JPS58172321A - Biologically active protein pr-a obtained from seed of peach and its preparation - Google Patents

Abstract

NEW MATERIAL:Biologically active protein PR-A[amorphous white powder; elemental analysis: C 47.8+ or -0.4%, H 6.8+ or -0.4%, and N 17.6+ or -0.4%; molecular weight: 200,000-400,000; carbonized at 260 deg.C; positive in ninhydrin reaction, etc.; soluble in water, insoluble in methanol, etc.; amino acid composition 0.6% lysine, 1.9% histidine, 2.9% ammonia, 11.0% alginine, 12.0% aspartic acid, 2.4% threonine, 4.4% serine, 30.4% glutamic acid, 3.8% proline, 5.2% glycine 4.4% alanine, 3.4% valine, 2.7% isoleucine, 7.3% leucine, 3.0% tyrosine, 4.8% phenylalanine, acidic substance]. USE:An antiphlogistic, and a remedy for cerebral thrombosis and myocardial infarction. PROCESS:Seeds of peach are extracted with water, the extracted solution is dialyzed or subjected to ultrafiltration, and biologically active protein PR-A having 200,000-400,000 molecular weight is separated and purified from a solution in the inner tube by chromatography.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a biologically active protein obtained from peach seeds and a method for producing the same.

Peach seeds are called tunin in Chinese medicine and are used to treat menopause due to accumulation of blood, pain caused by blood congestion due to bruises,
It is a type of Chinese medicine that has been used to treat pain caused by lumps caused by stagnation of postpartum ulcer blood, as well as joint pain caused by poor blood circulation.

In addition, it is also used as the main drug of Chinese herbal medicines such as Tokokujokito and Gekankechigan, which are often used as "proof of blood" in the pathological view unique to Chinese medicine, which has been attracting attention in recent years in the medical academic society. be. The anterior side of peach kernels has physiological activity that restores the uterine fundus, stops bleeding, and weakly inhibits blood coagulation, and amygdalin, a type of cyanogenic glycoside obtained from the alcohol extracted fraction of peach kernels, has pharmacological activity. It has been known for a long time to have weak analgesic and anti-inflammatory effects.

However, the above-mentioned effects reported for this side and amygdalin alone cannot explain the medicinal effects that peach kernel is said to have in Chinese medicine. Therefore, the present inventors developed peach (Prunus persica Batsch).
), and it was discovered that two types of biologically active proteins were present in the aqueous extract of the seeds. Until now, it has not been known that peach seeds contain biologically active proteins. The inventors conducted further research and completed the invention.

One protein of the present invention (hereinafter referred to as PR-A) is
It is stable in the state of amorphous white powder and in the state of aqueous solution, and has the following physical and chemical properties.

(a) Elemental analysis C: 47.8 ± 0.4%, H: 6.8 ± 0.4%, N:
17.6±04%.

810%, P: 0% ('b) Molecular weight (measured by gel filtration method) 200,000~, 4
00,000 (mainly around 10,000) (C1 melting point or decomposition point
-゛＼ Melting point unclear, carbonized at about 260℃ (d) Both ninhydrin reaction and biuret reaction positive (?) Infrared absorption spectrum as shown in Figure 1 (KBr method) (f) Ultraviolet absorption spectrum as shown in Figure 2 (aqueous solution (measured in water) (b)) Solubility Soluble in water. Insoluble in methanol, ethanol, acetone and propatool (h) Amino acid composition Lysine 0696 Histidine 1.9 Ammonia 29 Arginine 11.0 Aspartic acid 12.0 Threonine 2.4 Serine □' 4.4 Glutamic acid 30.4 Proline 38 Glycine 5 .2% Alanine 4.4 Cystine 0 Valine 3.4 Methionine 0 Inleucine 2.7 0 Isine 7.3 Tyrosine 30 Phenylalanine 48 (i) Acidic substance Another protein of the present invention (hereinafter referred to as PR-B) It is called.

) is stable in the state of amorphous white powder and in the state of aqueous solution, and has the following physical and chemical properties.

(a) Elemental analysis C: 42.6±0.4%, H: 6.2±04%, N:
16.6±04%.

S: 35±0.4%, P: 0% (b) Molecular weight (measured by gel filtration method) 5,000 to 30,000 (mainly around 10,000) (C) Melting point or decomposition point Melting point unclear. Carbonized at about 270°C (d) Both ninhydrin reaction and biuret reaction positive (el Infrared absorption spectrum as shown in Figure 3 (KBr method) (f) Ultraviolet absorption spectrum as shown in Figure 4 (measured in aqueous solution) (g) Dissolution Soluble in water.Insoluble in methanol, ethanol, acetone and hyperpanol (h) Amino acid composition 1 No. 2
．． 2% Histidine 1.3 Ammonia 3.1 Arginine 10.7 Aspartic acid 2.5 Threonine 1.0 Serine 27 Glutamic acid 50.1% Proline 1.6 Glycine 4.5 Alanine 0.5 Cystine 6.1 Valine 1.6 Methionine 3.2 Inleucine 0.7 0 Icine 1.5 Tyrosine 24 Phenylalanine 1.2 (i) Acidic substances Both of these two types of proteins have biological activity, but PR-A with a molecular weight of 200,000 to 400,000 Although the anti-inflammatory effect is weaker than that of PR-H, it has a strong anti-inflammatory effect.1. ．． On the other hand, PR-B with a molecular weight of 5,000 to 30,000 has strong anti-inflammatory and urokinase activating effects.

Both also have analgesic effects.

From this, the protein of the present invention is useful as an anti-inflammatory agent, and is also expected to be used as a therapeutic agent for cerebral thrombosis and myocardial infarction.

Next, the results of a biological activity test of the protein of the present invention will be shown.

(1) Anti-inflammatory effect Winter et al.'s method (Proc, Soc, Exp
, Biol, Med.

, Na' Yu 544 (1962)). The test substance solution was intravenously injected into Wistar male rats (body weight: 100-1409 kg), and 1 hour later, 1% lactin solution Q, 1 m/cm was injected subcutaneously into the rat's foot.

The volume of edema that occurred 2 hours and 3 hours after the injection of carrageen was measured, and the edema suppression rate was calculated using the following formula.

Note that 8 to 16 rats per group were used as controls. The results are shown in Table 1.

In Table 1, a significant difference was observed in the risk rate of 5%, #196, compared to the control group.

(2) Analgesic effect The method of Siegmund et al. (Proc, Soc,
&p, Biol, Med.

95, 729 (1957)).

The test substance was intravenously injected into dd-N female mice, and 30 minutes later, 0.1-/0.03% phenylquinone solution (0.5% ethanol solution) per 107 body weight was intraperitoneally injected. The number of Leidisig was counted for 15 minutes starting 5 minutes after the phenylquinone injection, and the inhibition rate relative to the control group was determined.

Note that 17 to 19 mice per group were used as controls. The results are shown in Table 2.

Table 2 shows a significant difference compared to the control group with a risk rate of 5%, -1%.

(3) Urokinase activation effect Add urokinase solution (10U) to 0.1mff of test substance solution.
/ne'') 0, 1me was mixed and left for 30 minutes, followed by the method of Noren et al. (Haemostasis 4
110 (1975)) was injected into the holes of a fibrin plate containing plasminogen, which was prepared in advance, and allowed to stand at 31°C for 15 hours. The area of the dissolved fibrin plate was measured, and the activation rate was determined using the following formula.

Note that water was used instead of the test substance solution as a control. The results are shown in Table 3.

Table 3 As is clear from Table 3, PR-A and PR-B have a urokinase activating effect, and PR-A in particular has a remarkable effect.

(4) Acute toxicity Std: ddY female mice (body weight 24-25y) were used in groups of 5 mice. Dissolve the test substance in distilled water,
Administered intravenously. Symptoms of toxicity were observed for one week after administration.

The results are shown in Table 4.

As shown in Table 4, PR-A and PR-B of the present invention are extremely safe.

The method for producing the proteins PR-A and PR-B of the present invention involves extracting peach seeds with water, (2) dialysis or ultrafiltration of the extract, and chromatography of the internal solution to produce PR-A.
PR-A is separated and purified from the precipitate produced by adding a hydrophilic organic solvent to the extract, and PR-A is purified from the supernatant.
It consists of obtaining R-B.

Peach seeds grown anywhere in the world can be used. Preferably fresh or preserved for 2-3 years.

You can either chop the seeds into pieces of about 2-5M in diameter and extract them directly with water, or crush the seeds with a mixer and pre-defatte them with a lipophilic organic solvent (e.g., acetone, ether, n-hexane, petroleum ether, etc.). However, it is preferable to crush the powder into fine powder and extract it with water because the yield is higher.

The extraction is carried out at a temperature of 10 to 60° C., preferably at room temperature, for 1 to 5 hours with or without stirring. If the extraction temperature is too high, the amount of unnecessary substances present in the seeds extracted will increase and PR-A and PR-B may be deactivated. If the extraction is allowed to take too long, especially in the summer at room temperature, the extraction mixture may ferment.

At the time of extraction, there is no particular limitation on the weight ratio of water to seed powder, but in consideration of subsequent processing, it is preferably 4 to 6:1.

Method (2) in which the extract is subjected to dialysis or ultrafiltration and then chromatography is as follows.

The dialysis may be carried out by a conventional method, for example, using a dialysis tube such as a 20/32 type Visking cellulose tube (manufactured by Visking, USA).

Ultrafiltration may also be carried out by a conventional method, and for example, an ultrafiltration membrane such as Diafilter' G-05T (sold by Bioengineering Co., Ltd.) may be used.

In order to isolate and purify PR-A and PRzB from the obtained internal solution, a conventional method such as column chromatography using a gel filtration agent as a carrier may be used after freeze-drying the internal solution.

As the gel filtration agent, for example, Sephadex "G-1"
5~G-200 (manufactured by Pharmacia, Sweden),
Sepharose” 2B to 6B (manufactured by the same company), Sephacryl f
``S-200 or S-300 (manufactured by the same company), Biogel'P-30 to P-300 (manufactured by Bio-Rad Laboratories, USA), Biogel eA (manufactured by the same company), Saga Panok 0 (manufactured by Therabank Laboratories, UK) The eluent may be water, but an appropriate buffer (e.g., NaH2PO4-Na2HPO4 buffer) may be used.
The degree of separation and purification is high.

PR-A and PR-B separated and purified in this way
can be further purified by further dialysis, centrifugation, and freeze-drying of the supernatant to remove inorganic substances contained in the buffer.

Method for separating and purifying by adding a hydrophilic organic solvent to the extract■
is as follows.

Add 30 to 55 vol of a hydrophilic organic solvent to the extract, such as acetone, ethanol, methanol, and ethanol.
/v%. The resulting precipitate contains only PR-A, and can be purified using the same chromatography described above to obtain PR-A.

On the other hand, the supernatant liquid contains PR-B, and the same hydrophilic organic solvent as above is added to this liquid at 60 to 85 v/v 9
PR-B can be obtained by adding the precipitate so as to obtain 6 and purifying the resulting precipitate using chromatography in the same manner.

Compared with method (2), which uses dialysis or ultrafiltration, method (2) has a slightly lower yield or can be processed in large quantities, so it is industrially superior.

Examples are shown below to explain the present invention in more detail.

Example 1 Peach seeds (produced in China, 1 kQ) were crushed with a mixer,
After standing in acetone (51) at room temperature for 5 hours with occasional stirring, the residue was removed from the mixture by vacuum filtration.

This operation was repeated 5 more times, the residue was dried with warm air for 10 hours, and the dried residue was crushed using a pulverizer (Pulverizer Model AP-8).
．． After making it into a fine powder (60 to 200 mesh) using Hosokawa Tekko KK), it was extracted with acetone three times (5 I!, 3 hours each), and the residue was extracted twice with methanol (51!, 3 hours each). , filtered under reduced pressure. The defatted white residue thus obtained (
Add this water (31 liters) and leave it for 5 hours at room temperature with occasional stirring to extract it, and centrifuge it (5000 rpm).
, p, m.

20 minutes) to separate the extract and the residue, and add the residue to water (11).
The washing solution was combined with the extract solution.

This extract solution Ovisking cellulose tuber (20
After dialysis under running water for a day and night, the liquid in the dialysis tube was collected, separated into a supernatant and a precipitate, and the supernatant was freeze-dried. white powder (28
5') was obtained. This powder (39) was mixed with 0.02M phosphoric acid.
Sodium-phosphate disodium; Noum buffer (pH 7,2
The solution was added to a column (φ4 x 102 cm) packed with Sepharose 6B, eluted with the same buffer (1200 i), fractionated into 24-
The sections from No. 4 to No. 33 and from No. 38 to No. 43 were combined and freeze-dried, and the white powder of crude PR-A (2,262y) was obtained from the former section and the white powder of crude PR-A (2,262y) from the latter section. A white powder (1,000y) of crude PR-H was obtained from the fraction.

In order to further purify these, first dissolve the crude PR-A powder (2,2y) in 002M sodium phosphate-disodium phosphate buffer (40me, pH 7,25) and fill it with Sephadex G-100. column (13,
8X 29cIn) and the same buffer (190me
), fractionated in 7-mi increments, and put the fractions from No. 15 to No. 25 together into a dialysis tube (Vuisking cellulose tube type 20/32), which was dialyzed under running water for one day and night. The internal solution was freeze-dried to obtain purified PR-A white powder (1,096y). The physicochemical and biological properties of this product are as described above.

Next, crude PR-H powder (0,980y) was added to 0.02M sodium phosphate-disodium phosphate buffer (30+
ne.

pH 7,25) and added to a column (13,5x28c+++) packed with Sephadex G-100,
Elute with the same buffer (300me), fractionate by Tme, combine the fractions from No. 25 to No. 36, put this into a dialysis tube (Dice King cellulose tube type 20/32), and place it under running water. After dialysis for one day and night, the internal solution was freeze-dried to obtain purified PR-H white powder (0,3009). The physicochemical and biological properties of this product are as described above.

Example 2 Peach seeds (North Korean degree, 500'!) were coarsely crushed in a mixer, and allowed to stand in n-hexane (31) at room temperature for 5 hours with occasional stirring, and then removed from the mixture by vacuum z-filtration. Take out the residue. Repeat this operation 5 times, dry the resulting degreased residue with hot air for 5 hours, and grind the dried residue into a fine powder (60~
200 mesh3), and then diluted with n-hexane three times (3 times each time).
(for 12 hours each) and the residue was dried with hot air.

Boil this powder with water (1.21 liters), extract for 2 hours while stirring, and centrifuge (5QQQr, p9m,
20 minutes) to separate the extract and the residue, and add the residue to water (250 minutes).
m), and the washing liquid was combined with the extract liquid.

Acetone (1.5 J) was added to this extract under stirring, and 30
After a few minutes, the mixture was centrifuged (10,000 r, p, m, for 20 min) and separated into a supernatant and a precipitate. This precipitate corresponds to crude PR-A, which was purified according to the method of treating with Sephadex G-100 etc. in Example 1, and purified PR-A (4, IF
) was obtained.

Add acetone (2 J) to the supernatant solution with stirring, and centrifuge after 30 minutes (10,000 r-p, m, 20 minutes).
The supernatant was separated into a supernatant and a precipitate (2,349). This precipitate corresponds to crude PR-H, and the precipitate (1y) was replaced with deionized water (I
on/), added to a column (φ2.2 x 56 cm) packed with Sephadex G-15, eluted with deionized water, fractionated into three fractions, and combined the fractions from No. 21 to No. 31. was freeze-dried to obtain purified PR-B white powder (
524#v) was obtained.

[Brief explanation of drawings]

Figures 1 and 2 show the infrared absorption spectrum and ultraviolet absorption spectrum, respectively, of PR-A.
The figure and FIG. 4 represent the infrared absorption spectrum and ultraviolet absorption spectrum, respectively, of PR-H. Patent Applicant Dainippon Pharmaceutical Co., Ltd. Attorney Kojima Akira Procedural Supplementary Letter (Method) (Spontaneous) Indication of the Case Patent Application No. 55765 of 1982 2, Title of Invention PR-B and Process 38 Amendment Relationship with the case involving a person who is making a patent application Address of the patent applicant: 3-25 Doshomachi, Higashi-ku, Osaka 5 Description subject to amendment 6 Contents of amendment Type of specification as shown in attached sheet (no change in content) (above)

Claims (1)

[Claims] (1) Molecular weight of 200,000 to 200,000 obtained by extracting peach seeds with water
Biologically active protein PR-A having a molecular weight of 400,000 and bioactive protein PR-B0 having a molecular weight of 5,000 to 30,000 (2) Extract peach seeds with water, dialysis or ultrafiltration of the extract, and extract from the internal liquid. PR-A and PR-H characterized by separating and purifying a biologically active protein PR-A having a molecular weight of 200,000 to 400,000 and a biologically active protein PR-B having a molecular weight of 50,000 to 30,000 by chromatography. manufacturing method. (3) Extract peach seeds with water, add a hydrophilic organic solvent to the extract, and extract bioactive protein PR-A with a molecular weight of 200,000 to 400,000 from the resulting precipitate. 3
1. A method for producing PR-A and PR-B, which is characterized in that a biologically active protein P'R, -B is obtained.