JPH04261122A - Angiotensinase activity inhibitor contained in leguminous plant seed - Google Patents

Abstract

PURPOSE:To provide the title inhibitor, an extract from leguminous plant seeds which gives hypotensive activity through angiotensinase inhibition. CONSTITUTION:The objective substance giving angiotensinase inhibitory activity can be obtained by purifying, using, e.g. ion exchange chromatography, an extract from seeds of leguminous plant(s) such as kidney beans, white beans, red beans, cowpeas, red peas, soybeans, black soybeans, etc. For the extract from each kind of the above seeds, the measurement of 50% inhibitory concentration for the angiotensinase of rabbit lung origin was 5-25mug/ml.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention is directed to the leguminous plant Phaseolus vulgaris (L.).
aris(L). Taisho Kintoki Bean, White Bean], Phaseolus coccineus (L)
coccineus (L). Tiger bean, purple-flowered bean, white-flowered bean], Phaseolus angularis. D'Avryu. F. Wight [Phaseolus angularis]
W. F. Wight, Azuki], Figna sinensis. Vigna sineusis Endl
．． Cowpea], Pisum sativum (L.)
sativum (L), red pea], glycine max, Merr
A crude product with angiotensin-converting enzyme inhibitory activity purified by ion-exchange chromatography from the aqueous extract of the seeds of Ill, Kurakake beans, Gankui beans, soybeans, hitashi beans (green soybeans), black soybeans, and Tamba Daikoku. Regarding.

0002

[Prior Art] Angiotensin-converting enzyme exists mainly in the lungs, vascular endothelial cells, renal proximal tubules, etc., and acts on angiotensin I to cleave a dipeptide from its C-terminus, resulting in angiotensin II, which has a strong pressor effect. It is an enzyme that converts This enzyme also has the effect of destroying and inactivating bradykinin, a hypotensive substance in living organisms, and is known to be doubly involved in the pressor system. It has been conventionally believed that inhibiting the activity of angiotensin-converting enzyme lowers blood pressure and is clinically effective in preventing or treating hypertension.

Captopril and enalapril were developed as pharmaceuticals based on this working hypothesis. These drugs are synthetic. On the other hand, angiotensin-converting enzyme inhibitors found in nature are extremely rare, and only a peptide substance (Protide, SQ2088) consisting of 9 amino acids obtained from snake venom from Brazil and Japan.
1) and the metabolite 1583 of actinomycetes belonging to the genus Streptomyces (Japanese Unexamined Patent Publication No. 177920/1983) are only known.

[0004] Recently, only peptides obtained by decomposing milk casein with trypsin have been known as angiotensin converting enzyme inhibitors obtained by treating proteins with proteases. (Unexamined Japanese Patent Publication No. 58
-10942, 59-44323, 59-44
No. 324, No. 61-36226, No. 61-36227
issue).

[0005]

[Problems to be Solved by the Invention] Based on the background of the prior art, the present inventors have developed a method for producing seeds of leguminous plants in their natural state without any enzyme treatment. When we searched for angiotensin-converting enzyme inhibiting substances, we found Taisho Kintoki beans, white beans, tiger beans, purple flower beans,
Substances that have angiotensin-converting enzyme inhibitory activity are present in aqueous extracts of white flower beans, azuki beans, cowpeas, red peas, black beans, hard beans, soybeans, hitashi beans (green soybeans), black beans, Tamba Daikoku, etc. As a result of this discovery and extensive research, we have completed the present invention.

[0006] That is, the present invention is an active ingredient contained in a commonly used food material, that is, seeds, which has few side effects, is easy to use, and can be ingested as a normal food.
Moreover, if only the active ingredients are extracted and purified, it can be provided as an excellent medicine.

[0007]

[Detailed Description of the Invention] Based on the above-mentioned state of the art, the present inventors searched widely for natural organic compounds and conducted intensive research in search of an angiotensin-converting enzyme inhibitor with few side effects. We discovered strong angiotensin-converting enzyme inhibitory activity in the aqueous extract of certain legume seeds.
It was discovered that the active ingredient has the property of being able to be adsorbed and desorbed by various ion exchange chromatography methods.

[0008] In general, the conventional method for separating and purifying active ingredients from natural products is to perform extraction and purification using the physicochemical properties of the active ingredients.

[0009] The present invention first uses Taisho Kintoki beans, white beans, tiger beans, purple flower beans, white flower beans, azuki beans, cowpeas, red peas, black beans, hard beans, soybeans, hitashi beans (green soybeans) Pulses such as , black soybeans, and Tamba Daikoku are ground separately in a mixer, and a certain amount of the ground material is immersed in an organic solvent that can be separated from water, such as n-hexane, petroleum ether, or chloroform, and thoroughly mixed to produce various beans. It is possible to remove the contained lipid components.

[0010] In addition, in order to remove protein from the above-mentioned legumes, usually a water-miscible solvent such as ethanol, methanol, acetone, etc., or a salting-out method in which precipitation is performed by adding inorganic salts, or trichloromethane are used. Any commonly used protein removal methods can be applied, such as adding acetic acid to remove precipitates.

The angiotensin-converting enzyme inhibitory activity of the aqueous extracts of the seeds of various leguminous plants that have been delipidated and deproteinized in this manner was determined by hip-lyr-histidyl-leucine (
Hip-His-Leu) was used as a substrate, and the Cushman method (D.
．． W. Cushman et al., Biochem.
Pharmacol. 20, 1637 (1971)], the results were as follows: Taisho Kintoki beans, white beans, tiger beans, purple flower beans, white flower beans, azuki beans, cowpeas,
Strong inhibitory activity was observed in all soybean seeds such as red peas, kurakake beans, gankui beans, soybeans, hitashi beans (green soybeans), black beans, and Tamba Daikoku.

Furthermore, aqueous extracts obtained from seeds of various leguminous plants are subjected to ion exchange chromatography, such as Amberlite IR-120 (as a cation exchange resin).
(manufactured by Rohm and Haas) DOWEX 50W (manufactured by Dow Chemical), Diaion SK1A (manufactured by Mitsubishi Kasei)
, and anion exchange resins such as Amberlite IRA402 and IRA68 (manufactured by Rohm and Haas)
, DOWEX 1 (manufactured by Dow Chemical Company), DIAION SA10B, PA-404, WA-30 (manufactured by Mitsubishi Chemical Corporation), etc., and easily adsorbed with an aqueous solution or buffer containing salt, alkali, or acid. In this way, the angiotensin converting enzyme inhibitor of the present invention is obtained. When the obtained fractions were measured using a partially modified measurement method of the above-mentioned Cushman method, Taisho Kintoki beans, white beans, tiger beans, purple flower beans, white flower beans, azuki beans, cowpeas, red peas, black beans, and ganku beans were detected. Strong inhibitory activity was observed in each of the crudely purified water extracts of beans, soybeans, hitashi beans (green soybeans), black soybeans, Tamba Daikoku, etc., each purified by ion exchange chromatography.

According to the present invention, Taisho Kintoki beans, white beans, tiger beans, purple flower beans, white flower beans, azuki beans, cowpeas, red peas, black beans, hard beans, soybeans, hitashi beans (green soybeans) Angiotensin-converting enzyme inhibitors, which are specifically contained in plant seeds such as , black soybeans, and Tamba Daikoku, can be efficiently separated from impurities that are present in large quantities.

The angiotensin-converting enzyme inhibitor of the present invention is stable to heat treatment at 90° C. for 30 minutes at pH 7.5, and is dialyzed through a cellophane membrane.

[0015]

[Effects of the invention] Taisho Kintoki beans, white beans,
Tiger beans, purple flower beans, white flower beans, azuki beans, cowpeas, red peas, black peas, hard beans, soybeans, hitashi beans (green soybeans)
Aqueous extracts obtained from the seeds of leguminous plants such as black soybeans and Tamba Daikoku, and crude extracts obtained by ion-exchange chromatography, exhibit strong angiotensin-converting enzyme inhibitory activity and have antihypertensive effects and bradykinin inhibition. It exhibits an activation-suppressing effect and is useful for the prevention and treatment of hypertension such as essential hypertension, renal hypertension, and adrenal hypertension, as a diagnostic agent for these diseases, and as a hypotensive agent used in various pathological conditions.

On the other hand, the present invention is extracted and purified from commonly used food materials, and is a food ingredient contained in common foods.From this point of view, it is expected that there will be very little influence from side effects. Ru.

[0017] Therefore, the aqueous extract and crudely purified product of the present invention have the important physiological activity of lowering blood pressure as shown above, and have the functional properties of food in leguminous plant seeds that are commonly eaten. Considering that it exhibits the following properties, it is also useful as an important food ingredient for functional foods.

[0018] The present invention will be explained in more detail with reference to Examples below.

[0019]

[Example] I. Obtaining water extracts Commercially available Taisho Kintoki beans, white beans, tiger beans, purple flower beans,
Grind 20g each of seeds such as white flower beans, azuki beans, cowpeas, red peas, black beans, soybeans, hitashi beans (green soybeans), black beans, Tamba Daikoku, etc. for 5 minutes with a mixer, and pass through a 30-mesh sieve. I got something.

8 ml of n-hexane was added to each of the pulverized products and stirred for 1 hour, and the n-hexane was filtered off. Again, 8 ml of n-hexane was added to each and the same operation was performed. Each residue was air-dried to obtain a defatted air-dried product.

Add 8 ml of water to each of the above air-dried products and mix well to obtain a yellowish brown to yellow viscous liquid.
When 8 ml of ethanol was added to these solutions and mixed well again, an insoluble portion and a white precipitate were generated. The resulting precipitate was heated to 10,000r. p. m. Centrifugation was performed for 20 minutes to obtain approximately 14 ml of non-viscous supernatant liquid. The supernatant thus obtained was concentrated under reduced pressure at 40° C. and then freeze-dried to obtain the respective aqueous extracts shown below. (Table 1) These aqueous extracts were examined for angiotensin converting enzyme inhibitory activity.

[0022]

[Table 1] II. Acquisition of crude refined products Water extraction of Taisho Kinton beans, white beans, tiger beans, purple flower beans, white flower beans, azuki beans, cowpeas, red peas, kurakake beans, soybeans, hitashi beans (green soybeans), black beans, Tamba Daikoku, etc. Dissolve 2 g of each in 20 ml of water, adjust the pH to 5.0 with IN hydrochloric acid, and pass through an ion exchange column chromatograph (column: diameter 1.5 cm x height 10 cm) of Amberlite IR-120 (H+ type). After adsorption, the mixture was thoroughly washed with 30 ml of water, and then eluted with 50 ml of 0.3N aqueous ammonia.

[0023] After recovering ammonia from each eluate under reduced pressure at F40°C, it was freeze-dried to obtain each crude product shown in Table 2.

[0024] These crude products were examined for angiotensin converting enzyme inhibitory activity.

[0025]

[Table 2] III. Experimental materials for measuring angiotensin-converting enzyme inhibitory activity (1) Angiotensin-converting enzyme: rabbit lung angiotensin-converting enzyme purchased from Sigma Chemical Co., Ltd. (2) Angiotensin-converting enzyme inhibitor 26 samples obtained in Examples I and II above ( 3) Enzyme substrate

[0026]

[Chemical formula 1] IV. Measurement of angiotensin converting enzyme inhibitory activity.

The method for measuring angiotensin converting enzyme inhibitory activity is to measure the enzyme activity of angiotensin converting enzyme in the presence and absence of each specimen, and from this and the determined inhibition rate, the inhibitory activity IC50 of each specimen is determined. was calculated.

[0028] The angiotensin converting enzyme activity was measured according to the method of Cushman and a modified method of Hayakari et al. [Analytical Biochemistry Vol. 84,
361 pages 1978; Anal. Biochem. 8
4,361 (1978)] was adopted as follows.

That is, 50mM Tris-HCl buffer (p
H8.2), 0.3M sodium chloride aqueous solution, 2mM
250 μl of enzyme reaction solution containing Hip-His-Leu substrate, rabbit lung-derived angiotensin converting enzyme (1-2 milliunits), and each sample was heated at 37°C.
Incubated for minutes.

Next, IN sodium hydroxide aqueous solution 15
The reaction was stopped by adding μl of 60mM sodium phosphate buffer (pH 7.2) and left at room temperature for 30 minutes.
1 ml of methyl cellosolve containing 1% cyanuric acid chloride was added thereto, and after being left for 15 minutes, the absorbance at 382 nm was measured.

[0031] The inhibition rate was calculated from the absorbance of the reaction solution containing each sample (a), the absorbance without the sample (b), and the absorbance of a blind sample that did not react to each (a' and b') using the following formula. .

[0032]

[Equation 1] Based on the above measurement method, the inhibition rate at several concentrations of the specimen was determined, and the 50% inhibitory concentration (IC50) was calculated.

[0033]V. Experimental results show that the Taisho Kintoki beans of the present invention,
Each water extract derived from white beans, tiger beans, purple flower beans, white flower beans, azuki beans, cowpeas, red peas, kurakake beans, hard beans, soybeans, hitashi beans (green soybeans), black beans, Tamba Daikoku and The inhibitory activity of each crude product against angiotensin converting enzyme is as shown in Table 3.
A 10- to 13-fold increase in activity was observed compared to the extract obtained in .

[0034]

[Table 3]

Claims (2)

[Claims] Claim 1: Phaseolus vulgaris, a leguminous plant that has angiotensin-converting enzyme inhibitory activity.
(L), Phaseolus coccineus (L), Phaseolus angularis (L). F. Wight, Figna sinensis. Aqueous extract from seeds of Endor, Pisum sativum (L.), Glycine max meryl. 2. The angiotensin according to claim 1, wherein impurities such as lipids and fatty acids are removed from the aqueous extract of each bean seed using an organic solvent, and then purified by ion exchange chromatography. A substance having converting enzyme inhibitory activity, that is, an angiotensin converting enzyme antihypertensive agent, and a method for producing the same.