DE1617510A1 - Process for the extraction of growth and metabolism promoting low molecular weight substances - Google Patents

Description

Process for obtaining growth and metabolism promoting low molecular weight substances. .; ;

In the German patent specification; 107688 $ describes a process for obtaining injectable atöiungs-promoting active substances from blood. According to this method, an extract is produced from defibrinated blood or fragments thereof, such as blood cells or blood plasma from animals for slaughter, especially young calves that are active in RES or such slaughter animals that have been converted into a state of changed activity according to German patent 1017744 Atation-promoting effect in the Warburg apparatus on mitochondria or liver comogenates can be demonstrated. up to C ₄ or carried out with acids. The higher molecular components are separated off by means of oilysis against water or diluted alcohol. The oil ysate obtained in this way is, if necessary, freed from the organic solvents used and brought to a concentration of 30-60 mg dry substance per ml.

It has been found that the ultrafiltration of hemolyzed blood cells from the blood of young animals, e.g. from calves, young sheep or young goats, but also from the blood of human newborns, such as is obtained in exchange transfusion, leads to a product which, after separation of ineffective accompanying substances (e.g. salts , free. Amino acids, etc.) by means of ion exchange or absorption columns, eg ion exchangers based on cellulose, dextran or resin, as well as hydroxyl apatite and / or preparative high-voltage electrophoresis, to isolate chemically pure substances with a high increase in metabolic activity. Auen from the blood cells of adult Tl era _f £ ■. D. Cattle, la «aar. »I clearly characteriele / ifc ^ xa. with the abovementioned closely related substances isolate the apparently age-related common of the abovementioned substances

Maximum absorption in the range of 290 * · 298 and from which by suitable chemical or biochemical 'methods the original substance represented semi-synthetically can be*

The substances mentioned are characterized by their UV absorption or infrared spectrum, through X-ray spectrograrran as well as by other physico-chemical sigfease-related, how they can be determined with electrophoresis, chromatography or analysis in the ultracentrifuge, etc. «

The measurement of the activity of the products isolated according to the invention is carried out using the Warburg apparatus on simple breathing models, preferably of obligate aerobic bacteria or of the growth rate of obligate aerobic microorganisms {bacteria ^ Yeasts, etc.) or the growth rate of plant seedlings.

The products obtained according to the invention should be more industrial Evaluation, e.g. for the promotion of hydroponic cultures, Enabling the cultivation of grain under unfavorable climatic conditions, increasing the yield rate of crops and in animal breeding and therapeutic applications in severe catabolic diseases in humans and animals; also to control the breakdown of orally or parenterally administered substances for nutritional or therapeutic purposes. The substances should continue to be used for improving si the growth of cells of animal and vegetable origin in cultures as well as to improve the survival time of isolated, - for the transplantation of certain tissues and organs or also single cells, e.g. sperm.

EXAMPLES:

* 1 liter of fresh calf blood becomes several times Washing a blood cell sediment with isotonic or hypertonic saline solution manufactured. The 400 ml of blood corpuscle sediment that accrues on average in this case are at least 800 ml of distilled water are added and the ultrafiltration is carried out in a high pressure membrane filter with a

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! Subject to permeability for molecules up to IO GOO molecular weight. It is filtered at a pressure of up to 30 atmospheres, the filter material must be stirred vigorously.

The filter cake still contains active substances, but is of economic reasons, not to be used for obtaining the products of the invention ι but he can serve as Aüsgangsmaterial for other products, for example for the production of specific antigens.

The ultrafiltration thus obtained approximately 1000 mL was concentrated to l / lO its volume by vacuum distillation at max + 1O ⁰ C, and then the support-free preparative Hochspannungselektrop "horese preferably using organic volatile buffer of pH-range 4.0 -. 10.0, Eg triethylamine acetic acid or formic acid or ethylene diamine formic acid or acetic acid, with the addition of equal parts of buffer / f to the filtrate. The electrophoretic separation of the filtrate takes place under the specified conditions at a voltage of preferably 2300 V and 200 mAmp The temperature to be maintained during the separation depends, among other things, on the load capacity of the separation chamber, but can range from -§-2 to + 40 ° C.

The electrophosis refractions obtained are preferred determined according to their UV absorption spectrum. Depending on the buffer used and the pH value during the separation (specific Salt formations) v / earth substances with absorption maxima im Range of 232-248, 252-262 and 275-29 obtained. Primary According to the invention, active substances are found under suitable conditions, especially in the short-wave range. fast-moving active fraction has e.g. an absorption maximum at pH 7.0 of 246, in the strongly acidic range at and in the strongly alkaline range at 290 ^, *,

By concentration by freeze drying or by vacuum distillation At temperatures below 20 ° C, the substances are enriched and the volatile buffer substances are removed. To

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Setting suitable concentrations and if necessary After sterile filtration, the substances obtained in this way are suitable for the applications according to the invention.

2.) An automatic production method of the substances according to the invention results in the following way with utilization of the above basic principles of electrophoretic Separation and separation according to molecular size by membrane filters

The blood cell lysate described under 1) is pumped through a multi-chamber high voltage electrodialysis system. The outer cells of the facility contain the dialysate as well one cathode each; they are separated from the inner cell by two membranes completed, which contains the anode and of distilled Water is flushed through. The membranes used are preferably ultrafilters with a molecular permeability for molecular weights up to Io ooo.

If the electrodialysis system does not use hemolysate but loaded with an ultrafiltrate from a lysate, so can instead of the ultrafilter membranes in the dialysis chamber too Ion exchange membranes are used.

A vacuum distillation system can be switched on in the circuit of the inner cell of the electrodialysis system and in the distillation unit piston the extracted substances are enriched, while the condensate again through the inner cell of the electrodialysis system flows and absorbs new substances.

An automatic control of the entire system can take place by means of a flow photometer, which is in the outlet of the Inner cell of the high-voltage dialysis system is switched. This automatically adjusts the absorption at the absorption maximum in the UV range of the critical substances can be controlled and as a signal for the automatic control of the throughput speed etc. serve to achieve maximum or economic utilization of the starting material.

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That in the inner cell of the high-voltage electrodialysis system or “protein-free raw material enriched in the downstream vacuum distillation flask, based on the starting material (Blood cell lysate or ultrafiltrate thereof) Concentrated at least 1/30 of the volume in the vacuum distillation, so that a viscous, yellow-white product is formed. The. Substances according to the invention are made from this raw concentrate taken up in ethanol * by the raw concentrate with the 5 times the volume of ethanol is stirred. The inventive Substances in ethanol in £ * b 'solution, while initially unused accompanying substances including inorganic salts etc. settle or throw off will. The obtained water-clear, yellowish alcoholic The solution is the purified crude product, which only a few, contains definable substances.

For use according to the invention, the product is preferably converted into water by removing the alcohol by vacuum distillation at temperatures as low as possible below 2 O ⁰ C until a syrupy product is formed which is completely water-soluble. The concentration of the essential products in the aqueous solution is adjusted by UV spectrophotometry at a wavelength of .246 ψφ. The degree of purity of the substances obtained in this way is sufficient for most of the applications according to the invention. Further work-up does not bring any advantages for most commercial areas of application, while the exact, quantitative determinability of the active substances is already guaranteed.

A preparation of the which can be used for therapeutic purposes substances according to the invention must have a concentration the 20-fold dilution with distilled water, measured against distilled water, at least one absorbance of 0.6 results in a layer thickness of 5 mm at 246

For special purposes, the single substances contained in the product mentioned above can be isolated. This is preferably done by using lower alcohols, such as

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181751 θ / -

e.g. isobutanol. The aqueous concentrate is vigorously mixed with twice the volume of isobutanol and separated into the aqueous and alcoholic phases at low temperatures or in the preparative ultracentrifuge at -20 - -30 ° G. The lower yellowish aqueous phase contains a substance according to the invention with an absorption maximum at 245 wj * _f a second substance according to the invention is obtained * when the isobutanol supernatant is frozen out at -80 ° C and the resulting white cloudiness is centrifuged off. This white sediment is freed from isobutanol in a vacuum and is then completely soluble in water. The absorption maximum of this substance is 247

A third substance is obtained in that the said: ieo-butanol fraction is evaporated in vacuo after the 247 fraction has been removed. What remains is a whale, water-soluble substance with an absorption maximum at 256 μm *. Two further, optically inactive impurities are not used any further.

3.) A particularly high concentration of the products according to the invention is obtained by the fact that the starting blood is in a strong dilution with distilled water is hemolyzed, one part of blood mixed with at least 20 parts of water will. This very thin hemolysate is subjected to a suitable spray drying process (e.g. in the spray drying tower) subjected; this creates a dry powder with extraordinarily large surface and the finest particle size, then that can be subjected to the specific, patent-compliant extraction with alcohols. This is done using the dry powder stirred directly in anhydrous ethanol »whereby the invention Substances in the ethanol go into solution. After centrifuging and / or filtering off the insoluble constituents becomes a protein-free concentrate according to the invention 'Substances obtained' from the dia after evaporation of the alcohol solution of the active ingredients with photometriach adjusted

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Claims (1)

! Specimen copy •. , - '} May not be changed claims? »- -— Process for the production of low molecular weight substances Blood to promote growth and metabolism in plants and animals and for therapeutic use in severe catabolic diseases in humans and animals, thereby characterized in that the blood of young animals is subjected to an ultrafiltration, the ultrafiltrate by means of ion exchange or absorption columns and / or preparative high voltage electrophoresis is separated into individual facts and the through various physicochemical properties, such as e.g. üV-Sbsorptioris ^ or infrared spectrum, through X-ray spectrography by electrophoresis, chromatography or Ultracentrifuge analysis characterized substances are isolated. Sub-claim 1) Method, characterized in that a blood cell - sediment from fresh calf blood with at least double that Amount of distilled water is added and the ultrafiltration is subjected to at least 30 Ätü. Dependent claim 2) Method according to dependent claim 1), characterized in that the ultrafiltrate for further purification with preferably volatile organic buffers in the pH range between 4.0 and 10.0 is added and separated by electrophoresis. Method according to 2), characterized in that the electrophoretic Separation at a voltage of preferably 2300 V and 200 mAmpr in a temperature range of 2-40 ° C he follows. Method according to the main claim, characterized in that a blood cell hemolysate or an ultrafiltrate thereof diner high-voltage electrodialysis is subjected to> lrd, where as membranes, preferably ultrafilter or ion exchange membranes used and an automatic control ' 10 9 81 Vi 18 8 3 ^ «« »jal» ^ _ the system can be done by means of a flow photometer. Method according to the main claim, characterized in that the blood of young animals is subjected to a strong dilution and in a spray drying tower into a product very small Grain size and thus extremely large surface is transferred, which the method according to 2) or the direct Is subjected to extraction in anhydrous alcohol. Method, characterized in that the erythrocytes of adult animals and humans using the method according to the invention w.erden w.erden and the resulting crude product to another biological or other synthetic Can be subject to change. Method, characterized in that the invention Substances for therapeutic purposes are ampouled in a concentration which under the given conditions a Gives an absorbance of at least 0.6. Considered publications: German patent specifications No. 1076888, 1017744. April 20, 1966 Wolfgang Helmbold, Heidelberg 10 98 i 1/18 8 3