JPH06247871A - Growth promoter - Google Patents

Abstract

PURPOSE:To obtain a growth promoter for accelerating growth of infants or young animals. CONSTITUTION:This growth promoter comprises angiogenin and/or its decomposition product as an active ingredient. The growth promoter is pharmaceutically manufactured to give a medicine or added as an additive to a food, a drink or a feed. Since angiogenin can be separated and produced from a milky raw material relatively readily, the growth promoter comprising angiogenin and/or its decomposition product as an active ingredient can be inexpensively supplied. Consequently, the growth promoter is useful as a material for a medicine, a food, a drink and a feed.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a growth promoter containing angiogenin and / or its degradation product as an active ingredient. This growth promoter is particularly useful for promoting the growth of infants and young animals.

[0002]

BACKGROUND OF THE INVENTION Angiogenin is a protein having an angiogenesis-inducing action and having a molecular weight of about 14 kDa, and was first isolated from a human intestine cancer cell culture medium [Biochem. , Second
4, 5480-5486, 1985], and then
It is also known to be isolated from normal human cells [B
iochem. 26, 5141-5146, 1
987]. And, human angiogenin is another protein that shows angiogenic action, acidica
nd basic fibroblast growt
h factor (FGF), transformin
g growth factor α (TGF-α), t
transforming growfactor
β (TGF-β), tumor necrosis f
It has been reported by Fett et al. that it exhibits an angiogenesis-inducing action on the order of picomoles as in the case of actor γ (TNF-γ) and the like [Biochem. , Volume 24,
5480-5486, 1985]. In addition, it was reported by Michael et al. That bovine diogenin exhibits an activity equivalent to that of human angiogenin [B
iochem. , Vol. 27, pp. 6282-6287, 1
988]. Therefore, angiogenin is used for its use as an angiogenic factor, especially for trauma, ulcers,
It is a substance expected to be used as a therapeutic agent for circulatory function and transplantation. A hair nourishing agent containing angiogenin as an active ingredient has also been proposed [JP-A-4-21].
No. 0618].

On the other hand, as a method for preparing angiogenin, a culture solution of human adenocarcinoma cell line HT-29 is acid-precipitated, filtered, and then treated with a cation exchanger (CM52, Whatman). The method of Fett et al. [Biochem. , Volume 24,
5480-5486, 1985], human plasma is treated with a cation exchanger, treated again with a cation exchanger, and then purified by reverse phase chromatography.
Ro et al. [Biochem. , Volume 26, 5141
Pp. 5146, 1987] is known. Also, B
ond et al., in a manner similar to the method of Shapiro et al.
It has been reported that a large amount of angiogenin was prepared from bovine serum [Biochem. , Vol. 27, 6282
~ 6287, 1988]. Furthermore, Spik et al.
Milk is a cation exchanger (SP-Sephadex C-5
No. 0, manufactured by Pharmacia), and again treated with a cation exchanger (S-Sepharose Fast Flow, manufactured by Pharmacia), and a method for producing angiogenin is proposed [JP-A-2-269000].

[0004]

DISCLOSURE OF THE INVENTION In the process of examining new uses of angiogenin, which is contained in milk in a particularly large amount, the present inventors show that angiogenin has some physiological action on infants and young animals. We suspected that this might be the case, and conducted an angiogenin administration test in rats, and found that the administration of angiogenin promoted the growth of young rats. In addition, when a similar test was performed on a decomposed product of angiogenin,
It was found that the same effect as that of angiogenin was exhibited, and it was revealed that the growth of rats was promoted, and the present invention was completed. Therefore, it is an object of the present invention to provide a growth promoter containing angiogenin and / or its degradation product as an active ingredient.

[0005]

The method of obtaining angiogenin used as an active ingredient of a growth promoting agent in the present invention is not particularly limited, and it is prepared from milk or blood of mammals such as humans, cows and pigs. Those produced, or those produced by gene recombination technology can be used. When the growth promoting agent of the present invention is used in foods, drinks, cosmetics or feeds, it is not necessary to use highly purified angiogenin, and there is no problem even if it contains lactoferrin.

As the decomposition product of angiogenin used as an active ingredient of the growth promoting agent in the present invention, those obtained by hydrolyzing angiogenin with a protease can be used. Proteases that can be used for the hydrolysis of angiogenin include trypsin, chymotrypsin,
Examples thereof include thermolysin, subtilisin, papain, pepsin, and the like, and these proteases are preferably used in a weight ratio of 1/30 to 1/200 with respect to the substrate. The substrate concentration is not particularly limited, but is preferably about 10% or less at which the viscosity does not increase so much. The enzyme reaction is usually carried out at 37 ° C, but when thermolysin is used, it is 40
It is better to carry out the enzyme reaction at ℃. In addition, these protease treatments can be performed in the range of 20 to 42 ° C. The pH range suitable for the enzymatic reaction is 7 to 8 when trypsin, chymotrypsin, thermolysin and subtilisin are used, 5 to 8 when papain is used, and 2 to 3 when pepsin is used. Regarding the time for carrying out the enzymatic reaction, if the time is too long, angiogenin will be extremely reduced in molecular weight, and therefore, it may be carried out within a range of 10 minutes to 10 hours, preferably 10 minutes to 4 hours.

The growth promoting agent of the present invention containing angiogenin and / or its degradation product as an active ingredient is orally administered as angiogenin and / or its degradation product as tablets such as sugar-coated tablets and tablets, granules, liquids or capsules. It can be used in a form that can be administered locally. Further, the growth promoting agent containing the angiogenin of the present invention and / or its decomposition product as an active ingredient may be added to various foods and drinks such as beverages, soups, jellies, breads, noodles, sausages and the like for use. Furthermore, the feed containing the growth promoting agent containing the angiogenin and / or its degradation product of the present invention as an active ingredient, cattle, horses, goats, pigs, sheep, domestic animals such as rabbits, pet animals such as dogs and cats, It can be administered to other domestic animals, experimental animals and the like.

When the growth promoter of the present invention is added to food or drink or feed, it is desirable to add angiogenin sterilized by a membrane, but it is also possible to heat sterilize the angiogenin under mild conditions. . When angiogenin is added to food or drink or feed and then heat-sterilized, it is necessary to consider that the activity of angiogenin is reduced to about 2/3.

In the present invention, the dose of angiogenin and / or its degradation product is not particularly limited, but the daily dose is 1 to 100 m / kg of body weight.
It is good to use g as a rough guide. That is, the dose is 1 m
When it is g / kg or less, almost no growth promoting effect is recognized,
Further, when the dose is 100 mg / kg or more, the growth-promoting effect is not significantly increased. Since angiogenin is a substance contained in milk in a large amount, it can be said that there is no problem in its safety. Next, an example of the present invention will be specifically described.

[0010]

[Example 1] Sulfonated chitopearl (Fuji Spinning Co., Ltd.)
Column made of 30 L (diameter 1.5 m x height 18)
cm), raw skim milk 3,000 kg, 1,800 kg / h
The liquid was repeatedly passed through at a flow rate of 3 times. After passing through the liquid, set the column to 3
100 L of hot water at 0 to 35 ° C is passed through to thoroughly wash, and
50 L of a 0.5 mM sodium hydrogen carbonate (pH 7.5) solution containing 7 M sodium chloride was passed through to collect 49 kg of an eluate. Next, this eluate was subjected to molecular weight cut-off of 50,000.
Concentrated and desalted with an ultrafiltration device equipped with the ultrafiltration membrane of
Lyophilization gave 263 g of a fraction containing angiogenin.

Further, in order to improve the purity, 720 mg of the angiogenin-containing fraction was added to a 10 mM Tris-hydrochloric acid buffer solution (pH 7.0) containing 3M sodium thiocyanate.
It was dissolved in 26 L and centrifuged at 1,000 rpm for 30 minutes to remove insoluble matter. Next, cold ethanol was added to this supernatant so that the ethanol concentration was 80%,
After standing at 40 ° C. for 4 hours, centrifugation was performed at 1,000 rpm for 30 minutes to remove the precipitate. Then, the supernatant was concentrated, desalted by an ultrafiltration device equipped with an ultrafiltration membrane having a molecular weight cut off of 2,000, and freeze-dried to obtain 10.3 g of angiogenin powder.

[0012]

Example 2 Angiogenin 3 g was added to 60 ml of 10 m.
It is dissolved in M phosphate-sodium phosphate buffer (pH 7.0), and 60 mg of trypsin (manufactured by Sigma) is added,
After the reaction was carried out at 37 ° C. for 1 hour, the reaction was stopped by performing a heat treatment at 90 ° C. for 10 minutes.
The precipitate was removed by centrifugation at 0 × G for 10 minutes. The reaction product thus obtained is freeze-dried,
2.6 g of angiogenin degradation product powder was obtained.

[0013]

[Test Example] The angiogenin prepared in Example 1 and the angiogenin degradation product prepared in Example 2 were tested for their growth promoting effect on rats. 16 years old for this test
Using Wistar rats weaned daily, 4 animals per group,
Group A did not receive angiogenin, and group B received angiogenin at a rate of 0.1 mg / day / kg, 1 a day.
1 group / mg of angiogenin in the group that was forcibly administered once
The group was forcibly administered once a day at a rate of 1 day / kg, the group D was forcibly administered angiogenin at a rate of 50 mg / day / kg once a day, and the group E was 200 mg for angiogenin /
The group was forcibly administered once a day at the rate of day / kg, and the group F was a group forcibly administered the angiogenin degradation product at the rate of 50 mg / day / kg once a day. In this test, casein 20% by weight, cellulose 5% by weight, corn starch 15% by weight, sucrose 45% by weight, vitamins 1
% By weight, 3.5% by weight of minerals, choline bitartrate 0.
2% by weight, DL-methionine 0.3% by weight and soybean oil 1
The diet adjusted to 0% by weight was administered. Also, water was freely available. Table 1 shows the body weights of the rats at the start of the test and 10 days after the start of the test.

[0014]

[Table 1] ──────────────────────────────── Group Start of test (g) 10 days after start of test (g ) ──────────────────────────────── A 36 ± 2 80 ± 2 B 35 ± 2 81 ± 3 C 36 ± 2 88 ± 3 * D 36 ± 2 94 ± 4 * E 37 ± 2 93 ± 4 * F 36 ± 3 91 ± 5 * ────────────────────── ─────────── Displayed as mean ± standard deviation, * indicates a significant difference with a risk rate of 5%. Significant increase in body weight in groups C, D, E and F compared to group A Was observed, and it was shown that angiogenin and angiogenin degradation products contribute to the growth promotion of juvenile animals.

[0015]

Example 3 Angiogenin 1 prepared in Example 1
g, sucrose 3.2 g, citric acid 53 g, sodium citrate 53 g, vitamin B ₂ 0.06 g, vitamin C
A composition obtained by mixing 0.37 g, folic acid 0.03 g, fragrance 6.7 g, 5-fold concentrated apple juice 134 g and water 3.5 kg was held at 63 ° C. for 30 minutes for heat sterilization, and then 5-1
It was cooled to 0 ° C. and stored in sterile containers. Next, this mixed solution was aseptically filled in a sterilized 200 ml paper container to produce a growth-promoting supplement beverage for infants.

[0016]

Example 4 Non-fat milk solids 11.5% and yeast extract 0.5
% Non-fat milk medium, Bifidobacterium longum SBT 2933R (Microtechnology Research Institute No. 8743),
Streptococcus thermophilus SBT 102
1A (Microtech Lab. No. 10658) and Lactobacillus acidophilus SBT 2062 (Microtech Lab. No. 1
No. 0730) was cultured at a temperature of 31 ° C. After culturing,
1.5 g of angiogenin prepared in Example 1 was added to give a complex bacterial starter.

On the other hand, 40 g of skim milk powder was added to 4.4 L of milk, stirred and mixed, and then the temperature was 60 ° C. to 70 ° C. and the homogenous pressure was 1
The raw material mix homogenized at 00 to 270 kg / cm ² was held at 90 to 95 ° C for 10 to 15 minutes for heat sterilization, and then cooled to 38 ° C. This raw material mix was inoculated with 500 ml of the above-described complex bacterial starter, fermented at 38 ° C for 5 hours, and then cooled to 10 ° C or lower. Sugar liquid mixture 1 in which 100 g of sugar, 5 g of xanthan gum and 5 g of orange flavor were mixed and dissolved in 5 L of the fermented milk thus obtained
After adding L and stirring, a homogeneous pressure of 100 to 270 kg /
Homogeneous treatment was carried out at cm ² , to produce a lactic acid bacterium beverage with a growth promoting effect for infants.

[0018]

Example 5 1,195 g of skim milk, 263 g of whey powder,
119 g of vegetable oil, 5 g of vitamins and minerals, and 2 g of angiogenin prepared in Example 1 were mixed to produce 1,584 g of infant formula milk powder having a growth-promoting effect.

[0019]

Example 6 Angiogenin 25 prepared in Example 1
0 mg, lactose 33.3 mg, corn starch 1
6.4 mg, carboxymethyl cellulose calcium 1
2.8 mg and 1.5 mg of magnesium stearate were blended to prepare a tablet growth promoter according to a conventional method.

[0020]

Example 7 Angiogenin 40 prepared in Example 1
0 mg, lactose 883 mg, corn starch 221
mg and hydroxypropyl cellulose 16 mg were blended to prepare a growth promoter for granules according to a conventional method.

[0021]

Example 8 Skim milk powder 60 g, WPC 14.3 g, fat 17.2 g, glucose 5.0 g, vitamins and minerals 2.5 g and angiogenin 1 prepared in Example 1
0 mg was compounded to produce a compounded feed for young animals.

[0022]

The growth promoting agent of the present invention containing angiogenin and / or its degradation product as an active ingredient is used not only as a medicine but also as a material capable of imparting a growth promoting effect to foods and drinks and feeds. be able to. Also,
Since angiogenin is contained in milk in a relatively large amount, it is possible to stably provide the growth promoting agent of the present invention.

Claims (1)

[Claims] 1. A growth promoter comprising angiogenin and / or its degradation product as an active ingredient.