JP2006342134A - Medicine given by using ank-khak and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a medicament containing a peptide derived from Monascus purpureus or Ank-Khak (Monascus purpureus-fermented rice) as an active ingredient, especially having blood pressure increase-inhibiting activity and erythrocyte deformability decrease-inhibiting activity, highly safe, efficacious when oral administered (ingested), and capable of being used as a pharmaceutical, a designated health food, a health food, etc., to provide a method for producing the same, and to provide a processed food of the same. <P>SOLUTION: An angiotensin 2 converting enzyme inhibitor contains a dipeptide Ile-Tyr or its salt, or contains a dipeptide Val-Trp or its salt, or contains a tripeptide Val-Val-Tyr or its salt as an active ingredient. The method for producing the peptide comprises preparing the Ang-Khak by using the Monascus purpureus, obtaining an extract by using the prepared Ang-Khak as a raw material, and treating the obtained extract by column chromatography. The extract or the processed food thereof is obtained by using the Monascus purpureus which produces the peptide and has the erythrocyte deformability decrease-inhibiting activity as a raw material. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a pharmaceutical agent comprising as an active ingredient a koji mold, that is, a peptide derived from nature, in particular, a blood pressure increase inhibitor having a blood pressure increase inhibitory effect, a method for producing the same, a pharmaceutical product having the effect, a food for specified health use, It relates to health foods.

  Angiotensin converting enzyme (hereinafter referred to as ACE), which has a high correlation with the onset of hypertension, mainly depends on lungs and vascular endothelial cells, acts on angiotensin 1 degraded by renin, and acts on vascular wall smooth muscle contraction. It is known that an angiotensin 2, which is an active peptide, has a strong blood pressure increasing effect.

  Furthermore, ACE has an action of degrading and inactivating bradykinin having an antihypertensive action.

  Thus, since ACE has the effect | action which raises a blood pressure, it is thought that the blood pressure rise can be suppressed by inhibiting this enzyme activity.

  Substances that act on such ACE inhibitory activity have already been found in many natural products and synthetic products, and some of these substances have been put to practical use as antihypertensive agents.

  For example, captolyl is famous as a chemically synthesized substance.

  However, such chemical compounds have the problem that safety issues must always be considered.

  On the other hand, it has been reported that natural components include various ACE-inhibiting peptides in milk-derived protein, plant-derived protein, or fish meat protein.

  These naturally-derived ACE inhibitors are being studied for practical use as hypotensive agents with low toxicity and high safety.

  In recent years, ACE inhibitors from lactic acid fermented milk have been reported (J. Dairy Sci. 78: 777-783, 78: 1253-1257).

  JP-A-6-40944 and JP-A-8-09994 are also disclosed.

  However, as the lactic acid fermentation proceeds, the amount of tripeptides having an ACE inhibitory activity is likely to vary depending on the fermentation conditions because it is produced by proteolytic enzymes produced by lactic acid bacteria, and it is difficult to obtain a constant content.

  Therefore, there is a demand for a blood pressure-lowering peptide derived from a natural substance that has a clear cause of blood pressure-lowering action, can be industrially produced stably, and is highly safe even at a small dose.

When a food-derived ACE inhibitor is orally ingested, the activity may decrease due to an action such as further degradation in the digestive tract.
J. et al. Dairy Sci. 78: 777-783, 78: 1253-1257 JP-A-6-40944 JP-A-8-99994

  An object of the present invention is to provide an antihypertensive agent that can be used for a natural product-derived pharmaceutical, a food for specified health use, a health food, or the like that is highly safe and effective by oral administration (intake), and a method for producing the same.

  In order to achieve the above-mentioned object, the present invention has been conducted through various studies, and as a result, the presence of an angiotensin converting enzyme inhibitory substance in the koji mold is presumed, and the angiotensin converting enzyme inhibitory substance in the koji mold is in reverse phase. By adsorbing to the partition resin and eluting with a water-containing organic solvent, it can be obtained in high yield and can be easily concentrated, and each component in the obtained inhibitor is inhibited using column chromatography. When a component having strong activity was isolated and IC50 and structural analysis of the component were performed, this component exhibited an angiotensin converting enzyme inhibitory action of the Ile-Tyr, Val-Trp, and Val-Val-Tyr sequences in the amino acid sequence. From the results of the digestion tolerance test, the ACE inhibitory effect was confirmed even after digestion and absorption in vivo. By but it was confirmed to be held, and completed the invention.

  According to the present invention, there is provided an angiotensin 2 converting enzyme inhibitor containing dipeptide Ile-Tyr, dipeptide Val-Trp, tripeptide Val-Val-Tyr and salts thereof as active ingredients.

  In addition, according to the present invention, there is provided a method for producing the above-mentioned dipeptide and tripeptide, which comprises producing koji mold and treating the extract obtained using the same as a raw material by column chromatography.

  In addition, according to the present invention, an extract obtained by using a koji mold containing the above-mentioned dipeptide and tripeptide as a raw material or a processed food thereof is provided.

  Moreover, according to this invention, the red blood cell deformability fall inhibitor using a red koji mold is provided.

  In addition, according to the present invention, there is provided an extract obtained by using a red koji mold having the above-described inhibitory effect on the decrease in red blood cell deformability, or a processed food thereof.

  The dipeptide and tripeptide of the present invention are those represented by the aforementioned amino acid sequences, and are Ile-Tyr, Val-Trp, and Val-Val-Tyr.

  The salts include pharmacologically acceptable salts, for example, inorganic acid salts such as hydrochloride, sulfate and phosphate, and citrate, maleate, fumarate, tartrate and lactate. Organic acid salts such as can be used.

  The dipeptide of the present invention can be prepared by purifying an extract obtained using red koji mold as a raw material by a known method such as reverse phase liquid column chromatography (hereinafter abbreviated as HPLC).

  Since the antihypertensive agent of the present invention contains the above-mentioned dipeptides, tripeptides and salts thereof having an ACE inhibitory activity and an antihypertensive action as active ingredients, treatment and prevention of hypertension in general mammals including humans. It is effective for.

  The administration (intake) method of the antihypertensive agent of the present invention can be carried out mainly by oral administration, intravenous injection or the like.

  As a form of the medicine, it can be used as a tablet, granule preparation, capsule preparation, etc., and further as a liquid preparation after making it into a dry powder.

  The dipeptides, tripeptides and salts thereof can be prepared and used in the various dosage forms themselves, but the dipeptides, tripeptides and salts thereof exhibit an antihypertensive effect even in trace amounts. Therefore, the purified product obtained by the column chromatography treatment, as it is, added with various nutrients or the like, or contained in foods and drinks, a functional food having a blood pressure lowering action and a function of preventing hypertension, specified health It can be used as a food for use and a health food.

  The erythrocyte deformability lowering inhibitor of the present invention contains, as an active ingredient, red yeast that has an effect of suppressing erythrocyte deformability lowering, which affects the viscosity of blood and makes it difficult to cause thrombus. It is effective in the treatment and prevention of hyperlipidemia.

  The administration (intake) method of the erythrocyte deformability lowering inhibitor of the present invention can be carried out mainly by oral administration, intravenous injection or the like.

  As a form of the medicine, it can be used as a tablet, granule preparation, capsule preparation, etc., and further as a liquid preparation after making it into a dry powder.

  The koji mold having the erythrocyte deformability lowering inhibitory effect can be prepared and used as such to form the various dosage forms per se, but since the erythrocyte deformability lowering inhibitory effect is exhibited even in a minute amount, Or, it can be used as a functional food, food for specified health use, health food, etc. with various nutrients added or contained in foods and drinks to have a function of suppressing the decrease in erythrocyte deformability and preventing hyperlipidemia. it can.

  The dipeptide having the Ile-Tyr sequence, the dipeptide having the Val-Trp sequence, the tripeptide having the Val-Val-Tyr sequence, and salts thereof according to the present invention have high safety that can be used as an antihypertensive agent having an ACE inhibitory action, etc. It is a novel peptide with no side effects, and can be expected to be used for various pharmaceuticals, foods for specified health use, functional foods and the like.

  Moreover, there is almost no influence by a digestive enzyme, and the ACE inhibitory effect is continuously maintained even after digestion and absorption in vivo.

  Moreover, since the blood pressure increase inhibitor by this invention contains the said dipeptide, the said tripeptide, and those salts as an active ingredient, it can reduce the blood pressure of a human and a mammal effectively.

  Dipeptides and tripeptides, which are active ingredients, are made from koji mold and can be easily and inexpensively produced by culturing.

  In addition, the erythrocyte deformability reduction inhibitor using the koji mold of the present invention suppresses erythrocyte deformability reduction even when a high-fat diet is ingested, suppresses blood lipid uptake, and prevents human and mammalian thrombus. It is less likely to occur and is effective in preventing hyperlipidemia.

  In addition, utilization of this koji mold can be expected for various pharmaceuticals having a function of suppressing a decrease in erythrocyte deformability, foods for specified health use, functional foods and the like.

  Next, embodiments of the present invention will be described in detail.

  Monascus is Purpureus IFO 4489 is used to culture to obtain red koji mold.

  Add 3 volumes of distilled water to the jar, stir for 5 minutes with an Automixer, shake at room temperature for 1 hour, centrifuge (7000 rpm, 15 min), and filter the resulting supernatant. The filtrate is boiled for 20 minutes and filtered again to obtain an extract.

  The red yeast rice extract is circulated through a column using a reverse phase partitioning resin to adsorb an angiotensin converting enzyme inhibitor and elute using a water-containing organic solvent such as water-containing acetonitrile.

  Even when elution is performed with this solvent, the substance that dissolves in water is eluted by supplying water before supplying the solvent in order to effectively perform the elution by the solvent of the adsorbed inhibitory substance. It is effective to perform the processing.

  An angiotensin converting enzyme inhibitor substance eluted with a water-containing organic solvent is a functional food, a specific health food having a hypertension preventing function mainly composed of an angiotensin converting enzyme inhibitory peptide by concentrating and lyophilizing the eluate. Products such as health foods can be obtained in powder form.

  In addition, the food material mainly composed of this angiotensin converting enzyme inhibitory peptide has the activity of inhibiting angiotensin converting enzyme by isolating the above extract using high performance liquid chromatography and elution with acetonitrile gradient. It can be in a form isolated and purified to a strong component.

[Example 1]
Angiotensin converting enzyme inhibitory activity was confirmed.

  As a test strain, Purpureus IFO4489 was used for pre-culture in a test tube medium for 7 days, and solid culture was performed in an Erlenmeyer flask for 7 days to obtain red yeast rice.

  3 ml of distilled water (180 ml) was added to this koji (60 g), permeated at room temperature for 1 hour, extracted and then filtered to obtain an extract.

  This red yeast rice extract was eluted with STEPABES SP-825 by a stepwise method in which the ethanol concentration was changed to 0%, 10%, 15%, 20%, 25%, 40%, 70%.

  As seen in FIG. 1, the 10% ethanol fraction had the highest activity and the protein content was high, so it was used in the next step.

  When the 10% ethanol fraction was passed through a Sephadex G-25 column, an elution pattern as shown in FIG. 2 was obtained.

  As a result of activity measurement and protein quantification with each peak being 1 to 5, fraction 3 with high inhibitory activity 3 was obtained (FIG. 3).

  These fractions 3 and 4 were purified by reverse phase HPLC.

  Reverse phase HPLC was performed on a linear gradient with 1% acetonitrile (containing 0.05% TFA) increased per minute.

  As a result of fractionating each peak and measuring the activity, in fraction 3, high AEC inhibitory activity was observed at peak 8 (compound A), peak 12 (compound B), and peak 15 (compound C) (Fig. 4).

  In fraction 4, high AEC inhibitory activity was observed in peak 11 (compound D), peak 14 (compound E), and peak 18 (compound F) (FIG. 5).

  These peaks were collected, rechromatographed and confirmed as a homogeneous material by HPLC, and the amino acid sequence was determined using a protein sequencer. The results are shown below. Compound D was excluded because it contains many impurities.

Fraction 3 Peak 8 Compound A Ile-Tyr
Fraction 3 Peak 12 Compound B Val-Val-Tyr
Fraction 3 Peak 15 Compound C Val-Phe
Fraction 4 Peak 14 Compound E Val-Trp
Fraction 4 Peak 18 Compound F Ile (amino acid)

  As described above, it was identified. About Val-Phe of the compound C of the said fraction 3, it is the peptide which this applicant has already disclosed by Unexamined-Japanese-Patent No. 2004-83529, and the compound F of the fraction 4 is an amino acid.

  In this example, the angiotensin converting enzyme inhibition rate was measured by a known angiotensin converting enzyme inhibitory activity measuring method (Yamamoto et al.'S improved Lieberman measuring method).

  Moreover, inhibitory activity (%) was calculated | required according to the following formula | equation.

  The IC50 value is a value when the inhibitor inhibits ACE by 50%.

Inhibition rate (%) = {(C-CB) / (S-SB)} / (C-CB) × 100
S: Sample absorbance, C: Control (buffer) absorbance SB: Sample blank absorbance, CB: Control blank absorbance

[Example 2]
An angiotensin converting enzyme inhibitory peptide was purified.

  3 kg of red koji mold was used as a raw material, 9 liters of 3 times its distilled water was added, extracted by shaking for 2 hours, and filtered to obtain an extract.

  This extract was supplied to reverse phase column chromatography for adsorption, and then the angiotensin converting enzyme-inhibiting peptide was eluted with 3 liters of water-containing acetonitrile.

  As the reverse phase resin used here, Cosmo Seal 5C18-AR300 (manufactured by Nacalai Tesque) was used.

Example 3
In order to predict the change of the red yeast rice-derived peptide obtained in Example 1 in vivo, a digestion tolerance test using pepsin, trypsin, and chymotrypsin was performed.

The results are shown below.
IC50 (μM)
Before digestion After digestion Compound A Ile-Tyr 3.99 3.74
Compound B Val-Val-Tyr 21.98 19.25
Compound C Val-Phe 49.69 51.24
Compound E Val-Trp 3.12 4.49

  From this result, it was confirmed that the ACE inhibitory effect was maintained even after being absorbed in vivo.

Example 4
We investigated the effect of red yeast on the reduction of erythrocyte deformability.

  Mice were ingested with a high fat diet containing 1% cholesterol and 15% butter to induce hyperlipidemia, and red blood sugar preparations were administered daily for 4 weeks with an oral sonde to examine the erythrocyte deformability of the mice.

  After feeding on Oriental Yeast's powdered feed MF for 2 weeks, a high fat dietary feed HFF of 1% cholesterol, 15% butter and 84% MF feed was freely fed.

  There were 5 mice in 1 group, and 3 groups (red bean group, sonication group, control group) were used.

  Oral administration of each group was as follows and was performed for 4 weeks.

1) Red potato group 3 g of red potato was suspended in 30 ml of physiological saline, and 0.5 ml per mouse was administered with an oral sonde.
2) Sonication group 3 g of red yeast rice was suspended in 30 ml of physiological saline, and then ultrasonic waves were applied for 10 minutes.
3) The control group physiological saline was administered at 0.5 ml per mouse by oral sonde.

  Four weeks later, the blood of mice was collected and the deformability of erythrocytes was measured.

  The deformability of erythrocytes was measured with a Laser-Assisted Optical Rotational Cell Analyzer (LORCA) manufactured by Mechatronics of the Netherlands after suspending erythrocytes in a high viscosity solution and approximating Newtonian fluid.

  The result is shown in FIG.

  The figure shows Elongation Index, Deformability data of erythrocytes.

  From the figure, the deformability of erythrocytes is decreased in the saline-administered group (control group), but the deformability is improved and increased in shear strain (Shear stress) in the erythema group and the ultrasonic treatment group. is doing.

  It is preferable to increase the absorbency by sonication.

Example 5
In 50 cc of the extract obtained in Example 1, 90 g of lactose and 17 g of corn starch were mixed, and this mixture was granulated with a paste prepared in advance from 7 g of corn starch.

  1 g of magnesium stearate was added to the obtained granules and mixed well, and the mixture was compressed by a compression tablet machine to produce 1000 tablets containing 50 mg of an angiotensin converting enzyme inhibitory component per tablet.

Example 6
200 g of 20% aqueous solution of the extract obtained in Example 1, 5 g of tocopherol acetate, 10 g of thiamine nitrate, 20 g of nicotinic acid amide, 50 g of anhydrous caffeine, benzoate and an appropriate amount of perfume were added to make 30 L and sterilized. Thereafter, 30 ml each was aseptically filled into a bottle to produce an internal solution.

Example 7
3500 g of raw red yeast rice was dried at 60 ° C. for 8 hours using a thermostatic device (WFO-601SD manufactured by EYELA) to make the water content 8% or less.

  The dried red yeast rice cake was passed through a 0.293 μm mesh with a pulverizer (SK100 / Crostfrei manufactured by Retsch) to obtain 2400 g of red yeast rice powder.

Example 8
Boiled rice is cooked in the same way as white rice, cooled to 70 ° C., put into a sterilized hot water bottle with 100 g of rice, 95 g of koji koji, 5 g of koji koji, and 100 g of 60 ° C. hot water and stir lightly.

  It was fermented with a 60 ° C. incubator for 8 hours to obtain a source of amazake.

  The amazake was fired to stop the fermentation, water was added to dilute it twice, and it was made into a paste with a mixer to obtain a red bean sweet liquor with a bright crimson and faint aroma.

Example 9
Boil 1 kg of soybeans, take 300 cc of broth, use as seed water, and grind soybeans.

  Spread 125g of miso soup on the bottom of the bowl.

  Mix 900g of yellow potato, 100g of red potato, and 490g of raw salt for preparation, add crushed soybeans and 125g of seed miso, and mix while adjusting with seed water.

  This was put into a koji and aged to obtain 4 kg of red koji miso.

Example 10
A mixture of 125 g of strong powder, 4 g of dry yeast, 20 g of sugar, and 25 g of red yeast rice powder obtained in Example 7 is mixed with 160 cc of water.

  Further, add 100 g of strong powder, 3.3 g of salt and 25 g of butter and mix well, and knead well until the gluten film is applied.

  The dough is rolled and allowed to stand at 40 ° C. for 30 minutes for primary fermentation.

  Apply wet cloth and let it rest for 10 minutes.

  Secondary fermentation is again performed at 40 ° C. for 30 minutes.

  Baked for 15 minutes in the oven.

  We obtained a deep red bread with a bright red color and a faint aroma.

Example 11
1 teaspoon of baking soda, 5 g of salt and 1 egg were mixed to make brine.

  Knead well while adding the above brine to 360 g of strong powder and 4 g of red yeast rice powder.

  Let sit for 2 hours at room temperature.

  35 g of potato starch was crushed and spread with a rolling pin, and the dough was cut at regular intervals.

  We obtained noodles of Okinawa soba with bright red color and faint scent.

  As described above, various pharmaceuticals, foods for specified health use, and health foods can be produced using the peptides of the present invention, salts thereof, and red koji molds.

It is a figure which shows the comparison table of the ACE activity by the elution ethanol concentration of a red yeast rice extract, and protein amount. It is a figure of the chromatograph which shows the elution pattern of a 10% ethanol fraction. It is a figure which shows the comparison table of the ACE activity and protein amount at the time of the peak in the elution pattern of a 10% ethanol fraction. FIG. 4 is a diagram showing a chromatograph of reverse phase HPLC results of fraction 3. FIG. 6 is a diagram showing a chromatograph of reverse phase HPLC results of fraction 4. It is a figure which shows the change of the erythrocyte deformability by the oral administration of the red koji mold sample.

Claims (7)

  An angiotensin converting enzyme inhibitor comprising a dipeptide having an Ile-Tyr sequence and a salt thereof as an active ingredient.   An angiotensin converting enzyme inhibitor comprising a dipeptide having a Val-Trp sequence and a salt thereof as an active ingredient.   An angiotensin converting enzyme inhibitor comprising a tripeptide having a Val-Val-Tyr sequence and a salt thereof as an active ingredient.   The method for producing a peptide according to any one of claims 1 to 3, which comprises producing red yeast rice cake and subjecting an extract obtained from the raw material to column chromatography.   An extract obtained by using as a raw material a koji mold that produces the peptide according to any one of claims 1 to 3, or a processed food thereof.   An inhibitor of erythrocyte deformability reduction using red koji mold.   An extract obtained by using the red koji mold according to claim 6 as a raw material or a processed food thereof.