WO2009057725A1

WO2009057725A1 - Protease-treated jew's mallow extract composition, method for production of the composition, and anti-hypertensive agent or beverage/food comprising the composition

Info

Publication number: WO2009057725A1
Application number: PCT/JP2008/069818
Authority: WO
Inventors: Yuji Kubota; Hideki Kano; Takanobu Takihara; Atsuhiro Mitomi
Original assignee: Ito En, Ltd.
Priority date: 2007-10-31
Filing date: 2008-10-24
Publication date: 2009-05-07

Abstract

Disclosed are: an anti-hypertensive agent which is effective for a patient suffering from hypertension, which has no adverse side effect, and which is highly safe and is therefore suitable for long-term administration; a food such as a health food, a a nutritional supplement and a food for specified health uses, which comprises the composition; and a beverage which comprises the composition. Specifically disclosed is a protease-treated Jew's marrow (Corchorus olitorius L.) extract composition which is produced by treating a Jew's marrow leaf, a Jew's marrow extract, a solution of a Jew's marrow extract, or a Jew's marrow puree with a protease. The composition has an excellent anti-hypertensive activity.

Description

The present invention relates to a proteolytic enzyme-treated Morohella extract composition, a method for producing the same, and an antihypertensive agent or food and drink containing the composition.

The present invention relates to a proteolytic enzyme-treated Morohella extract composition having a blood pressure lowering action, more specifically, a proteolytic enzyme-treated Morohaya obtained by subjecting Morohaya leaves, Morohaya extract or Morohaya puree to protease extraction. Extract composition, antihypertensive agent containing the proteolytic enzyme-treated moroheia extract composition as an active ingredient, food and drink comprising the proteolytic enzyme-treated morohea extract composition, and the proteolytic agent The present invention relates to a method for producing an enzyme-treated molhahea extract composition. Background art

1. About hypertension

In Japan, it is said that approximately 35 million people have hypertension. The second most common cause of death in Japan is heart disease and the third is cerebrovascular disease. Hypertension is deeply related to the cause of these diseases. As hypertension has no subjective symptoms, arteriosclerosis develops without being noticed, and this arteriosclerosis causes heart disease, angina pectoris, cerebral hemorrhage, cerebral infarction, kidney disease and other various complications.

Conventionally, general therapy and drug therapy have been used as methods for preventing and treating hypertension. General therapy is a method of treatment by exercise therapy such as weight loss, alcohol saving, aerobic exercise, etc., and dietary therapy such as salt reduction, and is the basis for treatment of essential hypertension. Although general therapy does not use drugs, there are advantages in terms of side effects or economic burden, but it depends on the individual's willingness and the cooperative system of the surroundings, and does not necessarily have a certain effect on improving hypertension. There are problems such as.

On the other hand, pharmacotherapy is a therapy used for hypertension in which blood pressure does not normalize by general therapy, such as when the effects of dietary therapy or exercise therapy are insufficient, or for severe hypertension. Drugs used in pharmacotherapy include calcium antagonists, angiotensin converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, diuretics, and sympathetic inhibitors And so on. However, since these drugs used as antihypertensive agents require long-term use, they generally have side effects such as digestive symptoms, orthostatic hypotension, and changes in metabolic function. In particular, ACE inhibitors are widely known as side effects such as dry cough, dizziness, standing up, nausea, roar, and excessive sedation, which lowers QOL and leads to reduced compliance. In addition, once medication is started, it needs to be continued, and it is not possible to stop or change the amount of medication at the patient's discretion, which increases the economic and mental burden. . Therefore, there has been a demand for an effective therapy that is free from side effects, has a low economic and mental burden, is safe, can be continued for a long period of time, and is effective.

2. Health foods, functional foods, health supplements, etc.

Therefore, what is attracting attention in order to solve the above problems is the treatment or prognosis with foods that can be said to be located between drug therapy with antihypertensive drugs and general therapy. It is a prevention method. Such foods are sometimes referred to as health foods, functional foods, health supplements, and foods for specified health use. Some foods that are taken on a daily basis contain substances that have a blood pressure lowering effect. .

For example, tea beverages containing plant-derived peptides are sold as beverages suitable for people with high blood pressure. For example, sesame protein degradation product (containing sesame peptide) L eu— V a 1-T yr (LVY), which is obtained by extracting the protein after defatted sesame and acting on the degrading enzyme, is the relevant component. Some have ACE inhibitory action and blood pressure lowering action. In addition, dairy lactic acid bacteria beverages with γ-aminobutyric acid (GABA) added are on the market. G A B A is a kind of amino acid, a typical inhibitory neurotransmitter, and has long been known to have a blood pressure lowering effect. Tea drinks for foods for specified health use that contain geniposide acid, the main component of the sugar cane glycosides contained in Tochu tea leaves, are also commercially available. Geniposide acid acts on the parasympathetic nerve and lowers blood pressure by smoothing blood flow. In addition, Naoyuki Yamamoto et al. Reported under the title “Lactic acid bacteria and fermented dairy products” that Jodalt produced by fermentation of lactic acid bacteria using milk as a raw material has a blood pressure lowering effect (see Patent Document 1). .

However, the causative substance of the blood pressure lowering effect is not clear. In addition, many health foods and beverages that have a blood pressure lowering effect are sold S, more effective products, products that can be taken for a long time with peace of mind, products with less economic burden However, there is always a need.

As other natural product ingredients, it has been reported that milk protein, soy protein, or fish protein contains various ACE inhibitory peptides. Practical use of antihypertensive agents with low toxicity and high safety is being investigated. However, it is included in these natural product-derived peptides The blood pressure-lowering peptide is very small, and there are many that cannot be expected to have a great effect with realistic oral intake, or that have a strong ACE inhibitory activity but a low blood pressure-lowering effect.

In recent years, fermented milk from lactic acid bacteria has been reported to have two kinds of tripeptides having strong ACE inhibitory activity, namely, Val_Pro_Pro and Ile_Pro-Pro (for example, non- (See Patent Document 1). Furthermore, there is a report that the strong blood pressure lowering effect of these tripeptides was confirmed in spontaneous hypertension rat (SHR) (for example, see Non-Patent Document 2). However, since tripeptide produced in lactic acid bacteria fermented milk is produced by proteolytic enzymes produced by lactic acid bacteria as lactic acid fermentation progresses, the amount of tripeptide is likely to fluctuate depending on the fermentation conditions, and should be obtained with a constant content. Is difficult.

As described above, the causative substances of blood pressure lowering effects are clear, industrially stable production is possible, and blood pressure lowering substances derived from natural products that are more effective and safer than conventional products and contain them Health foods and health drinks are required.

3. Morohaya

Morohaya (scientific name: Corchorus oitorius L.) is a kind of jute that originates in the Mediterranean region centering on Egypt, and is one of the food ingredients that has attracted particular attention in recent years due to its high nutritional value. Moroheiya, when minced or boiled, produces a unique jelly like Otara potato, which contains plant gum and viscous polysaccharides (mucopolysaccharides). Nutritionally, vitamins and minerals are abundant, and in particular, the total strength of rotin and calcium is high. Rotin is about 12 times that of Proccoli, Vitamin B 1 is about 3 times tomato, Vitamin B 2 is about 14 times pepper, It is said that um is about 2.4 times milk and dietary fiber is about 20 times lettuce. For this reason, it has recently been cultivated in Japan, and its dry powder along with fresh leaves is gaining attention as a food material.

To date, there have been many proposals for Morohaya, Morohaya powder and Morohaya extract, foods and compositions containing them, and methods for producing the same.

For example, Takashi Yamamoto and others have reported a production method for extracting a moroheiya extract by adding an organic acid for the purpose of lowering the viscosity of the moroheia extract for use in beverages (see Patent Document 2). . Moreover, Satoshi Inami and others have reported a composition containing an ethanol precipitate as an active ingredient in the water-soluble fraction of morohea leaves (see Patent Document 3). However, this active ingredient is dietary fiber.

In addition, Masayuki Yoshikawa et al., Under the title of “Composition containing aqueous extract of Morohaya,” extract gastric muyahea by heating with purified water, concentrate the extract and freeze-dry it. It has been reported on a composition comprising an aqueous extract of Morohaya having an essential component (see Patent Document 4). Furthermore, Yoshiyuki Masayuki et al. Describe a novel fatty acid or salt thereof, a fatty acid extract, a method for isolation thereof, and a pharmaceutical composition having an action of inhibiting nitric oxide (NO) production containing the fatty acid as an active ingredient. (See Patent Document 5).

4. Blood pressure lowering effect of Morohaya

As for the blood pressure lowering effect of Moguchihaya, the extract has been confirmed to have ACE inhibitory activity at the test tube level. Furthermore, nicotianamine has been isolated as an ACE inhibitory activity component of Morohaya extract (see Non-Patent Documents 3, 4, and 5). C

6 Norio Yamaguchi and others have proposed a method for efficiently producing nicotianamine from an aqueous soybean extract (see Patent Document 6). However, it has been pointed out that nicotianamine contains only a very small amount of morrohea, which requires a large amount of raw materials and a complicated purification process to actually be used as an antihypertensive agent. .

[Patent Document 1] Japanese Patent Laid-Open No. 7-1 2 3 9 7 7

[Patent Document 2] Japanese Patent Laid-Open No. 10-1 9 1 9 2 2

[Patent Document 3] Japanese Patent Laid-Open No. 7-6 9 9 1 0

[Patent Document 4] Japanese Patent Laid-Open No. 2 0 0 0-2 6 3 0 5

[Patent Document 5] Japanese Patent Application Laid-Open No. Hei 11 1 3 2 2 6 6 7

[Patent Document 6] Japanese Patent Laid-Open No. 2 0 0 3-2 3 1 6 7 5

[Non-Patent Document 1] J. Dairy Sci. 78: p.777-783

[Non-Patent Document 2] J. Dairy Sci. 78: 1253-1257

[Non-Patent Document 3] Food Testing and Research, No. 31, 7 8-7-9, 1 9 9 6 “Morrohair nutrients and angiotensin I-converting enzyme inhibitory activity”, Akihiko Arakawa

[Non-Patent Document 4] Bulletin of Tokyo Kasei University, Vol. 38 (2), 5 9— 6 3, 1 9 9 8 “Research on ACE inhibitors in foods”, Koichi Kimoto, Emiko Shimizu, Kuroda Yuko

[Non-Patent Document 5] Journal of the Japanese Society of Dietary Life, Vol. 10, No. 3, 2 0— 2 5, 1 9 9 9 “About antihypertensive substances in foods”, Koichi Kimoto Disclosure of the Invention

Problems to be solved by the invention

As mentioned above, for the improvement of hypertension, there is no fear of side effects, economical There has been a demand for safe and effective therapies with a light mental burden. In particular, health foods, functional foods, health supplements, etc. that have an effect of lowering blood pressure, which are located between drug therapy and general therapy, have been strongly demanded.

Therefore, the object of the present invention is effective for hypertensive patients, particularly essential hypertensive patients, which are said to account for 90%, and is derived from a safe food suitable for long-term use without side effects. It is intended to provide vasodilators or antihypertensive agents, foods and drinks containing these compositions, particularly foods such as health foods and health supplements, or beverages containing them. Furthermore, it is providing the manufacturing method of those compositions. Means for solving the problem

In order to solve the above-mentioned problems, the present inventors focused on Morohaya, which has been attracting attention as a food material with high nutritional value in recent years. Proteolytic enzyme treatment of leaves and the like makes it possible to produce a proteolytic enzyme-treated mochahae extract composition having a superior blood pressure lowering action, specifically, vasodilation or vascular relaxation. As a result, the present invention has been completed. Specifically, the present invention is as follows.

1. A proteolytic enzyme-treated morohella extract composition having a blood pressure lowering action obtained by treating morohaya with a protease.

2. The proteolytic enzyme-treated Morohella extract composition according to 1 above, wherein the protease is an endopeptidase.

3. The endopeptidase is an endopeptidase from Bac il lus. 3. The proteolytic enzyme-treated morohea extract composition according to item 2.

4. A method for producing a proteolytic enzyme-treated moroheiya extract composition comprising adding a protease to moroheiya hydrolyzate and applying enzyme treatment to inactivate the enzyme.

5. The moroheiya hydrolyzate is a morohea leaf solution obtained by adding morohae leaves to water or warm water, moroheiya extract, moroheiya extract solution or moroheia puree obtained by dissolving moroheiya extract powder in water or hot water Of Proteolytic Enzyme-treated Morohella Extract Composition.

6. An antihypertensive agent comprising the proteolytic enzyme-treated moroheiya extract composition as described in 1 to 3 above as an active ingredient.

7. A food or drink comprising the proteolytic enzyme-treated moroheiya extract composition described in 1 to 3 above. The invention's effect

Since the proteolytic enzyme treatment composition of the present invention is derived from Morohaya whose safety has been confirmed as food, it is extremely safe without side effects and can be taken on a daily basis. In addition, the production method is very easy because only protease (protein degrading enzyme) is allowed to act on the leaf of moroheiya, moroheiya extract or moroheia puree obtained by adding water or warm water. In addition to Morohaya itself as a vegetable, the blood pressure lowering action is remarkably superior to conventional ordinary extracts obtained without using a proteolytic enzyme. Furthermore, since Morohaya is a plant rich in vitamins, minerals, and dietary fiber and has high nutritional value, the proteolytic enzyme of the present invention can be used when compared with a single component that has a blood pressure lowering effect. If you ingest the treatment composition, It is possible to take multiple ingredients at the same time, and further effects can be expected. In addition, because it is made from the natural plant Moroheiya, it is inexpensive and highly safe, making it ideal for pharmaceuticals, foods for specified health use, health foods, beverages, etc. Brief Description of Drawings

FIG. 1 is a graph showing systolic blood pressure fluctuations caused by administration of a moroheia enzyme-treated extract. (Test Example 1)

FIG. 2 is a graph showing diastolic blood pressure fluctuations caused by administration of a moroheia enzyme-treated extract. (Test Example 1)

FIG. 3 is a graph showing heart rate variability due to administration of Morohaya enzyme-treated extract. (Test Example 1)

Fig. 4 is a graph showing fluctuations in mean blood pressure due to administration of a moroheiya enzyme-treated extract. (Test Example 1)

FIG. 5 is a diagram showing the diurnal variation in systolic blood pressure of the SHR rat. (Test example 1)

FIG. 6 is a graph showing the effect of morohaya enzyme-treated extract prepared from morohaya puree on blood pressure fluctuations. (Test Example 2)

FIG. 7 is a graph showing the results of measuring the ACE inhibitory activity of Morohaya enzyme-treated extract. (Test Example 3)

FIG. 8 is a diagram showing the vasorelaxant action of Morohaya enzyme-treated extract.

(Test Example 4) The best mode for carrying out the invention

The proteolytic enzyme-treated Morohella extract composition of the present invention is Proteolytic enzyme, ie, proteaase It can be obtained by acting and extracting. For example, in the case of Morohaya leaves, water or hot water is added to Morohaya leaves from which hard stem portions have been removed, and then steam heating is performed to crush them. Next, hot water of about 10 to 40 times, preferably about 20 to 30 times the weight of distilled water is added to the dried morohea leaves obtained by drying the crushed morohea leaves with hot air. After that, after heat sterilization at a temperature of 95 ° C or higher, cool to an appropriate temperature and maintain the liquid temperature in a water bath, and apply an appropriate amount of peptate dissolved in a small amount of water. An appropriate amount of zea is added to the dry leaf weight or dry leaf equivalent weight of the raw material. After adding the protease, stir every 10 minutes and hold for 15 minutes to 2 hours, preferably 45 to 90 minutes after adding the protease, and then hold at 95 ° C for 5 minutes. Proteolytic enzyme-treated Morohaya extract composition of the present invention is obtained by inactivating protease, filtering through a sieve having an opening of 85 5 μ ηι, and then suction filtration using No. 2 filter paper Can do. Treatment conditions such as solution temperature, enzyme concentration and pH for protease treatment may be adjusted so that the protease to be used works optimally.

The proteolytic enzyme-treated Morroheia extract composition thus obtained can be used as it is as a beverage, or in other fruit juices, vegetable juices, and mix juices. Further, it can be made into a concentrated liquid composition by concentration under reduced pressure to an appropriate concentration, and it can be freeze-dried and powdered according to the purpose. These concentrated liquid compositions and powdered enzyme treatment compositions can be used by adding to foods, beverages, etc. The

The morohaya hydrolyzate as the target of the enzyme treatment is an aqueous solution or hot water solution containing morohaya leaves or morohaya extract as a raw material, and is not particularly limited. Specifically, it is a morohella leaf solution in which morohea leaves are mixed in water or warm water, a morohaya extract solution, a morohaya extract solution or morohya puree in which morohaya extract powder is dissolved in water.

As the moroha leaves, any part of the above-ground part except the seeds may be used, but the part of the leaves from which the hard stem part is removed is particularly preferable. It is preferable to use dried leaves obtained by crushing them after steam heating and drying with hot air, etc., but the method for preparing the dried leaves is not particularly limited. Can also be used. It is desirable to treat the dried morohay leaves with water or warm water, more preferably after pulverization. The extraction time varies slightly depending on the extraction temperature, but is 15 minutes to 2 hours, preferably 45 minutes to 90 minutes. In addition to dried leaves, for example, Morohaya extract, which is an extract, Morohaya extract solution obtained by dissolving Morohaya extract powder in water, and Morohaya puree can also be used.

The protease enzyme used is not particularly limited, but is preferably an endopeptidase or an endopeptidase-based complex enzyme. A plurality of enzymes can also be used in combination.

The proteolytic enzyme or protease used in the present invention is an enzyme that catalyzes a peptide bond hydrolysis reaction. Although it is not particularly limited, it is preferably an end-type enzyme or an end-type complex enzyme. Of these, a proteolytic enzyme produced by Bacillus genus is more preferable. More preferably produced by Bac ill us subt ilis and Bac il lus thermoproteo lyt i cus 2 Proteolytic enzyme. It is also possible to use a combination of multiple enzymes.

The pH, treatment temperature, treatment time, and treatment concentration during protease treatment are not particularly limited, but can be appropriately selected depending on the enzyme used. ¾, ρ Η 5 · 0 to 8.5, more preferably ρ Η 5.5 to 7.0, treatment temperature 40 ° C to 70 ° C, preferably 50 ° C to 65 ° C The treatment time is 15 minutes to 2 hours, preferably 45 minutes to 90 minutes, and the preferred enzyme concentration is 0.001 to 3% by weight, preferably 0. 0 to 2% by weight, more preferably 0.1 to 1.5% by weight. However, it goes without saying that these conditions may be changed depending on the type of enzyme. For example, when using protease derived from Bacillus subtilis, pH 6.0 to 7.0, treatment temperature 55 ° C to 60 ° C, treatment time 1 hour to 2 hours, treatment concentration is 0.1 to 1.0 weight of the dry weight of the chamber. /. Is preferred. When using one of the proteases derived from Bacillus therraoproteolyticus, pH 6.0 to 7.0, treatment temperature 60 ° C to 70 ° C, treatment time 1 hour to 2 hours, treatment concentration is It is preferably 0.1 to 1.0% by weight of the dry weight of moroheiya.

After the enzyme treatment, the enzyme is deactivated by heating, and solid-liquid separation is performed by any method such as filtration, and the extract is recovered. The enzyme deactivation method is appropriately selected depending on the enzyme used, but can be performed by heating. For example, the enzyme may be left at 95 ° C. to boiling for 5 to 10 minutes.

“Proteolytic enzyme-treated morohea extract composition” means a composition obtained by treating morohea with proteolytic enzyme in water or warm water and extracting it. These proteolytic enzyme treatment compositions may be liquid or solid. Protein The white matter-degrading enzyme treatment solution itself, a proteolytic enzyme treatment solution obtained by solid-liquid separation of insoluble components such as stems, a concentrated concentrate obtained by concentrating it, and a powder obtained by freeze-drying them, or solidifying the powder It may be a tablet.

In the present specification, Morohaya hydrolyzate, that is, Morohaya leaf solution obtained by adding Morohaya leaves to water or hot water, Morohaya extract, Morohaya extract solution or Morohaya extract solution obtained by dissolving Morohaya extract powder in water or warm water. An extract obtained by treating le with a proteolytic enzyme is referred to as a morohea enzyme-treated extract or enzyme-treated extract.

The enzyme-treated solution and concentrated solution separated by solid-liquid can be used as beverages as they are or mixed with other soft drinks such as fruit juice vegetable juice. The powder or solid tablet itself can be used as a health food, functional food, health supplement, etc., and can also be used as a compounding ingredient for soft drinks and foods.

The proteolytic enzyme-treated moroheiya extract composition obtained in the present invention has a small taste that is peculiar to taste and odor, and therefore can be taken orally in liquid or solid form. The intake varies depending on the age and blood pressure level, but is usually 10 mg to 600 mg at a time in terms of powder, preferably 50 mg to 300 mg, more preferably once 10 mg to l 500 mg. The effect can be obtained by taking 1 to 3 times a day, but the number can be increased if necessary. In addition, when contained in a beverage, it is 50 mg to 600 mg, more preferably 50 mg to 400 mg, per 50 mL.

The proteolytic enzyme-treated morohea extract composition obtained in the present invention is itself dried, if necessary, or appropriately mixed with excipients or the like for convenience of formulation, such as powders, granules, tablets, and force-pellants. It can be administered in the form. Also, candy, jelly, tablet confectionery, beverage, soup, candy, rice cracker, Japanese confectionery, frozen confectionery, baked confectionery, etc. It is possible to mix and add to various foods and drinks. Preferably, it may be used by mixing with other foods and drinks, particularly preferably in beverages such as fruit drinks, vegetable juices, fruit vegetable juices, tea drinks, coffee drinks, sports drinks and the like. By doing so, not only the nutrients contained in vegetable juice etc. can be ingested on a daily basis, but also natural products having a blood pressure lowering effect can be ingested naturally for hypertensive consumers.

A preferred form of food or drink containing the proteolytic enzyme-treated morohea extract composition is a koji, jelly, tablet confectionery, beverage, soup, koji, rice cracker containing the proteolytic enzyme-treated moroheia extract composition. , Japanese confectionery, frozen confectionery, baked confectionery, etc., particularly preferably, fruit juice beverage, vegetable juice, fruit vegetable juice, tea beverage, coffee beverage, sports drink comprising the proteolytic enzyme-treated Morohaya extract composition And so on.

The examination of the blood pressure lowering effect of the proteolytic enzyme-treated moroheia extract composition of the present invention is to orally administer the moroheia extract of the present invention to a spontaneous hypertension rat and measure the blood pressure before and after administration. It went by.

Furthermore, the vasodilatory / relaxing action, that is, the blood pressure lowering action mechanism of the proteolytic enzyme-treated morohea extract composition of the present invention is considered to have been reported by nicotianamine contained in moroheia leaves that have been reported so far. It was also confirmed that it was not due to activity, but due to vasorelaxant action / vasodilatory action. That is, it was confirmed that the blood pressure lowering action of the proteolytic enzyme-treated Morohaya extract composition of the present invention was not due to nicotianamine contained in Morohaya leaves. The present invention has been completed based on these new findings.

Hereinafter, the present invention will be specifically described with reference to comparative examples and examples. In addition, this departure Ming is not limited to these examples.

[Comparative Example 1]

Preparation of Morohaya extract A (without protease treatment);

Morohaya leaves from which hard stems had been removed were steam-heated and then crushed and dried with hot air to obtain dried Morohaya leaves. Add 50 times the weight of distilled water of about 60 ° C to 50 g of the dried morohea leaves, heat to 95 ° C or higher, sterilize, cool to 60 ° C, Stirring was performed every 10 minutes while maintaining the liquid temperature at 60 ° C in a water bath. After 1 hour, hold at 95 ° C for 5 minutes, filter with a sieve with an opening of 8 50 μra, and then suction filter using No. 2 filter paper to obtain Morohaya extract . This extract was concentrated under reduced pressure to an appropriate concentration and then freeze-dried to obtain 6.8 g of a brown powder. Hereinafter, this powder is referred to as “A”. Example 1

Preparation of Morohaya enzyme-treated extract B (Protease N Amano G treatment); Morohaya leaves from which hard stems had been removed were steam-heated and then crushed and dried with hot air to obtain dried Morohaya leaves. Add 50 times the weight of distilled water of about 60 ° C to 50 g of dried moroheiya leaves, heat to 95 ° C or higher, sterilize, cool to 60 ° C, Keep the solution at 60 ° C in a water bath, and add 0.7% by weight of protease N Amano G (manufactured by Amano Enzyme Co., Ltd.) dissolved in a small amount of water to the raw dried Morohaya leaves. . Thereafter, stirring was performed every 10 minutes while the liquid temperature was kept at 60 ° C. 1 hour after addition of the enzyme, hold at 95 ° C for 5 minutes, filter through a sieve with an opening of 85 0 m, then N o. Suction filtration was performed using the filter paper 2 to obtain a moroheiya enzyme-treated extract. This enzyme-treated extract was concentrated under reduced pressure to an appropriate concentration and then freeze-dried to obtain 1 5 7 5 g of brown powder. . Hereinafter, this powder is referred to as “B”. Example 2 Preparation of Morohaya enzyme-treated extract B ′ (Samoase Y10 treatment);

Morohaya leaves from which hard stems had been removed were steam-heated and then crushed and dried with hot air to obtain dried Morohaya leaves. Add 50 times the weight of distilled water at around 60 ° C to 50 g of the dried moroheiya leaves, heat to 95 ° C or higher, sterilize, cool to 60 ° C, The solution temperature was kept at 60 ° C. in a water bath, and 0.7% by weight of Samoaise Y 10 (manufactured by Yamato Kasei) was dissolved in a small amount of water and added to the raw dried moroya leaves. Thereafter, stirring was performed every 10 minutes while the liquid temperature was kept at 60 ° C. 1 hour after addition of the enzyme, hold at 95 ° C for 5 minutes, filter with a sieve with an opening of 8 50 μm, and then suction filter using No. 2 filter paper. An enzyme-treated extract was obtained. This enzyme-treated extract was concentrated under reduced pressure to an appropriate concentration and then lyophilized to obtain 7 g of a brown powder. Hereinafter, this powder is referred to as “B ′”.

table 1

Sample Enzyme Optimal condition Type Origin Strength-Comparative Example 1 None-~~ Extract A

Example 1 T-mouth tear-se "PH 7.0-end Bacillus subtin ns Amano; D's thyme extract B 55 ° C

Example 2 Samoa-C 'Y10 pH7-8.5 End type Bad // us Daiwa Kasei Extract B' 65-70 ° C thermoproteolyticus

Rokko ^

[Test Example 1]

Evaluation test of blood pressure lowering effect;

We examined the antihypertensive effect of a single administration using spontaneous hypertension rat (SHR).

The powders A, B, B ′ obtained in Comparative Example 1 and Examples 1 and 2 were dissolved in water for injection so as to be 100 mg / kg (body weight of SHR). ) Was administered to the spontaneously hypertensive rat (S HRZHo, SPF) with a single gastric tube. Blood pressure was measured noninvasively before administration, 4 hours, 8 hours, and 24 hours after administration. The number of animals used in the test is shown in Table 2 below. In addition, although 7 mice were used for each group, data on individuals that clearly showed abnormal blood pressure fluctuations were deleted.

Table 2

B;

Figure 1 shows the changes in systolic blood pressure up to 24 hours after administration. Figure 2 shows changes in diastolic blood pressure up to 24 hours after administration. Figure 3 shows changes in heart rate up to 24 hours after administration. Figure 4 shows the change in mean blood pressure up to 24 hours after administration. Figure 5 shows the diurnal variation in systolic blood pressure of SHR up to 24 hours after administration.

It can be judged that the blood pressure fluctuation of the sputum is within the daily fluctuation range. On the other hand, B and B ′ showed strong blood pressure drop 4 hours after administration. Also in terms of mean blood pressure B and B ′ each showed a strong blood pressure drop 4 hours after administration.

As is clear from the test results of the morohaya enzyme-treated extract of Example 1 (B) and Example 2 (B ′) above, the enzyme-treated extract obtained by treating morohaya leaves with a proteolytic enzyme is an enzyme-treated extract. It exhibited a stronger blood pressure lowering effect than the normal excision without treatment. In each figure, each value represents an average value. Example 3

Preparation of Morohaya Enzyme-treated Extract C from Moguchiha Puree;

After steam blanching Moroheiya leaf stems with hard stems removed, 1.

5 times the amount of water was added and crushed and stored frozen in a pure state. This frozen puree was thawed in running water, 1.5 times the weight of distilled water (1500 g) was added to 100 g of puree, and mixed uniformly with a juicer mixer. This was sterilized by heating to 95 ° C or higher, cooled to 60 ° C, kept at a liquid temperature of 60 ° C in a water bath, and 0.0% of the initial puree weight (10 00 g). 5% by weight of protease N Amano G (manufactured by Amano Enzym Co., Ltd.) was dissolved in a small amount of water and added. Thereafter, stirring was performed every 10 minutes. One hour after the addition of the enzyme, it was kept at 95 ° C. for 5 minutes and treated with a centrifuge to remove puree solids. This was filtered through a sieve having an opening of 8500 μm, and then suction filtration was performed using a No. 2 filter paper to obtain an enzyme-treated extract. The extract was concentrated under reduced pressure to an appropriate concentration and then lyophilized to obtain 23 g of a brown powder. Hereinafter, this powder is referred to as “C”.

[Test Example 2] Antihypertensive effect after a single administration to spontaneous hypertension rat _ (hereinafter referred to as SHR); the obtained powder C was dissolved in injection water so as to be 100 mg Z kg (body weight of SHR) 1 A single oral dose was administered to an 8 week old (male) spontaneous hypertension rat (SHRZHos, SPF) using a gastric tube. Blood pressure was measured noninvasively before administration, 4 hours after administration, 8 hours, and 24 hours after administration.

Table 3

result;

Figure 6 shows changes in systolic blood pressure and diastolic blood pressure up to 4 hours after administration.

Both systolic blood pressure and diastolic blood pressure were confirmed to be statistically significant at 8 hours after administration compared to the pre-dose measurements. It was confirmed that the blood pressure lowering action of the morohaya puree raw material was enhanced by the protease enzyme treatment.

[Test Example 3]

Confirmation of A C E inhibitory activity;

In order to investigate the action mechanism of blood pressure lowering of the morohea enzyme-treated extract of the present invention, the test was conducted from the viewpoint of the ACE inhibitory activity. .

A 7- to 10-week-old male SD rat was anesthetized with pentobarbitanore (50 m / kg, ip), and a body surface electrocardiogram (lead II) was recorded. Cannula to femoral artery A whole body blood pressure was measured by inserting one. A cannula for vasopressor administration was inserted into the femoral vein, and a catheter for sample injection was placed in the duodenum. The state of anesthesia was maintained by pentobarbital administration as appropriate subcutaneously (guideline: 10 to 15 mgZkg / hr). When the circulatory dynamics was stabilized, angiotensin I (1 g / kg, iv) was intravenously administered. Then, angiotensin I changed to angiotensin II by the action of angiotensin converting enzyme (ACE) in the living body, and increased blood pressure. When blood pressure recovered, the morohea enzyme-treated extract obtained in the same manner as in Example 1 was further purified and administered into the duodenum (50 mg / kg), at 30 and 60 minutes after administration. The pressor response was confirmed by intravenous administration of the same dose of angiotensin I. The enzyme-treated extract was purified by carrying out an adsorption treatment with a synthetic adsorbent, collecting the adsorbed fraction, treating it with a cation exchange resin, and then collecting the adsorbed fraction. The results are shown in Fig. 7. Each value represents mean value + standard error. N is 6.

Result;

The pressor response by administration of angiotensin I at the time of administration of the enzyme-treated extract (Control) was +56.2 mmHg. When angiotensin I was administered 30 minutes and 60 minutes after administration, the pressor response was +57.7 mm Hg, +58.2 mmHg, and +58.2 mmHg, respectively. No change was observed. That is, the effect of inhibiting the conversion of angiotensin I to angiotensin II by administration of the moroheia enzyme-treated extract could not be confirmed. Therefore, it was confirmed that Moroheiya enzyme-treated extract does not have an AC E inhibitory action as already reported, but lowers blood pressure by another action mechanism. [Test Example 4]

Confirmation of vasorelaxant action;

It was confirmed in Test Example 3 that the antihypertensive action of the moroheiya enzyme-treated extract of the present invention was not due to the conventionally reported ACE inhibitory action. Therefore, further studies were conducted to elucidate the mechanism of blood pressure lowering.

A ring-shaped specimen was prepared using the thoracic aorta extracted from a male SD rat, and the blood vessel tension was measured in oxygenated 37 ° C Tyrode solution. After the blood vessels were contracted with a vasoconstrictor (phenylephrine) (P he O. 1 μM), the morohea enzyme-treated extract obtained in the same manner as in Example 1 was further purified. 0, 1 00 0, 3 00 μg / mL was added, and the presence or absence of a vasorelaxant action was observed. The tension when Phenylephrine was added was 100%, and when the tension was lowered, the blood vessel was relaxed. The enzyme-treated extract was purified by adsorption treatment with a synthetic resin adsorbent. The results are shown in Fig. 8. Each value represents the average soil standard error. N is 4. 'Result;

As shown in FIG. 8, the moroheia enzyme-treated extract of the present invention showed a statistically significant relaxing action at 1 000 μg / mL or more. Therefore, it has been clarified that the action mechanism of the blood pressure lowering action of the extract of the present invention treated with the molohair enzyme is based on the vasorelaxant action. This is new knowledge about Morohaya. Industrial applicability

The morohae enzyme-treated extract of the present invention, that is, the proteolytic enzyme-treated morohae extract composition, has an excellent blood pressure lowering action and can be ingested by oral administration because it has less peculiar taste to taste and odor. And as a medicine, It can be used effectively for various foods and drinks, especially health foods, health drinks and foods for specified health use. Furthermore, there is no fear of side effects and the economic and mental burden is light, so it can be taken safely and continuously for a long time, and is suitable as a prophylactic or therapeutic agent for hypertension. In addition, the blood pressure lowering action and blood vessel relaxing action of the Morohaya enzyme-treated extract of the present invention are based on a mechanism of action different from that of nicotianamine contained in Morohaya, and new effects are expected. is there.

Claims

The scope of the claims

2. The protein-degrading enzyme-treated Morohella extract composition according to claim 1, wherein the protease is endopeptidase.

3. The proteolytic enzyme-treated Morohella extract composition according to claim 2, wherein the endopeptidase is an endopeptidase derived from Bacillus.

4. A method for producing a proteolytic enzyme-treated Morohella extract composition, which comprises subjecting a Morohaya water solution to protease treatment and inactivating the enzyme.

5. The morohaya hydrolyzate is a morohaya leaf solution obtained by adding morohaya leaves to water or hot water, a morohaya extract, or a morohaya extract solution or morohaya puree obtained by dissolving morohaya extract powder in water or warm water. A method for producing a proteolytic enzyme-treated Morohella extract composition according to claim 1.

6. An antihypertensive agent comprising the proteolytic enzyme-treated Morohella extract composition according to any one of claims 1 to 3 as an active ingredient.

7. A food or drink comprising the proteolytic enzyme-treated morohea extract composition according to any one of claims 1 to 3.