JPS6366124A - Production of deproteinized thymus extract having metabolic activity - Google Patents

Abstract

PURPOSE:To obtain the titled extract, by dialyzing a crude extract and by extraction from a thymus and enzyme treatment to separate a deproteinized part and protein-containing part, hydrolyzing the latter part to a peptide fraction by an enzyme, removing a protein and treating a product obtained by putting the remainder and above-mentioned deproteinized part together. CONSTITUTION:An aninal- or human-derived thymus is divided or homogenized, defatted at -15 deg.C and ether-treated and a buffer solution having 6.8-7.9pH and an organic acid having <=3pH are added in turn to separate and extract, and a protease is added to a residue to provide a crude extract, which is then dialyzed (at 4-5 deg.C, for 24-96hr) to separate a deproteinized part and a protein- containing part. The latter part is concentrated or subjected to lyophilization and hydrolyzed into a peptide fraction by an enzyme and the remaining protein is removed by adding an organic acid or ethanol and the resultant remainder and the above-mentioned deproteinized thymus are put together and concentrated to a desired dry residue content (6-15%) under vacuum and adjusted to 7.0pH and filtered under asptic condition to afford the aimed deproteinized thymus extract.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a deproteinized extract having metabolic activity from animal organs.

Deproteinized extracts and protein-containing extracts obtained from organs derived from animals or humans are used as original medical products 4 and raw materials for cosmetics. As a medical product, it must be completely deproteinized and contain no pyrogens.

Water-soluble metabolically active extracts obtained from organs are classified as follows.

Protein-containing extracts are evaluated for metabolic activity, enzyme content, collagen content, etc. Deproteinized extracts are also evaluated based on metabolic activity, peptide components, and other factors.

The basis of the metabolic activity already mentioned is as follows.

Growth test, wound healing test, REs activity, Warburg tissue respiration y1 factor (liver homogenate, mitochondria), etc.

These are in all cases the most important criteria for judging the quality and applicability of metabolically active extracts. The same applies to cosmetics.

The present invention is a deproteinized extract from the organs of animals, etc., which has a highly metabolic activation effect (tissue respiration increase rate with Wahlpurgutist is usually 300% or more), and is rich in peptides and nucleic acid-related components. The basic feature of the present invention is to provide a method for producing an extract containing protein, which consists in first separating the obtained crude extract into a protein-free part and a protein-containing part by dialysis; In the process of producing the crude extract, the protein-containing portion obtained by dialysis is also subjected to enzyme treatment to enrich it with peptides.

First, to remove the lipoid and hormone parts,
The organs are treated with acetone, followed by ether treatment at low temperatures (below -15°C, for more than 4 days''), and the extract is then treated with a buffer solution to pH 6-8. p) 13
Treat with organic acid below. (Acid component 8% or less, 0.0
Added less than 1% zinc salt or magnesium salt, organic acid of citric acid cycle), ? ! ) The extracted extract is sterile filtered and stored at 4°C until dialysis. The residue is treated with an enzyme (protease) (from the optimum temperature of oxygen).
The extract thus obtained, containing partially deproteinized bebutide, is optionally treated with preservatives. In addition, after adjusting the pH to 7.0, it is treated with aldonic acid, centrifuged, sterile filtered, combined with the protein-removed extraction part, and dialysis is performed. Dialysis is performed at low temperature, but dialysis tubes of 70 to 71 are used. p) 17. into the dialysis tube under sterile conditions, preferably. Add purified water to which methylparaben has been added. In addition to methyl paraben, preservatives include sodium sorbate, chlorethone, benzic acid, and benzyl alcohol. Dialysis time is 24~
After 96 hours of dialysis at a temperature of 4-5°C, the protein-free part and the protein-containing part were obtained, and the protein-free part and the protein-containing part were stored at 4°C through a sterile filter. Gonagi is concentrated by freezing and dry cooking, then subjected to enzyme treatment, and residual protein is removed by adding organic acid or ethanol.
After adjusting the pH to 16, 2 to 75, the protein-free extract obtained by combining the two is concentrated in vacuum (11), and if necessary, a preservative is added for adjustment. Concentration in vacuo is carried out until the amount of dry residue is between 6 and 15%, depending on the product in each case6Measurement of the metabolic activity is carried out at the end of the concentration as well as at the end of each individual production step18.
! The Hg ratio of the Warburg oxygen consumption for a liver homogenate of a thymus extract containing 10% dry residue, performed every 7 J, is 3.0 or more.1 The metabolically active extract prepared according to the present invention is extremely Low toxicity, e.g. LD50 of thymus extract (5-10% dry residue) is 20 g/K
g or more (mouse) for 1 minute, 5 minutes, 15 minutes, 1119, 6
Observations after 24 hours and 24 hours showed normal values. As a result of conducting a skin secondary irritation test (rabbit) and an ocular mucosa test (rabbit) using the thymus extract, no side effects were observed. Also, thymus extract does not contain hormones and has no side effects that can be attributed to hormone-mimetic effects (liposide hormones). Breasts as medicine +111! The extract must be freed of histamine and histamine-like substances, for example by treatment with diaminooxidase.

Metabolic activity is enhanced when an aqueous or alcoholic solution or lyophilized thymus extract is used in combination with a facial fluid extract or a placenta extract.

Thymus extract stimulates cell division, promotes growth, and increases resistance and immunity to infectious agents and toxins. The thymus gland shrinks with age and even disappears as the child grows older. At the same time, the number of substances that act on living organisms continues to decrease. In other words, by administering insulin to a diabetic patient due to pancreatic disease, life can be maintained, and by administering thymus extract to an elderly or convalescent person, it is possible to supplement the insufficient active substance. Similarly, it is possible to apply it to cosmetics to rejuvenate the ever-growing skin.In other words, the amount of 0 contained in the thymus extract (see Table 1) (damage healing)
] Cosmetics using a combination of an extract containing a component that promotes cellular respiration and a thymus extract have made it possible to improve skin protection. Anti-aging +
l- (medicine or chemical product) thymus gland, protonin (A
In addition to the theory of internal respiration by cellular respiration-promoting factors, oxygen by A (M, von Arde's theory)
It is well known that the thymus extract according to the present invention also has an extremely high effect on cellular respiration (+4.2).

Male Wistar rats weighing 225-250 g are anesthetized with ether and the scalp is shaved clean. Make a 4cm long cut along the center of the part. Make a total of 3 cuts at leg intervals. 6 days after treatment, I
I day mouth 16 days 1. On day 21, open the injured area and take 3 sections of skin in a direction perpendicular to the wound line. (A 1 cm wide cut is made around the wound line.) The wound area is removed and the distension force is measured. Thymus extract was administered subcutaneously at 25 mg/Kg per day, and physiological saline 25 I1g was administered to the control group.
/Kg was administered in a similar manner.

The number of animals used in the experiment was 20 in each group.

The healing promoting effects of thymus and blood extracts on injured ft3 in rats are shown in Table 1.
11 Extract>Blood extract was effective in this order, and it was found that combination use with blood extract was most effective. The thymus gland extract and the extracts from fish eggs, uterus, and cock's comb according to the present invention exhibit excellent effects on improving skin structure and are extremely effective as cosmetic warming agents.

The experimental results for rough skin using the Padberg method are shown in Table 2.

In the Padberg method, since the amount of methylene blue adsorbed varies depending on the condition of the skin, determination is made by measuring the amount of methylene blue adsorbed. In other words, many experiments over many years have shown that ``perceived rough skin'' or ``skin damaged by surfactants'' absorbs more methylene blue in proportion to the degree of damage. The adsorption amount of methylene blue on skin well-treated with cream showed a small value.

The Padberg method is tested as follows.

1. Determination of test site 2. IT1 treatment with a 1% aqueous solution of nonionic surfactant.

3. Rinsing with warm water at 35°C 4, wiping the test site 5. Acclimating the subject for 30 minutes at 23°C 1 60% relative temperature.

6. Apply staining solution 2011n for 30 seconds.

(This staining solution = 20% 0.5% methylene blue and 80% 1% nonionic surfactant) 7. Allow the dye to dry for 30 seconds.

8. Remove excess dye with 0.25% nonionic surfactant solution.

Elute for 60 seconds each.

Note 1 Sodium nilauryl sulfate 2% purified ice
48% Isopropyl alcohol 50% 10. Bf (Onm) of the eluate was measured using a Zeiss spectrophotometer.
Measure the absorbance at

+1. Result = absorbance of untreated skin.

12. Be sure to apply the sample on the designated area twice in the morning and evening for 20 days! σ. The use of other cosmetics is prohibited for 3 days before the start of the test and during the test period.

13.1-9 operations 211] [+The final operation is performed 4 hours before the test.

Results - Absorbance after use The numbers in Table 2 are the ratio of absorbance in % compared to the initial state and at the end of the test. Very good results were obtained with the addition of T.

(Example 1) A method for producing a deproteinized thymus extract having a cell activating effect from the thymus of a calf. Immediately after slaughtering a healthy calf, the thymus was frozen and fragmented, 50 grams of acetone was added thereto, left for 48 hours, and then acetone was added. Add 25 grams and leave for 72 hours to remove the fat-soluble portion. After separating the acetone layer, 15 grams of ethyl ether and 5 to 10 grams of purified water were added to the residue, and the mixture was heated at -15°C to -2
After standing at 5°C for more than 100 hours, centrifuge. Add buffer to the residue and extract twice in neutral water (■,
If), add a solution containing #1-8% of the residual acid cycle and (11) 1-0.001% zinc and extract twice at pH 3 or lower (m, rv). Pronase is added to the residue and J-reacted for 48 hours, followed by addition of aldonic acid to remove protein. Further, the extract (V) that was centrifuged for 1 to 2 hours was combined with the previously obtained extracts I to ①, and after adding paraben and adjusting the pH to 7.0, dialysis was performed. After dialysis, a protein content test was performed. After confirming complete protein removal, sterile filter and store at 4°C (Extract Vl)
,! The white content is concentrated by freeze-drying, then hydrolyzed with enzymes, deproteinized twice, centrifuged, and the resulting protein-depleted extraction? Then, combine with the protein-free extract ① and concentrate, add a preservative, and after adjusting the pH, concentrate until it contains about 10% solids. The analytical values of the thymus extract are shown in Tables 3 and 4.

(￥ Absolutely 2) Deproteinized extract with metabolic activation effect obtained from frog eggs, Xenopus Iaevis D, a type of frog
After homogenizing 100 kg of AUD-IN eggs, ethyl ether 201 was added, a preservative was added to the n1 extract obtained by centrifugation, and the pH was adjusted to 7.8. Decomposes amines.

After adjusting the thus obtained deodorized crude extract to pHB 9,
Dialysis is carried out as in Example 1, yielding 11 kg of deproteinized extract from frog eggs containing 5% residual dry matter and 0.7% peptide. The oxygen consumption increase rate of this extract is about 3.5.

(Example 3) Pig or sheep uterus or! Deproteinized extract with metabolic activation effect obtained from Maru.

25kg pig's round or uterus or sheep's round or uterus 10
0 kg was cut into small pieces, homogenized, defatted with acetone, hormones were separated, 30 kg of ethyl ether was added, and the crude extract obtained by centrifugation was adjusted to pH in the same manner as in Example 1.
7.0, dialysis, and 18 kg of extract containing about 3% dry residue (enzyme consumption increase rate 3.7,
Contains 1.4% peptide).

Table 1: Effects of various extracts on wound fh healing rI Number Treatment group Control group Δ Difference (25m/Kg) (1) Blood extract 6216±18103±30113 11 489±25 401±216818
810±50 720±298021 18
37±55 1422±59215 (2) Thymus extract θ 230±1488±33 131
1+ 490±28 418±247
418 803±44 701±37
10222 ° 1703±49 1441±5
1 282 (3) Blood extract + thymus extract 6250±18 105±29 14511
530shi31 430±21
10018 901±48 740±33
18121 1809±49 14B6±
55 343P (0,05 Table 2: Rough skin level test using W10 type cream containing thymus extract (↑) according to Example 1) (Pad-berg
method) Test period = 20 days l) Subject age 2) Placebo 3) T2 additive 4
7 7B, 7
70.44 0 9
7.13 [10,8487θ,
j 84.54 2
90.4 ・55.
86 1 B4.9
73.04 6
91.8 40.74 2
79.0 47.
85 9 95.0
58.84 3
91.4 59.35 0
92.2 8
2.0 Mean value 84.5 59.3 Table 3: Analysis of thymus Jtltlj according to example 1 PH6,9
0 Dry residue 9.85% total nitrogen content
0.88% JLC (aminochuu 0 Figure 1 JLC (diffusion and diffusion-related substances a) Figure 2 Oxygen consumption rate
4.2 Viable bacteria count test
Sterility Table 4: Veptide content of thymus extract Standard peptide 0.050 20.000
Solution 1■/intestine 0.050 20.0000
．． 050 20. OQO standard peptide dissolved KN 2tng/ml O,09?
20.6190.098 20.408
20.48B 0.43480.098
20.40B Standard peptide solution 3@glWA statement 0.142 21.12
70.143 20.979 0.144 20.833 Srel (%) 2,122 Procedural amendment April 15, 1988 1. Indication of case 1989 Patent application No. 209550 2. Name of the invention Metabolic activity Process for producing deproteinized thymus extract 3, relationship with the amended case Patent applicant address: 3-28, Kanda Sakuma-cho, Chiyoda-ku, Tokyo Name: Yamakawa Trading Co., Ltd. Representative: Hiroshi Kuriyama 4, Agent 5. Date of amendment order (voluntary) 6. Subject of amendment 7. Contents of amendment (1) Page 14 of the specification, line 2, "rPad-berg"
is corrected to "rPadberg".

(2) In the third and fourth lines from the bottom of the same page, the words rJLcJ are corrected to rTLcJ, respectively, and "diffusion and diffusion-related substances" in the third line from the bottom is corrected to "nucleic acids and nucleic acid-related substances."

(3) Delete lines 3 and 4 from the bottom of page 15.

that's all

Claims (2)

[Claims] (1) After subdividing or homogenizing the thymus gland of animal or human origin, it is defatted at -15°C, treated with ether, and then adjusted to pH 6.
After separation and extraction by adding a buffer solution having a pH of 8 to 7.9 and an organic acid having a pH of 3 or lower, protease is added to the residue to obtain a crude extract. This crude extract is separated into a protein-free part and a protein-containing part by dialysis, and the latter is concentrated or freeze-dried, then hydrolyzed with enzymes to the peptide fraction, residual protein is removed by acid/ethanol precipitation, and the latter is concentrated or freeze-dried. Concentrate in vacuo to the desired dry residue content and pH
A method for producing a deproteinized thymus extract, which comprises adjusting the protein to 7.0 and then sterile filtration. (2) The production according to claim 1, in which the acid of the citric acid cycle is added in a total concentration up to 8% with addition of 0.01% zinc salt for extraction at pH 3 or less. Law.