DE940850C - Process for the production of products with vitamin B activity - Google Patents

Description

Process for the manufacture of products with vitamin Bo2 activity the Invention relates to the manufacture and isolation of therapeutically useful Substances, especially of products, which are used as therapeutically effective components contain a previously unknown chemical compound known as vitamin B. shall be.

The new compound "Vitamin B12" is a powerful growth factor for the microorganism "Lactobacillus lactis Dorner" (LLD) and has pronounced effective effects in the therapeutic treatment of pernicious anemia.

LLD-active raw materials are used as starting materials, i. H. Fabrics which contain a substance capable of stimulating the growth of Lactobacillus lactis Dorner favor. LLD-active raw materials can e.g. B. from commercially available Liver preparations or by cultivating the microorganism Streptomyces griseus a suitable nutrient medium by adsorption of the active material from the culture broth, Extraction of the adsorbate by means of an aqueous solution of pyridine or alkyl-substituted Pyridine and conversion of the extract into a concentrate can be obtained.

It has already become known that antipernicious substances from the liver are found with the usual working methods for isolating active substances methods found use in extraction, precipitation, adsorption and the like; these known Processes are characterized by the fact that they are cumbersome and time consuming. the Liver preparations obtained in this way, however, usually contain 99.98% inert material and contain only traces of vitamin B12 according to the usual methods can hardly be isolated from this.

Generally speaking, the manufacture of products can be more pronounced According to the invention, vitamin B12 activity takes place in such a way that an LLD-active raw material, z. B. a liver preparation, or a Streptomyces griseus containing as the main ingredient Raw concentrate is extracted with the help of a solvent and by introduction the solution thus obtained in a column charged with adsorbent material a chromatographic fractionation is effected, whereupon the column for the purpose of fractionated Extraction of the active substance from the adsorbent with a suitable solvent is washed out and the extract is converted into a vitamin Bl2 concentrate.

According to one embodiment of the invention, a fraction is a Liver preparation, which has at least 10000 units img LLD, as the starting material used. Such a fraction can be obtained from commercially available liver preparations will. So you can z. B. according to the method of Daking and Weet (Journ. Biol. Chem. 109, 489 [1935]) from an aqueous liver extract one soluble in 70% ethanol Win faction. The solid starting material can be used in aqueous solution of the chromatographic Treatment in a column charged with charcoal and that active material with an organic solvent, e.g. B. 5001, pure ethanol, pulled out will.

Other useful extractants are e.g. B.

5001 acetone above, water saturated with butanol, with benzyl alcohol saturated water and glacial acetic acid.

According to another embodiment, the by extraction of the alcoholic obtained solid starting material with a lower aliphatic alcohol Solution subjected to chromatographic treatment in a column which is active Contains clay and the active material extracted with methanol or aqueous methanol.

The extracts are obtained in small fractions, and the activity of the individual fractions by microbiological testing with the help of Lactobacillus lactis Dorner noted. The high activity fractions will either dried in the frozen state or concentrated to a low volume.

The z. B. solid or emerged from the alumina treatment concentrated intermediates are extracted with ethanol and those in ethanol insoluble solids eliminated. The ethanol solution is brought to dryness, the residue is dissolved in methanol and then by adding acetone or ether a precipitate is produced which, after separation from the liquid, is dissolved in water will. A precipitate is formed again by adding acetone to the aqueous solution generated, which is dissolved in methanol, whereupon several parts of the space acetone is added will. The resulting precipitate is after separation from the liquid dissolved in water and, by careful addition of acetone, crystallizes until it becomes cloudy second hand. The obtained crystals. are washed with acetone and from a water-acetone mixture recrystallized.

According to one embodiment, the vitamin Bl2 concentrates are after the dissolution and precipitation processes of a further purification with the help of a known one Countercurrent distribution between water and a mixture of 75% toluene with 25 Subjected to 01o o-cresol and the product from the best fractions by crystallization won (cf. Craig: J. Biol. Chem. 16I, 32I to 332 [1945] "Identification of Organic Compounds by Distillation, Studies «).

If desired, those obtained by treatment with activated carbon can be used Concentrates can be dissolved in a lower aliphatic alcohol and the alcoholic Solution of the chromatographic treatment in one charged with active clay Column are subjected.

According to another embodiment of the invention, a predominantly Raw concentrate consisting of Streptomyces griseus with water or a low aliphatic alcohol, such as methyl alcohol, extracted. The selection of the extraction liquid depends on the adsorbent material that is used to carry out the chromatographic Fractionation is used. As an adsorbent, activated carbon, active alumina can be used and the like. Use. Active clay has been found to be particularly suitable.

When working in columns that contain activated carbon, has the use of aqueous extracts when working with those charged with active clay Columns the use of alcoholic extracts has proven advantageous. The column is preferably loaded wet, e.g. B. such that it is with the adsorbent and the solvent used for the extraction is filled and the solvent Drained until its level is the surface of the adsorbent reached in the column.

The alcoholic extract is on the surface of the column The active clay is poured on and flows through the adsorption material by its natural weight or with the help of pressure. The column will then are charged with fresh solvent, and appropriate fractions of the extract are made won. The fractions exhibiting sufficient activities can be microbiologically or can be determined by their pink color and concentrated individually or after association will. The concentrated solution is mixed with a solvent, in which the active substance is insoluble, e.g. B. acetone, added and this resulting red precipitate recovered. The precipitate is dissolved in water and then acetone is added, whereby red crystals of vitamin B1 are excreted.

The work-up methods used according to the invention, such as, for example the chromatographic fractionation, are already on preparations that the end obtained from the liver have been applied, as, however, just as the example of the Determination of lipochromes in human liver (cf.

Zechmeister and von Cholnoky, The chromatographic adsorption method [1937]) shows, even with knowledge of these methods, it is not yet possible Develop a method for obtaining vitamin B12 from the liver. The liver is dehydrated by repeated treatment with alcohol according to this publication, then extracted with ether and the obtained extract with concentrated alcoholic K O H 16 hydrolyzed for up to 20 hours; however, a liver preparation treated in this way can no longer contain vitamin B12, as considerable due to alcohol treatment Amounts of vitamin B12 are removed from the liver, moreover vitamin B12 in ether is insoluble and finally, through the KOH treatment, the vitamin B12, if at all which is present in the ether extract is destroyed.

The vitamin Bla obtained according to the invention in a substantially pure form is a red connection. Analysis of two in vacuum at 1000 for 2 hours dried samples gave the following values: C .................. 56.35% 56, II% H .................. 6.72% 6.72% N .................. I4.5I% I4 , 76% P ................... 2.24% 2.27 0 / o Co ................. 4.42% 4.58% It can be assumed that the slight differences between the sum of the percentages and 100% based on the presence of oxygen.

According to the above analysis, vitamin B12 should be roughly empirical Formula C616iHs69iNi4OiiPCo. Vitamin B12 is soluble in water, methyl alcohol, Ethyl alcohol and phenol and essentially insoluble in acetone, ether and chloroform. Vitamin B12 has a partition coefficient of about 1.46 in the 75 01o system Toluene-25 01o o-cresol-water and a partition coefficient of about 1.2 in the water-benzyl alcohol system. The compound crystallizes from suitable solutions, such as B. aqueous acetone, in the form of red crystals having the following crystallographic Have intermediate properties: refractive index sa I, 6I9, n I, 649, nγ I, 659. Vitamin Bl2 crystals do not have a definite melting point; they darken about 210 to 2200.

The absorption spectrum of vitamin B12 in aqueous solution is indicated by maxima of 2780 A (E11, "= 62.7).

The spectrum does not change noticeably when the pE value changes. In acidic solution the intensity of 3610 Å is reduced by about 10%; in alkaline Solution, other minor changes show up, and the fine structure becomes less pronounced.

The infrared absorption spectrum was measured with a carefully calibrated Perkin-Elmer I2 A spectrometer determined. The following absorption maxima were found. In the following combination, "st" means strong, "m" means and "schw" weak absorption intensities.

Wavelength in My (IO-4 cm) 3.05 st 8, I5 st 4.62 black 8.67 st 5.98 st 9.36 st 6, I3 m IO, OO st 6.70 st 10.80 black 7, I4 st II, IO black 7.35 st II, 8I m 7.60 black I2.35 m It was found that vitamin produced according to the invention B12 has such a biological activity that O, OOOOI3y per ccm of culture medium is sufficient to under half the maximum growth of Lactobacillus lactis Dorner common conditions. Vitamin B12 possesses a high therapeutic level Of value in the treatment of pernicious anemia and other macrocytic anemias. The dosages required will vary depending on the individual cases and are depending on the severity and duration of the pathological conditions and the therapeutic Resilience of the patient. The therapeutically effective minimum dose is likely when administered intramuscularly in the form of an aqueous saline solution or isotonic Solution about 1 g per day or a multiple of this amount, e.g. B. about 7 e6g in the Week when administered in larger intervals.

Publications on the results of clinical trials of vitamin Bl2 therapies in the treatment of macrocytic anemias have occurred several times. It will, inter alia. referenced on T. D. Spies, R. E. Stone, G. Carcia Lopez, F. Milanes, R. Lopez Toca and T. Arambu, Folic Acid and Vitamin B12 in »Nutritional Macorcytic Anemia, Tropical Sprue, and Pernicious Anemia ", Lancet, 2, 519-522, October 2, 1948; B. R. Hall and D. C.

Campbell, “Effect of Vitamin B12 On the Hematopoietic and Nervous Systems in Addisonian Pernicious Anemia. "Journal of Laboratory and Clinical Medicine, 33, 1646, December 1948.

Vitamin B12 is also capable of making young chickens grow considerably to promote. In comparison tests where the basic food, the purified Contained (so-called vitamin-free) casein as a source of protein, 30 parts of vitamin B12 per trillion were added, the chickens fed it showed after 16 days a weight of I27 g while those with the same but vitamin BI2-free food fed chickens weighed an average of only 99 g.

The activity of the products mentioned in this application was by microbiological experiments using Lactobacillus lactis Dorner determined as test material.

Example I 1 g of a liver concentrate with a microbiological Potency of 5200 LLD units / mg were dissolved in 10 ccm of water and placed in a column, the one with 10 g preferably acid washed charcoal loaded was treated chromatographically. The column was then washed thoroughly with water washed and the active material extracted with 50% ethanol. The extract was collected in a number of fractions of 10 cc each. The factions which had the highest potency in microbiological tests, were combined and dried in the freezer state. The dried material (Ir5mg) was microbiological Potency of about 10,000 units / mg, corresponding to a yield of about 60%.

Example 2 I 4.6 g of a concentrate with 6000 LLD units / mg liver concentrate, containing a total of 87000000 LLD units were three times for about 30 minutes extracted with 200 cc of methanol while stirring. The combined extracts were passed through a column about 7.5 cm in diameter and 600 g of active Contained clay. After the remainder of the solution had penetrated into the clay, was Added methanol and filtered through the column for chromatographic treatment calmly.

During this operation, a pink ribbon moved downward through the column, while the brownish-yellow color of the original solution is essentially remained in the top of the column. The effluent was in a number of fractions collected. The fractions which showed a pronounced pink color were separated and united with one another. The solution thus obtained, which according to microbiological Testing approximately 65,000,000 LLD units was carried out in vacuum at one temperature evaporated below 250 to a thin syrup, the pE value obtained during evaporation has a tendency to increase, was determined by the addition of dilute hydrochloric acid (2.5 olo N) held at about 6 to 7; The residue was at low pressure (r mm of mercury) dried at room temperature.

Example 3 245 mg of the red residue from the chromatographic Treatment with clay, which was obtained according to Example 2, was with 15 ccm absolute Ethanol extracted, leaving 54 mg of a colorless insoluble residue and a red solution were obtained. The red solution provided 218 mg of a red oil which was dissolved in 0.5 cc of methanol. A red precipitate was formed by adding acetone, while the solution turned yellow. The precipitate (31 mg) had a Value from I to 7000000 LLD units / mg and an absorption maximum of 3600 Å, E: e, = 26.9. It was reprecipitated from water (0.75 cc) by acetone (15 cc). The red oil obtained (I2.8 mg) was again obtained from a methanol solution with acetone down with no further weight loss. The product showed an absorption maximum at 3611 Å, Elq, 6 "= 51.1. About 12 mg of this material were dissolved in 0.7 ccm of water and acetone (3 to 4 ccm) added until it became cloudy. At the Standing overnight, dark red needle-like crystals peel off. After 2 days the mother liquor was removed and the crystals washed with acetone and dried. The yield was 2 mg; a further 1.4 mg of crystals were obtained from the mother liquor won.

Example 4 6 mg of the vitamin Bl2 concentrate obtained according to Example 3 with an activity of about 1 to 2 times Io6 units / mg in a ten-plate countercurrent distributor with water and a mixture of 75 0/0 Toluene and 250% ortho-cresol fractionated. 2 cc of each phase were per Centrifuge tube used and the lower (aqueous) phases transferred. To The products were terminated by adding 8 cc of chloroform displaced per centrifuge tube in the aqueous phases. The aqueous phases from the Centrifuge tubes 2 to 5 were combined with each other; washed with chloroform and subjected to freeze-drying. The yield was 1.1 mg of red amorphous Vitamin Bl2 concentrate of 7 X 106 units / mg of microbiological LLD activity. Recrystallization in water with the addition of acetone gives 0.6 mg of crystalline material Get vitamin B12.

Example 5 3.2 mg of vitamin B12 once crystallized from liver were dissolved in methanol, filtered and the filtrate brought to dryness in vacuo. The residue was dissolved in 0.15 cc of water and about 1 cc of acetone until it became cloudy added. The solution was inoculated and left at room temperature overnight. The crystals thus formed were separated from the mother liquor with acetone washed and dried. The yield of crystallized vitamin B12 was about 2 mg.

Example 6 By growing a strain of Streptomyces griseus in a suitable nutrient medium, extraction of the active material by adsorption the culture broth, extraction of the adsorbate with an aqueous solution of pyridine or an alkyl-substituted pyndine, a crude concentrate was obtained, which in when dry, a microbiological potency of about 1500 to 2000 LLD units / mg exhibited.

750 g of the dried concentrate were dissolved in 2.5 1 of dry methyl alcohol entered, the mixture stirred for about 30 minutes and then filtered. Then 2.5 liters of dry methyl alcohol was added to the solid residue and the mixture stirred and filtered. This procedure was carried out using 2.5 liters of dry each Methyl alcohol repeatedly. The combined filtrates were microbiological Activity of approximately 1.2 trillion LLD units.

The combined filtrates were fractionated by chromatography adsorption using active clay moistened with methyl alcohol.

Methyl alcohol was then allowed to seep through the column and a number of factions of extract won. These factions, which have sufficient activity (generally at least 1,000 LLD units / cc exhibited) were combined and at a temperature below;, on at reduced Pressure concentrated to about a tenth of the original volume. If this is Forms a small amount of white insoluble material, that becomes concentrated Filtered mixed. The filtrate is then concentrated in vacuo to a thick syrup, absolute ethyl alcohol added and the resulting white precipitate separated from the solution.

By adding acetone to the clear alcoholic solution, a red flaky precipitate is produced, which is dissolved in about 2 ccm of water and passed through Addition of about 24 cc of acetone is excreted in the form of a red oil. That The oil is separated off, dissolved in about 1 cc of water and the solution is treated with acetone, until turbidity occurs. When standing, vitamin B, 2 becomes more in the form of small red needle-shaped Crystals excreted.

Example 7 In an alternative way of working, a red one becomes more flaky Precipitation, which by adding acetone to the clear red ethyl alcoholic Solution has arisen as obtained in Example 6. By washing the precipitate with acetone and drying, obtaining a substance that has a microbiological Has a potency of about 4 million LLD units / mg. A second crop of the same potency can be obtained by adding acetone to the mother liquor. Further addition of acetone to the mother liquor results in a precipitate red oil. By treating this oil with a mixture of ethyl alcohol and acetone an amorphous red solid body is obtained, which has a microbiological potency of about 1.5 million LLD units / mg.

The various dried fractions insoluble in acetone are combined with one another and dissolved in about 4 cc of methyl alcohol. The alcoholic Solution becomes more active in a column containing IO g moistened with methyl alcohol Alumina is charged, adsorbed. The column is then charged with methyl alcohol, with a dark red band moving through the column. Once the red color is in that Excerpt appears, Io-ccm fractions are successively removed, until the drain is essentially colorless. The first three fractions included the greater proportion of the red dye; they are united with one another and with evaporated to dryness at a temperature below 250 and reduced pressure. Of the Dry residue is dissolved with about 2 ccm of methyl alcohol and the solution with about 16 cc of acetone treated, whereby a red amorphous precipitate is formed, which by Centrifugation can be separated. This precipitate becomes the same again Subjected to treatment with alcohol and acetone. This is where part of the material becomes (5 mg of 2I, 6 mg) dissolved in about 5 cc of water and the solution with about 5 cc of acetone treated.

When left standing, clumps of red needle-shaped crystals separate from vitamin Bu, which has a microbiological potency of about II million LLD units / mg exhibit.

Example 8 About 1880 grams of a dried concentrate of a Streptomyces griseus preparation with a microbiological potency of around 2000 units / mg will be around 16 1 dry methyl alcohol entered, the mixture stirred for about 30 minutes and filtered.

The filtrate is about 15 kg of active clay made with methyl alcohol is moistened, sucked up in a chromatographic column, the column is then charged with methyl alcohol and a number of fractions of the Outflow obtained from the lower part of the column. The chromatographic fractions, which have the most pronounced microbiological activity are poured together and concentrated in vacuo to about a tenth of the original volume. The concentrate is filtered and the filtrate is further concentrated in vacuo to a thick syrup. Then alcohol is added, filtered again and the filtrate with stirring mixed with so much acetone that a complete precipitate of what is present therein red material takes place. The red precipitate is recovered and returned to 100 ccm of methyl alcohol dissolved.

This solution will slowly flow into about 1 liter of acetone with stirring left, forming a light red, flaky precipitate which is separated off, washed and dried. 7.8 g of a red powder with a microbiological one are obtained Potency of about 65,000 LLD units / mg or about 0.6% of vitamin B12.

In this example, the vitamin Bl2 content is that of the chromatographic Process stage obtained product (o, 6 0 / o vitamin B12) much lower than in the other examples and much less than is necessary. Even if they is not realized under conditions which products of maximum potency result, yields of products are obtained, the potency of which is considerably higher than that of the previously known concentrates for combating pernicious anemia.

The example is only intended to illustrate the fact that the Invention, even if carried out under conditions in which products are obtained that do not have maximum potency, at least deliver concentrates, which are considerably more effective than those previously known, for combating pernicious Concentrates used for anemia and which, if desired, without further concentration can be used for clinical purposes.

Example 9 10 g of a concentrate of that described in Example 8 Species with an average potency of 80 ovo LLD units / mg are in methyl alcohol dissolved and the solution in about 500 g of active moistened with methyl alcohol Alumina soaked in a chromatographic column. Then the Column operated and separated by feeding methyl alcohol into the upper part Fractions of the extract flowing down below won. Those Parliamentary groups, that have pronounced microbiological activity are selected, brought together and evaporated at reduced atmospheric pressure. The residue is with absolute Ethyl alcohol extracted and the white substance, which does not go into solution, separated. The red ethyl alcoholic solution is mixed with an excess of acetone, whereby an insoluble red oil is excreted. This oil is separated and dissolved in about 10 cc of water. The active material that with low inert accompanying substances is added, is precipitated again as oil by adding excess acetone. The highly active red material is dissolved again in about 3 ccm of water and the Solution treated with acetone until a faint cloudiness appears. When left standing red, needle-like crystals of vitamin B12 separate from the solution.

Claims (7)

PATENT CLAIMS I. Process for the manufacture of products with pronounced Vitamin B1 activity, characterized in that a Lactobacillus Lactis Dorner (LLD) active raw material with a solvent, e.g. B. a lower aliphatic Alcohol, such as methyl alcohol, is extracted by introducing the solution into a a chromatographic fractionation column charged with an adsorbent is subjected, whereupon the column for the purpose of fractional extraction of the active Substance from the adsorbent with a solvent; z. B. a lower one Aliphatic alcohol, such as methyl alcohol, is washed out and extracted from the extract Vitamin Bl2 concentrate is obtained. 2. The method according to claim I, characterized in that a liver fraction with an LLD activity greater than 10000 units / mg was used as the starting material will. 3. The method according to claim I, characterized in that a predominantly A raw concentrate consisting of a work-up product of Streptomyces griseus is used as the starting material. 4. Process according to Claims I to 3, characterized in that activated clay is used as an adsorbent. 5. Process according to Claims I to 4, characterized in that the chromatographic fractionation is carried out in two or more stages, with the extract of the previous chromatographic fractionation as the starting material is used for the following. 6. Process according to Claims I to 5, characterized in that the vitamin Bl2 concentrate obtained from the alcoholic extract in a lower aliphatic alcohol, e.g. B. methyl alcohol, is dissolved and the active material by adding a solvent miscible with the alcohol, e.g. B. acetone, excreted will. 7. Further development of the method according to claim 6, characterized in that that the excreted active material is dissolved in water and then again by addition of acetone is excreted to the aqueous solution, the process of dissolving in water and the precipitation of the active material with the aid of acetone, if necessary can be carried out several times. Referred to: Vitamins and Hormons, I945, 5. 259, 257, 243, 288, 258, 245 and 270 / 27I; Zechmeister-Cholnoky, The chromatographic adsorption method, I937, 5/8/9, IO5, Io8 and I20 / I2I; Biochemical Journal, 1944, p. 231.