DE911325C - Method of extracting oxytocin and vasopressin from the posterior lobe of the pituitary and separating them from one another - Google Patents

Description

Process for the extraction of oxytocin and vasopressin from the Posterior pituitary lobes and for separation from one another The invention relates to a method for the extraction of oxytocin and vasopressin from the posterior lobe of the pituitary gland and to separate them from one another.

The important role played by the posterior pituitary hormones in therapy Vasopressin and oxytocin play, which are extracted from the posterior pituitary gland of the animals, z. B. the cattle, where they occur together, are extracted, is known. It is very desirable to be able to separate these two hormones from each other because hers Effectiveness varies, and their effects can at times be opposite be. So it is the case with the use of oxytocin for the purpose of women to provide relief during childbirth, with the vasopressin as a result of it Dangerously interferes with effect on blood pressure. All previously known methods Separating these hormones from each other allow only the isolation of the one the same (of the oxytocin or the vasopressin), the other goes in the course of the Separation treatment is lost or destroyed in the process. This represents at the high Price of these hormones is a very noticeable disadvantage. On the other hand, they are the hormones thus obtained are never completely pure, for each of them is not through negligible amounts of the other or by accompanying hormones, which in the Posterior pituitary lobes occur, such as B. the intermediate contaminated.

These disadvantages are now eliminated by the method according to the invention, which allows the oxytocin and the vasopressin by means of a single step both to extract and to separate from each other with very good yield. By the process according to the invention, up to 50% and more of the total hormone activity can be achieved recover the starting material.

The method according to the invention consists essentially in the treatment the posterior lobe of the pituitary gland of an animal or a dry extract of this organ with an aqueous weak solution of an organic acid, being a solution which contains all the hormones of the posterior lobe of the pituitary gland, whereupon these hormones are adsorbed from this solution by active silicon dioxide and the silicon dioxide thus obtained with an aqueous weak solution of the above treated with organic acid. In this way you get an extract on the one hand, so-called eluate, which has almost all of the oxytocin, and on the other hand a silica residue which contains almost all of the vasopressin. In the end we deal with the last-mentioned extract and the last-mentioned residue mentioned separately for the purpose of extracting the oxytocin and the vasopressin in almost pure form Form from the same. Among the organic acids that are responsible for dissolving of hormones and treatment of silicon dioxide after adsorption of hormones suitable, should preferably be mentioned acetic acid, but also propionic acid and tartaric acid. The organic acid is of one concentration in aqueous solution in both operations below -%, preferably 0.5% used to reduce the hormones against acid are sensitive not to destroy.

The dissolving out of the hormones and the treatment of the silicon dioxide with the aqueous solution of an organic acid take place in the heat in order to exhaust the treated substances as far as possible. At the same well, the resulting solutions are immediately cooled to a temperature below 2O ° C, preferably from about o ° C to not to jeopardize the hormones by decomposition. Incidentally, as is always the case with hormones, this precaution should be observed at all stages of the procedure.

That is to adsorb the hormones from their acidic aqueous solutions used active silica is advantageously a silica, which, for short summarized, is prepared so that from a solution of an alkali silicate by an organic acid, such as. B. acetic acid, tartaric acid, salicylic acid od. Dgi., the silicon dioxide precipitates, the latter at a temperature excellently below It dries from i2o ° C and activates it by adding it to a weakly concentrated solution Boil acetic acid, wash it with water and dry it again.

An example execution of the production is activated further below Given silicon dioxide according to this procedure. The extraction of the oxytocin from the eluate can be extracted in the classical way by extraction with n-butanol, excellent after alkalizing it with ammonia. This butyl alcoholic solution is concentrated in vacuo and then finally with an aqueous solution of a organic acid, e.g. B. acetic acid extracted. In this form the product can in therapy, if necessary with the addition of a preservative such as trichlorobutyl alcohol, Find application.

However, it is preferable to use the oxytocin solution prior to extraction with n-butanol with a suitable solvent, such as. B. secondary octanol, Chloroform od. Like. To treat the lipids and pigments, which from the Originating from raw material and contaminating the oxytocin, leaching out and so on to remove.

The octanol extract is separated off and the remaining aqueous Phase subjected to extraction using n-butanol.

According to a characteristic feature of the invention, the oxytocin eluate can also of ferrous ions, which are contained in it and the destruction of the hormones catalytically speed up being freed by using it prior to extraction with n-butanol treated with a substance that is suitable for being non-ionized with the ferrous ions To form complexes, e.g. B. a-dipyridyl, copper tuna, sulfocyanate, oxyquinolein and the like.

This is done in such a way that you get to the acidic solution of the Oxytocins e.g. B. adding a-dipyridyl, then the excess of dipyridyl and one Part of the organic acid is extracted from the solution, which then, as in the previous one described, is treated exhaustively with n-butanol.

Extraction of vasopressin from the residue of silicon dioxide can be carried out by treating the latter with alkalis, the pn should nevertheless remain below io to the proteins through which the vasopressin is contaminated, to remove, whereupon the silicon dioxide with an aqueous solution an electrolyte that contains an organic acid in low concentration, is eluted.

Suitable electrolytes for elution are the alkali salts, e.g. B. sodium chloride or sodium sulfate, but especially the latter, given its poor solubility in organic solvents. A circumstance which precludes its removal by precipitation using an organic solvent, e.g. B. by means of ethyl alcohol. In this way, after the solvent has been distilled off, an aqueous solution of vasopressin is obtained which is free from mineral salts and can be used in this state or after renewed purification. The organic acid to be used here is one of the acids mentioned above, e.g. B. acetic acid with concentrations which do not exceed 2 % and are preferably below 0.5% .

After another important feature for the invention, one adds Eluate traces of a surfactant such as B. alkyl sulfonate, sulforicin oleate or the like, added to increase the degree of extraction. A mixture of high molecular weight Dimethylbenzyl-alkyl-ammonium-chloride (the alkyl radical contains 12 to 18 carbon atoms) and a polyoxyethylene derivative of sorbitol mono-oleate have proven to be special for this purpose proven effective. For the purpose of Purification of the aqueous eluate from vasopressin one can proceed according to the invention in the following way: The eluate is mixed with a solution an aromatic acid, which is sparingly soluble in water, in a solvent containing Water is miscible, treated. One can use a solution of salicylic acid in acetone use. In contact with the water of the eluate, the aromatic acid falls into very fine form with adsorption of the vasopressin. After being filtered off the acid crystals z. B. exhausted with a mixture of water and ether and the the aqueous phase containing vasopressin obtained. You can do it by repeating it multiple times this operation in this way contained 87% of what was originally in the saline solution regain previous vasopressin. The aqueous vasopressin solution thus obtained can finally be brought to a very high degree of purity that they are countercurrent in a suitable apparatus, such as. B. that of Craig, extracted using n-butanol containing water and salicylic acid. The pure vasopressin accumulates in the aqueous phase.

The following table shows schematically the course of the process according to the invention. The arrows provided with dashed lines denote those operations which are not absolutely necessary unless one wishes to obtain products of very high purity. Posterior lobe of the pituitary gland Vasopressin -f- oxytocin aqueous solution organic acid Oxytocin + vasopressin solution Adsorption on activated Si 0 2 Eluting with aqueous solution organic acid r Oxytocin eluate Si 02+ vasopressin Dipyridyl ammonia p $ io w aqueous solution removal of proteins Fe1 + ion complexes aqueous solution Octanol 'electrolyte -1-- or- ganic acid aqueous solution aqueous vasopressin freed from pigments eluate aromatic Acid + orga- n-butanol; niche solution medium and Extraction with water w butanolic solution of purified aqueous of oxytocin vasopressin solution aqueous solution ; Extraction in organic acid 1 i countercurrent Y'Y Purified aqueous aqueous vasopressin Oxytocin solution Solution of great purity For the purpose of a better explanation of the method according to the invention, a detailed example is given below.

Example A. Preparation of activated silica 7.5 l sodium silicate from 35 to 37 ° B6 (clear, colorless, iron-free) are mixed with 2.51 of distilled water stirred until a very homogeneous mass is achieved.

Pour with the help of a separating funnel and with constant movement one slowly. 50% acetic acid is added. Gradually a solid mass is formed. Care must be taken to break the thick lumps that gradually form. At the moment when the whole thing thickens into a mass and you start moving the same can no longer be maintained, the lumps formed are broken into Pieces, continuing with the addition of 50% acetic acid.

After a certain time, the mass liquefies again. Man then resumes stirring and, without adding acid, sets it a further 1J2 Hour away. Check for an acidic reaction using litmus paper. If those persists after 1/2 hour, the mass is left for about 1/2 hour rest before decanting the liquid.

The mass is then three times in a row, initially with ordinary, then washed with distilled water, always carefully decanting. : Ulan needs about 31.5% acetic acid for this treatment.

For the final wash with distilled water, it is recommended that the silicon dioxide in small batches in a mortar in the presence of distilled Crush water.

It is then filtered through a Buchner funnel, washed and dried for 24 hours at 110 ° C. The product is finally passed through a sieve 1r.50 (made of plastic material), the coarse particles being comminuted. The yield of crude silica is 1500 g. For the purpose of activation, 1 kg of crude dry silicon dioxide obtained in this way is brought to the boil for 1/2 hour in 10 1 10% acetic acid with constant stirring. After decanting, it is washed three times cold with 10 liters of distilled water, each time being decanted. It is filtered through a Buchner funnel and washed three more times until the filtrates react neutrally to litmus. After testing (by means of silver nitrate in nitric acid solution) the last wash water for the absence of chlorine and for the absence of iron (using potassium ferrocyanide), the silicon dioxide is allowed to dry at 120 ° in a drying cabinet. The silica produced in this way is carefully stored in well-closed bottles. Before it is used as an adsorbent, the silicon dioxide is crushed in a mortar or in a mill with porcelain balls and sieved on a No. 8o sieve made of plastic material. B. Separation and extraction of the posterior pituitary hormones i. Phase: extraction of all the hormones from the posterior pituitary gland. One starts from 18 g of a powder which is obtained by acetone extraction from the posterior lobes of the bovine pituitary glands, each containing about 18,000 IU of each of the two hormones oxytocin and vasopressin. An extract is first prepared by boiling with the aid of 180o ccm of 0.3% acetic acid and the mixture is filtered through a suction filter into an ice-cold container. To the filtrate, which has a pH of about q. first 50 g, another 50 g and finally 100 g activated silicon dioxide prepared as described above are added at intervals of 11/2 hours without filtering, the whole thing being added for a total of q. Hours 30 minutes, namely at a temperature of -f- 2 to 3 ° C is stirred.

It is filtered and this treatment is repeated with two further portions of 50 g of silicon dioxide by stirring the mixture for 1 hour 30 minutes stirs. It is then filtered and the two fractions obtained, silica, are combined. which contain all of the hormonal activity adsorbed.

200 cc of 0.5% acetic acid is then brought to the boil and added while stirring, add the adsorbate obtained as described above. Cooking will maintained for 5 minutes. It is then filtered into a vacuum using a vacuum o ° cooled bottle.

This treatment is repeated twice with 100 cc acetic acid each time repeated. The combined extracts contain 80 to 85 per cent of what was initially available been oxytocins and are suitable for the isolation of this substance.

The suctioned silicon dioxide, which has a weight of about 60o g is used to extract vasopressin.

2nd phase: separation and purification of the oxytocin. About the iron ions to be converted into a complex compound, o, oo2 is added to the combined extracts ° / o a-Dipyridyl added. These extracts are concentrated in a carbonic acid atmosphere at a temperature below or at most 5 ° C, until a volume of 400 cc received. The concentrate is extracted with peroxide-free ethyl ether in order to to remove the a dipyridyl until the acetic acid content falls to about 0.2 per cent has decreased.

The aqueous phase is then cleaned three times with 50 cc secondary octanol extracted. The separated from the octanol and in this way The aqueous phase freed from pigments and proteins then becomes light with ammonia made alkaline, kept cold and then ten times in a row with 50 cc each previously extracted with n-butanol saturated with water.

The butanolic extracts obtained in this way contain the entirety of oxytocin. Almost all of the water is removed by freezing at -60 ° C in a mixture of carbonated snow and acetone and then centrifuged in the Cold. The withdrawn water is discarded. One then frees the Butanol extract of excess ammonia by removing the former while passing it through treated with a stream of nitrogen at ordinary temperature in vacuo. Then drive away the butanol by distillation on a water bath at 25 to 30 ° C while passing through of nitrogen and at a pressure of 1 / 1o to 1 / "mm until you get a concentrate small Volume.

The entire concentrate then undergoes six consecutive extractions with in each case 50 cc of 0.1% acetic acid. The acetic acid extract that which contains pure oxytocin is used three times at ordinary temperature each time Washed 50 cc of chloroform to remove traces of butanol.

The last traces of chloroform are bubbled through removed from nitrogen. In this way, a solution of oxytocin in dilute acetic acid. A preservative, e.g. B. Trichlorobutanol, and sterilize it by filtering. It is then ready to be bottled on ampoules ready.

The yield is around 50% of that in the starting powder Oxytocin activity that has been present.

3rd phase: separation and purification of the vasopressin. Only the last third of the silica obtained at the end of the first phase, say 200 g, is used for this operation. This amount contains approximately 5,000 units of vasopressin. It is suspended in 300 cc of 0.5% ammonia, and concentrated ammonia is added until it has an alkaline reaction to phenolphthalein, the pH still remaining below 10. The whole is stirred at 0 ° for 20 minutes, then filtered, and the silicon dioxide is washed with 3 ° acetic acid. The filtrate is discarded.

The silicon dioxide obtained in this way is eluted in a column with 1.5 l of a solution containing 2.5% anhydrous sodium sulfate, 2. 1o-5 ° ,. o Dimethylbenzyl-alkyl-ammonium chloride, the alkyl radical of which has 12 to 18 carbon atoms and 0.1% o pure acetic acid, all calculated in percent by weight. The eluate is concentrated in vacuo at low temperature to 1/10 of its initial volume and the sodium sulfate which has precipitated out is removed by filtration. 50 cc of an acetone solution of salicylic acid (30%) are added to the filtrate.

The mixture is stirred in the cold for 15 hours and then the microcrystals of the precipitated salicylic acid are filtered. The mother liquors are subjected again to a similar treatment. The combined salicylic acid, which contains almost all of the vasopressin, is dissolved in 30 cc of ethyl ether in the presence of 50 cc of distilled water.

The separated aqueous phase contains the vasopressin, which on this way with a yield of 45%, based on the content of the pituitary starting material, is won. This solution can be sterilized by filtration in the cold be filled into ampoules and then serve therapeutic use. Man can also obtain a further purification of the same by the fact that after the Previous solution obtained concentrated, with the remaining traces of sodium sulfate with 3 volumes of ethyl alcohol can be precipitated in the cold. To Removal of the alcohol by distillation in vacuo creates the vasopressin solution an extraction in a countercurrent apparatus, e.g. B. that of Craig, subject to for which one uses n-butanol which is saturated with water and which contains 1% salicylic acid. In this way, a product is obtained which has an activity of 1,000 U.I. per milligram contains.

The method according to the invention thus allows both an extraction of oxytocin and vasopressin from the posterior lobe of the pituitary gland as well as the simultaneous Separate these hormones from each other with good yield without losing any of the Hormones must be sacrificed to cleanse the other.

Claims (7)

PATENT CLAIMS: i. Process for obtaining oxytocin and vasopressin from the animal posterior pituitary gland by extraction of both hormones and Separation of the same, characterized in that the animal posterior pituitary lobes or a dry extract obtained from them after treatment with a weak one aqueous solution of an organic acid and subjected to the thus obtained, the The solution containing all of the hormones of the posterior lobe of the pituitary gland, the hormones adsorbed by means of activated silicon dioxide and then the silicon dioxide obtained in this way treated with a weak aqueous solution of said organic acid, whereupon on the one hand the eluate containing almost all of the oxytocin and on the other hand the residue containing almost all of the vasopressin for recovery an almost pure oxytocin or vasopressin of an extracting treatment is subjected. 2. The method according to claim i, characterized in that as organic acid acetic acid, propionic acid, tartaric acid or the like, advantageously in aqueous Solution and with a concentration below 2%, preferably such of the order of 0.5 ° / o is used. 3. The method according to claim i or 2, characterized by the use of a silicon dioxide as an adsorbent, which has been activated by means of an organic acid. q .. Procedure according to Claim i to 3, characterized in that the oxytocin from its eluate by Extraction is separated using n-butanol, whereupon the butanolic solution in turn extracted by means of an aqueous solution of an organic acid, wherein Advantageously, extraction with n-butanol to remove lipids and pigments an extraction using secondary octanol, chloroform or the like, suitable solvent precedes. 5. The method according to claim q., Characterized in that that traces of a substance were added to this eluate before extraction with n-butanol which binds the iron ions to form complexes, e.g. B. a-Dipyridyl, copper tuna, a sulfocyanate, an oxyquinoline or the like. 6. The method according to claim i to 5, characterized characterized by the fact that the vasopressin is obtained from the residual silicon dioxide it is obtained that the latter can be reduced to a pH below in order to remove the proteins io alkaline and the silicon dioxide with the aqueous solution of an electrolyte, such as B. sodium chloride or sulfate, which is an organic acid, e.g. B. acetic acid of weak concentration elutes. 7. The method according to claim 1 to 6, characterized in that a surface-active agent is added to the eluate of the silicon dioxide. B. The method according to claim i to 7, characterized in that the eluate of the vasopressin, which originates from the silicon dioxide, with a solution of an aromatic acid sparingly soluble in water, e.g. B. salicylic acid, in an organic , water-miscible solvent, z. B. acetone, is treated, whereupon the precipitated in microcrystals aromatic acid, which contains the vasopressin adsorbed, separated and treated with a mixture of water, in which the vasopressin accumulates, and a solvent, such as. B. ether, in which the aromatic acid accumulates, is treated. G. Process according to Claims 1 to 8, characterized in that the eluate of the vasopressin is purified by extraction in countercurrent using an n-butanol containing water and salicylic acid.