JPS5959622A - Bioactive protein obtained from stone of almond, apricot or japanese bush cherry - Google Patents

Abstract

PURPOSE:To obtain a protein having strong antiphlogistic and analgesic activities and useful as an antiphlogistic agent, by extracting the stone of almond, apricot (Pruns armeniaca Linn. var. ansu Maxim.) or Japanese bush cherry (Prunus japonica Thunb.) with water. CONSTITUTION:The stones of almond, apricot or Japanese bush cherry are roughly crushed with a mixer, defatted with an oleophilic organic solvent, pulverized, and extracted with water at 10-60 deg.C, preferably at room temperature, for 1-5hr. The extract liquid is dialyzed or subjected to ultrafiltration, and two kinds of proteins are separated and purified from the remaining liquid by chromatography, or the extract liquid is added with a hydrophilic organic solvent, and a protein having high molecular weight is separated from the produced precipitate, and a protein having low molecular weight is obtained from the supernatant liquid. PR-AA having a molecular weight of 300,000-350,000 and PR-BA (10,000-15,000) are obtained from almond, PR-AB (250,000-350,000) and PR-BB (10,000-30,000) from apricot, and PR-AC (250,000-350,000) and PR- BC (10,000-15,000) from Japanese bush cherry.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to biologically active proteins obtained from almond, anse or Japanese plum seeds.

The present inventors have discovered that peach seeds are used in Chinese medicine to treat menopause due to accumulation of clotted blood and ulcer pain due to 4'J bruises.

Focusing on the fact that it is being applied to the pain caused by stagnant postpartum clotted blood and joint pain due to poor blood circulation, we conducted research on its active ingredients and found that its water extract is an anti-inflammatory, Discovered a protein with analgesic and urokinase activating effects and filed a patent application (Japanese Patent Application No. 57-557
65).

This time, the present inventors have discovered that almonds, which are closely related to peaches,
Assuming that proteins with similar biological activity exist in the seeds of Anse and Niwaume, they conducted intensive research and discovered the relevant proteins from these seeds. Hitherto, it has not been known that these seeds contain biologically active proteins. The inventors conducted further research and completed the invention.

All proteins of the present invention are stable in the form of amorphous white powder and aqueous solution, and have the physicochemical properties shown in Table 1 below. In addition, in the following, among the proteins obtained from almonds, those with a molecular weight of 1v5 are referred to as PR-AA,
The low molecular weight protein is called PR-BA, the protein obtained from Ansu is called PR-AB, and the protein obtained from PR-BB Japanese plum is called PR-AC and PR-BC.

3Q,',! - Stinging) Table 1 *1 Measured by gel p-filtration method *ZKBr method Table 1 (continued) *3 Measured in aqueous solution The protein of the present invention has strong anti-inflammatory and analgesic effects, It is useful as an inflammatory agent and is also effective for cerebral blood
It is also expected to be a therapeutic agent for 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, IEx
p, Biol, Med.

111544 (1962)). Wistar male rats (body weight 100-140 y) were intravenously injected with a test substance solution, and 1 hour later, a 1% carrageenan solution was administered at 0.5%. +-me was injected subcutaneously into the paw of the rat.

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.

In addition, 1 group of 8 to 164 Rano] was used as a control. The results are shown in Table 2.

Table 2 A significant difference was observed compared to the control group with a risk rate of *5% and *1%.

(2) Analgesic effect Siegmund et al.'s method (Proc, Soc,
'EXI), Biol.

Med, 95. 729 (1,957)).

The test substance was intravenously injected into dd-N female mice, and 30 minutes later, 0.1 me per 10 y of body weight of a 0.03% phenylquinone solution (05% ethanol solution) was injected intraperitoneally. The number of syringes was determined over a period of 15 minutes starting 5 minutes after the phenylquinone injection, and the inhibition rate was determined relative to the control group.

Note that 17 to 19 mice per group were used as controls. The results are shown in Table 3.

Table 3 Table 3 (Continued) A significant difference was observed compared to the control group with a risk rate of *5% and **1%.

(3) Acutely toxic ddN female mice (body weight 24-25y) were used in groups of five. The test substance was dissolved in physiological saline and administered intravenously. When we observed blue symptoms for one week after administration, the LDso values for all substances were 1600.
(■/Ky') or more.

Thus, the protein of the present invention has extremely high safety.

The method for producing the protein of the present invention involves extracting either almond, apricot, or Japanese plum seeds with water, dialysis or ultrafiltration of the extract, and separating two types of proteins from the internal solution by chromatography. (1) Obtain high molecular weight proteins from the precipitate produced by adding a hydrophilic organic solvent to the extract, and obtain low molecular weight proteins from the supernatant.

Almond, apricot, or Japanese plum seeds grown anywhere in the world can be used. Fresh ones or ones that have been stored for two or three years are best.

You can chop the seeds into pieces with a diameter of 2 to 5 pieces and extract them directly with water, but you can also crush the seeds with a mixer and extract with a lipophilic organic solvent (e.g., acetone, ether, n-hexane, petroleum ether, etc.). It is preferable to degrease the powder in advance, crush it into a fine powder, and extract it with water, since 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 coexisting in the seeds extracted may increase, and two types of proteins may be inactivated. If the extraction is allowed to take too long at room temperature, especially in the summer, the mixture may ferment.

During extraction, the mixture of water and seed powder is M, I'L;l'j,
'There is no particular limitation, but considering the subsequent processing, 4 to 6:
1 is preferred.

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

Dialysis may be carried out by a conventional method, and for example, a dialysis tube such as Dice King 0 Cellulose Tube Model 20/32 (manufactured by Dice King, USA) may be used.

The ultraf-filtration may also be carried out by a conventional method, for example, an ultraf-filtration membrane such as Diafilter ""G-05T (sold by Bio-Engineering Co., Ltd.) is used.

In order to purify the two types of proteins from the obtained internal fluid, the internal fluid may be freeze-dried and then a conventional method such as column chromatography using a gel membrane as a carrier may be used. As the gel filtration agent, for example, Sephatechno G-1
5~G-200 (manufactured by Pharmacia, Sweden),
Sepharose 02B-6B (F1 Company &), Heptaphryl S-200 or S-300 (manufactured by the same company), Biogel
I'-30 to P-300 (manufactured by BioRad Laboratories, USA), Bioge/l10A (manufactured by the same company),
Zagabac 0 (Ikiris, manufactured by Cerabac Laboratories) etc. can be opened. The eluent may be water, but an appropriate buffer (e.g., NaH2PO4-Na2HPO4%
II solution) provides a higher degree of separation and purification.

The two proteins thus separated and purified can be further purified by further dialysis, centrifugation, and lyophilization of the supernatant to remove inorganic substances contained in the buffer solution.

〉I Part 1) The good is the JCjC Ikuo Ochidai, and the separation is made.
Add a hydrophilic organic solvent such as acetone, ethanol, methanol, furopanol, etc. to the extract to a concentration of 30 to 55% v/v. The resulting precipitate contains only high molecular weight proteins, and can be purified using the same chromatography described above.

On the other hand, since the supernatant liquid contains low molecular weight proteins, the same hydrophilic organic solvent as above was added to this liquid at 60-85v.
/v%, and the resulting precipitate is similarly purified using chromatography.

Illegal method (2) has a slightly lower yield than the above-mentioned method (2) of dialysis or ultra-II filtration, but it can be processed at a rate of 1:1.
Industrially superior.

EXAMPLES In order to explain the present invention in more detail, examples are shown below.

Example 1 Almond seeds (produced in California, 500 y) were crushed using a grinder, and after standing in acetone (2.5 t) at room temperature for 5 hours with occasional stirring, the residue was taken out from the mixture by filtration under reduced pressure. This operation was repeated 5 more times, the residue was dried with hot air for 10 hours, and the dried residue was crushed into fine powder (60~
200 mesh), and then 3 times with acetone (2.5 mesh each time).
t, 3 h each time), and the residue was extracted twice with methanol (
The mixture was extracted for 2.51.3 hours each and subjected to vacuum once.

Water (12) was added to the defatted white residue thus obtained, and the mixture was allowed to stand for 5 hours at room temperature with occasional stirring for extraction, followed by centrifugation (5000 r, p, m).

The remaining 114i' was washed with water (300 mf), and the washing liquid was combined with the extract.

Transfer this extract to a VISKING cellulose tube (20/
After dialysis under running water for one day, the liquid in the dialysis tube was collected, centrifuged, separated into supernatant and precipitate, the precipitate was suspended in water, and this was freeze-dried. and coarse P
A white powder (41,2y"l) of R-AA was obtained, and the supernatant liquid was freeze-dried to obtain a white powder (11,4y). Supernatant liquid freezing: i3 dry powder (3,5y) was .02M-phosphoric acid-strium-nitric acid phosphate, thorium buffer (pH 7,25
, 35+++f'), and the solution was added to a column (φ6 x 5Qcm) packed with Sepharose 613, eluted with the same buffer solution (1600+++e), and 10
The fraction was fractionated by me, and the fractions from No. 131 to No. 150 were freeze-dried to obtain a white powder (1,42y') of phase PR-BA.

In order to further purify these, first dissolve the crude PR-BA powder (1,4y) in 0.02M sodium phosphate-disodium phosphate buffer (15me, pH 7,25) and add it to Sephadex G-100. Column packed with (
φ3.8 x 29 cm) and added the same buffer solution (3.8
50 me ), fractionated into 7 me increments, and the fractions No. 31 to No. 41 were combined to obtain a dialysis tube (Vuisking cellulose tube 20/32 m). The physicochemical and biological properties of this product are as described above.

Next, crude PR-AA powder (3.0P) was added to 002M sodium phosphate-disodium phosphate buffer (30meS
pH 7,25), packed with Sephadex G-100, added to this column (φ5
Fractionate each me, combine the fractions from No. 31 to No. 40, put this into a dialysis tube (Dice King cellulose tube type 20/32), dialyze it under running water for one day, and then drain the internal solution. Lyophilization was performed to obtain purified 1) R-AA white powder (1,74y). The physicochemical and biological properties of this product are as described above.

Example 2 Amp seeds (produced in China, 5ooy) were coarsely crushed in a mixer, left in acetone (2,57) at room temperature for 5 hours with occasional stirring, and the residue was removed from the mixture by vacuum δj filtration. put out. This operation was repeated 5 more times, the residue was dried with hot air for 10 hours, and the dried residue was ground into fine powder (60-W20
After soaking with acetone 3 times (2.57 mm each).
3 hours each time), and the residue was extracted twice with methanol (2 times each time).
5 tons, 3 hours each) and passed under reduced pressure once. Water (1 liter) was added to the defatted white residue obtained in this way, and the mixture was left to stand for 5 hours at room temperature with occasional stirring, followed by centrifugation (5000 r, p, m, 20 minutes) to extract the extract. Separate the residue, wash the residue with water (300m), and combine the washings with the extract.

Transfer this extract to a Guisking cellulose tube (20/
After dialysis under running water for a day and night, the liquid in the dialysis tube was collected, centrifuged, separated into a supernatant and a precipitate, and the supernatant was freeze-dried to form a white powder ( 59,0P
) was obtained. This powder (10y) was added to 0. ．． 02 I+4 phosphate-sodium phosphate-disodium phosphate buffer (pH 7
, 25,100n+F), and the solution was poured into a column packed with Sepharose 613 (φ6 x 50c+n'
) and eluted with the same buffer (1500 ml).
The fractions 80 to 110 and 1 to 135 were combined and lyophilized separately, and crude PR-AB was obtained from the former fraction.
white powder (5,62y), white powder (5,62y) from the latter category
4,14y) was obtained. This latter white powder (4y) was packed again with Sepharose 6B and column (φ6x50cm)
) and eluted with the same buffer (1600 m('),
The mixture was fractionated by 1 orne, and fractions No. 115 to No. 144 were combined and freeze-dried to obtain a white powder of crude pR-13x3 (2,26P').

In order to further purify these, first crude 1) R-AB
The powder (2y) was dissolved in 0.02M sodium phosphate-disodium phosphate buffer (20me, pH 7,25), and the solution was added to a column packed with Sephadex G-100 (
φ5×32Cm) and the same buffer (300me
), fractionate each S me, combine the fractions from No. 30 to No. 40, put them into a dialysis tube (Vuisking cellulose tube 20/:32 type) K, and dialyze it under running water for one day and night. After that, the internal solution was lyophilized and purified PR
-AB white powder (J, 34y) was obtained. The physicochemical and biological properties of this product are as described above.

Next, the crude PR-BH powder (2,2y) was added to a 0.02M phosphoric acid-disodium phosphate buffer (22M
e, pH 7,25) and Sephadex G-1
Add it to a column (φ5X300111) packed with 00, elute with the same buffer solution (60 (h++e), fractionate 2.5t, and combine the fractions from No. 75' to No. 1.05. After placing it in a Leo dialysis tube (Dice King cellulose tube type 20/32) and dialyzing it under running water for one day and night, the inner solution was freeze-dried and a white powder of purified PR-BB (
341nw). The physical, chemical and biological properties of this product are described in 1. That's right.

Example 3 Japanese plum seeds (produced in China, 500p) were crushed using a mixer, left in acetone (2,5A') at room temperature for 5 hours with occasional stirring, and the remaining seeds were removed from the mixture by vacuum filtration. put out. This operation was repeated 5 more times, the remaining residue was dried with hot air for 10 hours, and the dried residue was crushed into fine powder (60 to 200
3 times with acetone (2.5t each, 3 times
the residue was extracted with methanol twice (2.5 h each).
A 13 hours each) and filtered under reduced pressure. Water (11) was added to the defatted white residue obtained in this way, and the mixture was extracted by leaving it for 5 hours at room temperature with occasional stirring. Separate the extract and the residue, and wash the residue with water (300m).
.

The washing liquid was combined with the extract liquid.

Dice this extract into a cellulose tube (20/
After dialysis under running water for a day and night, the liquid in the dialysis tube was collected, centrifuged and separated into supernatant and precipitate, and the supernatant was freeze-dried to form a white powder (3s , 4y)
I'm H. This powder (5y) total 0.02M phosphate-sodium-difluorium-1-lium phosphate mild mj solution (pH 7.2
Sepharose 6
B was added to a column (φ6 x 50 C1i) packed with B, and eluted with the same buffer (1600me) for 10 minutes.
? Fractionate each ILe, and combine the divisions from No. 80 to No. 110 and from No. 121 to No. 130, respectively.
They were freeze-dried separately to obtain crude PR-AC white powder (3,59y) from the former section and crude PR-BC white powder (1,16g) from the latter section.

In order to further purify these, the coarse PR-AC powder (1,2y) was added to a 0.02M phosphate-sodium-disodium phosphate buffer (12me, pi-17,25
') and added to a column (φ3.8 x 29 cm) packed with Sephadex G-100, eluted with the same buffer (200211e), and 7.3
Fractionate each tube into dialysis tubes (Dice King Cellulose Tube 2).
After dialyzing it under running water for one day and night, the internal solution was freeze-dried to obtain purified PR-AC white powder (type 0/3.2).
60fnf/') was obtained. The physicochemical and biological properties of this product are as described above.

Next, phase PR-BC powder (1,1y) was added to 002M sodium phosphate-disodium phosphate buffer (11, m
e, pH 7,25), Cef7,
Column packed with G-100 (φ3.8X23cm
) and eluted with the same buffer (300me).
．． Fractionate 57ne each, put the fractions 27 to 42 together, put this into a dialysis tube (Dice King cellulose tube type 20/32), dialyze it under running water for one day, and then freeze the internal solution. It was dried to obtain a white powder (210ff1g) of purified PR-BC. The physicochemical and biological properties of this product are as described above.

[Brief explanation of drawings]

Figure 1 shows PR-AA, Figure 2 shows PR-BA, Figure 3 shows PR-AB, APR-BB, and Figure 5 shows PR-A.
FIG. 6 of C shows the infrared absorption spectrum of PR=BC. Figure 7 is of PR-AA, Figure 8 is of PR-BA, Figure 9 is of PR-AB, Figure 10 is of PR-B 13,
Figure 1 shows the ultraviolet absorption spectrum of PR-AC, and Figure 12 shows the ultraviolet absorption spectrum of PR7BC. Patent Applicant Dainippon Pharmaceutical Co., Ltd. Agent Akira Kojima Figure 8 Figure 9 Wavelength Figure 10; Ij, Long Figure 11 Procedure Supplement 1 Document (method) (spontaneous) 1. Indication of the incident Showa 1957 Patent Application No. 170439 2. Name of the invention: Biologically active protein derived from seeds of almonds, amphibians or Japanese plums. Noro, contents of amendment As shown in attached sheet, type engraving of specification 'B-'j 'ILh
)'s child name (no change in content) (hereinafter) Procedure - Mountain (F Needle (spontaneous) 1 Indication of incident 1982 Patent Application No. 170439 2 Name of invention Bioactive protein 3 Supplementary IE Relationship with the patent applicant case Tyr 25 Doshomachi 3-chome, Higashi-ku, Osaka Name 2
91 Dainippon Pharmaceutical Co., Ltd. W"1" deceased President Tomio Fujiwara 4 Agent 5 Column 6 of "Name of the invention -" of the specification subject to amendment 1, the invention on page 1 of the specification of the contents of the amendment In the name column, 1-Biologically active protein obtained from almonds, apricots, or Japanese plums 4'' is written in application 1 as 11A, ``Bioactive protein obtained from almonds, apricots, or Japanese plum seeds.'' and correct it. that's all

Claims (3)

[Claims] (1) Biological part 441 “Protein PR” with a molecular weight of 300,000 to 350,000 obtained by extracting almond seeds with water
- AA and a bioactive protein PR-BA0 with a molecular weight of 10,000 to L5″15 (2) Biologically active protein P R-A B with a molecular weight of 250,000 to 350,000 obtained by extracting Anse seeds with water
and biologically active protein R-BB with a molecular weight of 10,000 to 30,000.
0 (3) Biologically active protein PR-AC having a molecular weight of 250,000 to 350,000 and bioactive protein PR- having a molecular weight of 10,000 to 15,000 obtained by extracting Japanese plum seeds with water.
BC8