

[0005]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors have searched for a fibrin aggregation inhibitor in plants that are routinely used as food. As a result, the present inventors have found that a perilla plant belonging to the Labiatae family contains a blood coagulation inhibitor having an activity of inhibiting fibrin aggregation, which is effective as a therapeutic agent for thrombosis, and completed the present invention.

That is, the present invention extracts perilla with hot water, separates solid and liquid, and selectively separates one of a plurality of fractions having fibrin aggregation inhibitory activity from the resulting aqueous solution.
To provide a blood coagulation inhibitor by purification and
One of the fractions is selectively separated, purified and filtered.
To provide a perilla hot water extract having a brin aggregation inhibitory activity
It is characterized by the following. Blood coagulation inhibitor of the present invention and perilla
Hot water extracts are used as active ingredients to inhibit blood coagulation.
Agent.

[0007] The blood coagulation inhibitor of the present invention is a polysaccharide having a molecular weight of 30,000 to 100,000 and having a fibrin aggregation inhibitory activity obtained from a perilla hot water extract . In addition, the filter of the present invention
Perilla hot water extract with brin aggregation inhibitory activity is polyfe
It is a knol . Blood coagulation inhibitor of the present invention
Perilla hydrothermal water with harmful substances or fibrin aggregation inhibitory activity
The extract was separated into solid and liquid from the perilla hot water extract, the resulting aqueous solution was precipitated with a precipitant, the water-soluble components were eluted, and the eluted components were separated by column chromatography and purified. It is possible to get.

The blood coagulation inhibitor of the present invention and fibrin
Perilla as a raw material of a perilla hot water extract having an aggregation-inhibiting activity is a plant of the Labiatae family and its closely related plants. , Mint,
Swordfish, perilla, swordfish, catfish, naginata kouju,
Hikikoshi, Sorghum, Sage, Thyme, Basil, Mint, Lavender, Oregano, Sevoli, Lemon Palm,
Rosemary, catnip, hyssop, bergamot and the like can be mentioned. The leaves, stems, and seeds of these raw materials are used, and can be used for both raw and dried plants. Dried ones are convenient raw materials in terms of their excellent preservation.

[0009] The method from the Labiatae extracts hot water of the active ingredient can be carried out in an appropriate condition, in general, for example, water was added to the dried perilla leaves, boiled for 15 minutes that Done by

The further separation and purification of the active ingredient from the hot water extract obtained by the above-mentioned boiling treatment can be carried out in accordance with the usual purification method from natural products. For example, after completion of hot water extraction, a solid and a liquid are separated by filtration, centrifugation, or the like, and the resulting aqueous solution is precipitated by using a precipitant such as cetylpyridinium chloride to elute water-soluble components from the precipitate. And column chromatography [for example, chromatography using DEAE ion exchange resin (manufactured by Tosoh Corporation) and the like]. Component having fibrin aggregation inhibitory activity of the present invention
The (blood coagulation inhibitor comprising a polysaccharide) inhibiting blood coagulation component I, blood coagulation inhibitory components II, blood coagulation inhibitory components III
(Perilla fever having inhibitory activity on each purified fibrin aggregation
Water extract ).

The fraction having fibrin aggregation inhibitory activity and the blood coagulation inhibitory components I, II, and III, which are fractionated from the perilla hot water extract in the present invention, have a fibrin aggregation inhibitory activity, and are used for pharmaceuticals and functions. Used for sex foods.
A fraction having fibrin aggregation inhibitory activity in the present invention,
In order to use the blood coagulation inhibiting components I, II, and III for medicines and functional foods, any purification stage can be used. For example, in functional foods,
Functional foods having a fibrin aggregation inhibitory action can be obtained by adding various purification stages to various foods, for example, black tea, soft drinks, juices, candy, starchy foods, various processed foods, and the like. .

The blood coagulation inhibitor according to the present invention and
The perilla hot water extract having fibrin aggregation inhibitory activity can be used in a form containing other blood coagulation inhibitors or other medicaments compatible therewith.

Molecular weight : Each of the blood coagulation inhibiting components I, II, and III was measured for molecular weight by an ultrafiltration membrane method (Centreprep manufactured by Amicon), and passed through a filter having a molecular weight of 100,000. Since it does not pass through the filter, it is confirmed that the blood coagulation inhibiting components I, II and III have a molecular weight of 30,000 or more and 100,000 or less, respectively.

Solubility : The blood coagulation inhibitor I is readily soluble in water and methanol.

Both the blood coagulation inhibiting components II and III are slightly soluble in water.

None of the blood coagulation inhibiting components I, II and III are extracted with neutral ethyl acetate. None of the blood coagulation inhibiting components I, II and III are extracted with neutral n-butanol.

In various ion exchange chromatography
Adsorption characteristics : The blood coagulation inhibiting components I, II and III all adsorb to the DEAE anion exchange column. The blood coagulation inhibitor I is C
It is an acidic substance because it does not adsorb to M cation exchange resin. The blood coagulation inhibiting component I is a 1M NaCl solution,
Inhibiting blood coagulation component II is a 50% acetone solution and by inhibiting blood coagulation component III is 0.1N HCl containing 50% acetone solution, is sequentially eluted respectively. On the other hand, none of the blood coagulation inhibiting components I, II and III are adsorbed on the CM-Toyopearl cation exchange column.

Thermal stability : The blood coagulation inhibiting components I, II and III are all at 100 ° C.
It is stable by heat treatment for 0 minutes and does not inactivate the activity of inhibiting fibrin aggregation.

Enzyme treatment stability : The blood coagulation inhibiting components I, II and III were all treated at 30 ° C.
The trypsin treatment for 5 minutes does not inactivate the fibrin aggregation inhibitory activity.

Color reaction : The blood coagulation inhibitory component I is a saccharide because the color reaction by the phenol-sulfuric acid method is positive.

Since the blood coagulation inhibitory component I shows a positive result by the carbazole-sulfuric acid method, it can be understood from the above properties that it is a polysaccharide containing uronic acid.

The blood coagulation-inhibiting components II and III precipitate when treated with polycral AT, and are understood to be polyphenols in view of the above properties.

Color of the substance : The blood coagulation inhibitory component I is brown.

Specific rotation : [α] _D = −0.4 (0.
25, H ₂ O) absorption spectrum : FIG. 1 shows a chart of the purified blood coagulation inhibitory component I.

Infrared absorption spectrum : FIG. 2 shows a chart of the purified blood coagulation inhibitor I.

LD _{50 The} purified blood coagulation inhibitory components I, II, and III each have an LD ₅₀ of 2 g / Kg or more.

[0027]

EXAMPLES Example 1 Production Method of Blood Coagulation Inhibiting Component from Perilla Leaves One liter of water was added to 100 g of dried perilla leaves, and the mixture was boiled for 15 minutes. Next, after cooling the hot water extract, suction filtration was performed under reduced pressure to separate solid and liquid. The resulting aqueous solution 500
A precipitate was formed by adding 0.5 g of cetylpyridinium chloride to the mixture and leaving the mixture at 30 ° C. for 12 hours. The precipitate was washed with 100 ml of 15% ethanol, and after removing the reagent by centrifugation at 10,000 rpm for 20 minutes, 50 ml of water was added to the precipitate to elute the water-soluble active ingredient.

The obtained aqueous solution was packed in a DEAE-Toyopearl column (manufactured by Tosoh Corporation) equilibrated with 50 mM borate buffer (pH 7.0), washed with the same buffer, and then used as an eluate in a 1 M NaCl solution. , 0.1N HC
1 solution, 50% acetone solution, containing 0.1N HCl 5
Elution was performed sequentially with a 0% acetone solution. The collected fractions were dried under reduced pressure to obtain a blood coagulation inhibitor I (DEAE).
Fraction I), blood coagulation inhibitor II (DEAE fraction II), and blood coagulation inhibitor III (DEAE fraction III). Further, in order to purify each of these dried products, 0.01% of 3-
Blood coagulation inhibitor I purified by Cellulofine GC-700 gel filtration column chromatography using an aqueous solution of [(3-cholamidopropyl) -dimethylammoniol] -1-propanesulfonate]
(Purified fraction I), blood coagulation inhibitory component II (purified fraction II),
A blood coagulation inhibitory component III (purified fraction III) was obtained.

The purified fraction I, purified fraction II obtained above,
A blood coagulation test was performed using the purified fraction III. The results are shown in FIG. 3, in which the vertical axis represents the fibrin aggregation time (clotting time) and the horizontal axis represents the fraction number DEA.
3 shows an E-Toyopearl column chromatogram. The blood coagulation time was measured up to 50 minutes. According to FIG. 3, the blood in which the blood coagulation inhibiting components I, II and III of the present invention are present
Even after 50 minutes, the blood did not coagulate, indicating that there was a fibrin aggregation inhibitory activity.

Example 2 Measurement of Fibrin Aggregation Inhibitory Activity The perilla hot water extract obtained in Example 1 above, purified fraction I,
The fibrin aggregation inhibitory activity of DEAE fraction II and DEAE fraction III was determined by the method of Maruyama et al. (Biochem. Biophys. Act).
a, Vol. 1164, pp. 215-218 (1993)).

Bovine fibrinogen (Sigma)
2.5 mg of buffer (20 mM Tris-HCl, pH
7.42 ml of a solution dissolved in 1 ml of 130 mM NaCl) and 0.12 ml of various test liquids were mixed,
It was kept at 37 degrees for 1 minute. 0.5 U / ml to the reaction solution
20 mM Tris-HCl buffer (260 mM N
0.12 ml of a bovine thrombin solution in which aCl, containing 15 mM Cacl2, pH 7.4) was added, and 37
The clotting time was measured. The results are shown in the graph of FIG. 4 in which the vertical axis is the fibrin aggregation time and the horizontal axis is the various test liquids.

As a test solution, a perilla hot water extract (3
50 μg / ml), purified fraction I (25 μg / ml), D
EAE fraction II (120 μg / ml), DEAE fraction III
(120 μg / ml) and water and heparin (250 μg / ml) as controls.

FIG. 4 shows that the perilla hot water extract, the purified fraction I, the DEAE fraction II, and the DEAE fraction III exhibit fibrin aggregation inhibitory activity.

Example 3 LD ₅₀ Purified blood coagulation inhibitory component I (purified fraction I) and blood coagulation inhibitory component II (purified fraction I) obtained in Example 1 above.
When an acute toxicity test was performed on I) and the blood coagulation inhibitory component III (purified fraction III) using ICR mice (male), there were no deaths due to oral administration of 2 g / Kg of these substances. Therefore, it can be seen that the LD ₅₀ of each of the purified fractions I, II, and III is 2 g / Kg or more.

Example 4 Commercially available black tea leaves were pulverized, and the blood coagulation inhibitory component I of the present invention was added to the pulverized material so as to be 3% by weight and mixed well. These three types of mixtures were packed in tea bags to obtain tea bags to which the blood coagulation inhibitory component I was added. By exuding this tea bag with boiling water,
The blood coagulation inhibitor I was eluted and mixed into the black tea, and black tea having fibrin aggregation inhibitory activity was obtained.

Example 5 Three 1 kg of commercially available orange juice were prepared. To each of the orange juices, one of the blood coagulation inhibitory components I, II, and III of the present invention was added in an amount of 3% by weight and mixed well to obtain an orange juice having a fibrin aggregation inhibitory activity. .

[0037]

EFFECTS OF THE INVENTION Perilla hot water extract obtained by the present invention
Yes La purified molecular weight having a fibrin aggregation inhibitory activity comprising the 30000 to 100000 polysaccharide consisting of blood coagulation inhibitor and / or purification process is subjected fibrin aggregation inhibitory activity
Perilla hot water extract, specifically, fibrin aggregation inhibition
Perilla hot water extract consisting of active polyphenols
Exhibit excellent fibrin aggregation inhibitory activity as described herein. These substances and each purified perilla hot water extract are :
It has a high intake capacity and is useful as a blood coagulation inhibitor or a functional food to which the substance or component is added.

[Brief description of the drawings]

FIG. 1 shows a chart of an absorption spectrum of a purified blood coagulation inhibitory component I (a blood coagulation inhibitor composed of a polysaccharide).

FIG. 2 shows a chart of an infrared absorption spectrum of purified blood coagulation inhibitor I (a blood coagulation inhibitor composed of a polysaccharide).

FIG. 3 shows DEAE- of the purified fraction I, the purified fraction II, and the purified fraction III, in which the ordinate represents the fibrin aggregation time (clotting time) and the abscissa represents the fraction number.
The Toyopearl column chromatogram is shown.

FIG. 4 is a graph in which the vertical axis is the fibrin aggregation time and the horizontal axis is various test solutions.

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Kazuya Nakagome 1-1-3 Higashi, Tsukuba City, Ibaraki Prefecture Within the Institute of Biotechnology, Industrial Technology Institute (72) Inventor Susumu Maruyama 1-3-1 Higashi 1-3 Tsukuba City, Ibaraki Industry (72) Inventor Tomoko Okada 1-1-3 Higashi, Tsukuba, Ibaraki Pref., Japan Institute of Biotechnology Industrial Technology (72) Inventor, Eiko Kuroda 1-1-1, Higashi, Tsukuba, Ibaraki Pref. 3 Within the Institute of Biotechnology, Industrial Technology Institute (72) Inventor Hiroaki Okuno 1-3-1, Higashi, Tsukuba, Ibaraki Prefecture Within the Institute of Biotechnology, Industrial Technology Institute (72) Inventor, Noboru Tomizuka 1-Higashi, Tsukuba, Ibaraki No. 1-3 Inside the National Institute of Advanced Industrial Science and Technology (72) Inventor Yasunori Fukumori 1 Kita 4 Jo Nishi, Chuo-ku, Sapporo, Hokkaido Chome-3, Hokuren Agricultural Cooperative Federation Examiner Mika Fuji (56) References JP-A-7-223962 (JP, A) JP-A-5-58904 (JP, A)

Claims (4)

(57) [Claims] 1. Extracting perilla with hot water, separating solid and liquid,
The resulting aqueous solution was obtained from a plurality of fractions, one fraction having different and <br/> physicochemical properties have fibrin aggregation inhibitory activity from, (at 1) Molecular weight 30,000 to 100,000, (2 ) Easily soluble in water and methanol, (3) not extracted with neutral ethyl acetate, not extracted with neutral n-butanol, (4) adsorbed on a DEAE anion exchange column and
Eluted with Cl solution, (5) did not adsorb to CM cation exchange resin, (6) did not inactivate fibrin aggregation inhibitory activity by heat treatment at 100 ° C for 30 minutes, (7) fibrin aggregated by 15 minutes of trypsin treatment Inhibitory activity
Sex not deactivated, (8) a phenol - a positive color reaction with sulfuric acid method, (9) carbazole - a reaction with sulfuric acid method is positive (10) and the dryness things brown ( 11) Specific rotation is [α] _D = -0.4 (0.25, H
A ₂ O), blood coagulation inhibitor comprising a polysaccharide having a property. 2. Extracting perilla with hot water, separating solid and liquid,
Having fibrin aggregation inhibitory activity from the obtained aqueous solution;
Select one fraction of multiple fractions with different physicochemical properties
Separated and purified, the following physicochemical (1) to (11)
Obtaining a blood coagulation inhibitor comprising a polysaccharide having properties;
Add a blood coagulation inhibitor to food to achieve blood coagulation inhibitory function
Functional foods that are given to foods. (1) It has a molecular weight of 30,000 or more and 100,000 or less, (2) It is easily soluble in water and methanol, (3) Neutral n-butanol, not extracted with neutral ethyl acetate
Is not extracted by (4) Adsorbed on a DEAE anion exchange column,
Eluted with Cl solution, (5) Not adsorbed on CM cation exchange resin, (6) Fibrin aggregation inhibitory activity by heat treatment at 100 ° C for 30 minutes
Sex does not deactivate, (7) fibrin aggregation inhibitory activity by 15 minutes of trypsin treatment
Sex does not deactivate, (8) The color reaction by the phenol-sulfuric acid method is positive, (9) The reaction by the carbazole-sulfuric acid method is positive, (10) The dried product is brown, (11) Specific rotation is [α] _D = −0.4 (0.25, H
_Two O). 3. Extracting perilla with hot water, separating solid and liquid,
Having fibrin aggregation inhibitory activity from the obtained aqueous solution;
Select one fraction of multiple fractions with different physicochemical properties
Separation and purification give the following physicochemical properties (1) to (8)
Perilla hot water extract consisting of polyphenols having properties
And adding the perilla hot water extract to food to inhibit blood coagulation
Functional foods characterized by giving functions to foods. (1) It has a molecular weight of 30,000 or more and 100,000 or less, (2) slightly soluble in water, (3) Neutral n-butanol, not extracted with neutral ethyl acetate
Is not extracted by (4) Adsorbed on a DEAE anion exchange column,
Not eluted with Cl solution, eluted with 50% acetone solution, (5) Not adsorbed on CM cation exchange resin, (6) Fibrin aggregation inhibitory activity by heat treatment at 100 ° C for 30 minutes
Sex does not deactivate, (7) fibrin aggregation inhibitory activity by 15 minutes of trypsin treatment
Sex does not deactivate, (8) Precipitates when treated with polycral AT. 4. Extracting perilla with hot water, separating solid and liquid,
Having fibrin aggregation inhibitory activity from the obtained aqueous solution;
Select one fraction of multiple fractions with different physicochemical properties
Separation and purification give the following physicochemical properties (1) to (8)
Perilla hot water extract consisting of polyphenols having properties
And adding the perilla hot water extract to food to inhibit blood coagulation
Functional foods characterized by giving functions to foods. (1) has a molecular weight of 30,000 or more and 100,000 or less, (2) is slightly soluble in water, (3) is not extracted with neutral ethyl acetate, and is neutral n-butanol.
In not extracted, adsorbed to (4) DEAE anion exchange column, 1M Na
Not eluted with Cl solution, eluted with 50% acetone solution
Eluted with 50% acetone solution containing 0.1N HCl
And, (5) not adsorbed to the CM cation exchange resin, (6) a fibrin aggregation inhibitory activity in heat treatment at 100 ° C. 30 minutes
Sex not deactivated, fibrin aggregation inhibitory activity with trypsin treatment (7) 15 minutes
Sex not inactivated, precipitated with handles (8) Porikuraru AT.