DE3902980A1 - METHOD FOR PRODUCING VINCAMINE AND EPIVINCAMINE BY BREEDING VINCA MINOR CELL CULTURES - Google Patents

Abstract

Vincamine and epivincamine are produced by culturing cells of the culture FICE-V3 (DSM 4365) under aerobic conditions in a liquid medium containing a source of assimilable carbon, a source of assimilable nitrogen and mineral salts. They may be incorporated into pharmaceutical compositions.

Description

The invention relates to a method for producing Vincamin and Epivincamin.

The production of vinca alkaloids by fermentation Process in an artificial medium has been around for several Years. FR-A-23 00 131 describes a process for the preparation of a mixture of undefined Vinca-Alka described loide. There the production of a com complex alkaloid mixture after more than 20 days of fermentation tion of cell suspension cultures and preferably with con continuous lighting with yields of around 20-45 mg / l described.

It has surprisingly been found that according to the invention a new cell culture derived from Vinca minor L. Vincamin and his isomeric epivamin without further Alka can form loide in good yields. The present Er The invention thus relates to a method for producing Vincamine and epivamin, which is characterized by that cells of the culture FICE-V3 (DSM 4365) in aerobic loading conditions in a liquid medium, which is an assimi carbon source, an assimilable nitrogen contains source and mineral salts, are grown.

It is thus possible to yield vincamine and epivamine of about 1g / l or above in a liquid culture hold regardless of whether the culture is illuminated or be shines or not. The high yield at the Her position, the simplicity of the procedure, which in ex industrial fermentation devices  and the fact that only two isomers, Vin camin and epivamin, which are formed, are the essential ones Lichsten advantages according to the present invention and represent an improvement over the extraction ver drive from alkaloids from Vinca minor plants and the semi-synthetic manufacturing process of FR-A-23 00 131 represents.

The so formed vincamine and epivine can from the Cells and / or the culture broth are isolated. You can be separated from each other. Vincamin and Epivin camin differ from each other in their stereochemistry in the 16 position. It is known from the literature that the Conversion of vincamine to epivamin easily with high levels Yields can take place in acid solution (G. Szantay et al., Tetrahedron Lett. (1973) p. 191). That according to the invention Provided vincamine can therefore be converted into epivamine will.

The new culture that forms vincamine and epivamin, is called strain FICE-V3. It was on December 23 About 1987 at the German Collection for Microorganisms (DSM) under the deposit number DSM 4365. The morphological properties of FICE V3 cells are the following:

Cultures grown on a solid medium (Callus-Kultu ren) are colorless, easily disassembled cell masses. The cell len have a round to elliptical shape with a through knife from 50-100 µm. With the tested cultural conditions the callus cultures show no organogenesis or anything a differentiation. When exposed to light with 2000 lux discolour the callus cultures due to  chlorophyll biosynthesis green. However, that affects Exposure to light does not affect alkaloid biosynthesis.

When growing in liquid media do not grow differentiated cultures as small aggregates from 5 to 50 Cells. The cells have the same shape and the same Dimensions like those grown on solid media the.

Another characteristic is the formation of the two alka loide vincamine and epivamin in essentially pure Form, which in the following simply as "raw Vincamin" be be drawn.

The new crop was made from a Vinca minor plant that near Lecco (Como, Italy). Pflan parts (leaves, shoots and roots) were washed with 70% ethanol for 5 minutes, 2% sodium hyperchlorite for 2 minutes and 5% mercury (II) chloride sterilized for 45 seconds. After every They were washed with sterile distilled water rinsed.

The leaves, shoots and roots were then cut into pieces cut and aseptically in Gamborg B5 medium (O. L. Gam borg et al., Exp. Cell Res. 50, p. 151 (1968)), supplemen mented with 1 mg / ml naphthalene acetic acid (NAA) and 7 g / l Agar, fixed and kept in the dark at 28 ° C. To An undifferentiated tissue (callus) started for 10 to 15 days to grow and after 20 to 30 days it became sloping on agar plates transferred with the same medium. Usually it takes about 20 days at 28 ° C in the dark for that Cultural growth has ended. After 2 or 3 transfers who  preserved cultures and as an inoculum in Schüt telkolben used.

About 1-2 g fresh weight of the Callus are with a glass homogenized and placed in a 300 ml Erlenmeyer flask give which 50 ml of liquid Gamborg medium, supplemen animals with 1 mg / l NAA. After 7 days of incubation at 28 ° C in the dark on a rotary shaker at 120 rpm, the dry weight of the culture is et wa 10 mg / ml, and 5 ml of vegetative culture become inoku a 300 ml Erlenmeyer flask, which 45 ml Gamborg B5 medium with 30 g / l sucrose and 1 mg / l Contains NAA (G30 medium). These cultures are grown at 23 ° C in the dark on a rotary shaker grown at 120 rpm for 10-14 days.

The FICE-V3- Cells grown in a liquid medium. Piston or Fermentors made of glass or other commonly used Materials such as stainless steel can be used. The liquid medium can be a nutrient solution, which is a assimilable organic carbon source, an assimi adjustable organic or inorganic nitrogen source, inorganic salts and optionally plant hormones and / or contains vitamins. The assimilable organic coals can be from carbohydrates such as sucrose, glucose, Fructose, starch, dextrin, glycerin, mannitol and mannose stand. The assimilable organic or inorganic Nitrogen source can be made from amino acids or their mixtures, Peptides and proteins or their hydrolyzates, casein hydro lysate, water-soluble fractions of cereals like that Residues from corn distillation or wheat from the Alcohol production, or yeast and also from inorganic Ni  occurred and inorganic ammonium salts exist.

The method according to the invention is typically described in Sus board culture, for example in a flask with stirring Ren or in a vented fermentor at a pH in the loading range from 4.4 to 7.0, preferably 6.5, and at a tem temperature in the range from 18 to 36 ° C, preferably 23 ° C, carried out. In general, preferred crops are conditions working in the dark at a pH of 5.1 to 6.8 at a temperature of 18 to 30 ° C and from 8 to 16 days. The production of the raw vincamine begins after 2 to 3 days of growth and reaches its maximum after 10 to 14 days.

The extraction of the raw vincamine can be done from the cell len as well as from the culture broth. From the gefil The raw vincamine is typically included in the cells a water-miscible organic solvent, such as Methanol, ethanol or acetone extracted. From culture broth that has been separated from the cells and up to one pH of 8-9 has been made alkaline, the raw vin camin generally with a water-immiscible solder solvents such as chloroform, methylene dichloride or ethyl ether, extracted.

The vincamine or epivincamine thus formed can be combined in one pharmaceutical preparation to a pharmaceutical accept bar carrier or diluent can be processed.

The following examples illustrate the invention.

example 1

The procedure is carried out in 30 ml Erlenmeyer flasks containing 50 ml Contain nutrient medium G30 and its pH to 6.5 with diluted ammonia was carried out. The Pistons are placed on a rotary shaker (120 rpm; eccentric path: 8 cm) shaken. The opti male incubation temperature is 23 ° C. The pistons who inoculated with cells made from 14 day old FICE- V3 cultures were obtained in B5 agar medium.

After 14 days the production is about 750 mg / l Vinca min and 340 mg / l epiviramine, determined by HPLC. ten Liters of cell suspension cultures, obtained from 200 flasks, are centrifuged and the cells and the supernatant are extracted separately. The supernatant is brought to pH 9 brought with sodium carbonate and twice with 10 liters Extracted methylene chloride. The organic extracts who which in turn with a 2% aqueous tartaric acid solution solution extracted. The acidic solution is brought to pH 9-10 adjusted and extracted with methylene chloride.

The centrifuged cells are washed with a 50% aq Rigen acetone solution, which contains 2% tartaric acid, homogeneous nized and filtered. The filtrate is concentrated in vacuo triert and the resulting aqueous solution becomes alkaline made, pH 9-10, and extracted with methylene chloride.

The organic extracts of cells and over are combined and evaporated to dryness. 8.8 g of crude vincamine, which consists of 6.0 g of vincamine and 2.8 g Epivincamine, determined by HPLC, are obtained. The two alkaloids are determined by column chromatography  Silica gel in methylene chloride with increasing amounts of etha nol separated. 5.6 g of pure vincamine are removed from the column and 2.5 g of epivamine isolated.

Example 2

The process is carried out in flasks according to a two-stage Fer mentation procedures carried out. First the wax tum on Murashige medium (T. Murashige et al., Physiol. Plans. 15, (1962) p. 473) with 20 g / l glucose, 1 mg / l NAA and carried out under the conditions as in Example 1. After 8 days the dry weight reaches 10-13 mg / l. The Cultures are centrifuged aseptically and the cells in Production medium G30 resuspended. After 6-8 fermenta days of production of crude vincamine is 1050 mg / l. HPLC analysis corresponds to 73% vincamine and 25% Epivaminamine, based on the total alkaloids. By Procedure in 100 Erlenmeyer flasks, as in Example 1 described, 3.6 g of pure vincamine and 1.0 g of epivamine isolated. It will be almost the same Yields obtained when glucose or maltose in the medium can be used instead of glucose.

Example 3

The process is carried out in 10 liter fermenters which contain 6 l of nutrient medium G30 included. A Vinca culture minor FICE-V3 is, as described in Example 1, in one 30 ml Erlenmeyer flask made with 50 ml medium B5. After 7 to 10 days (dry weight 12 mg / ml) the Culture for inoculating a 2000 ml round bottom flask, wel contains 450 ml of medium B5. The culture will on a rotary shaker at 100 rpm in the  Incubate darkness at 28 ° C. After 7 to 10 days the fully grown culture (dry weight 10 mg / ml) as an inoculum for a 10 liter fermentor containing 6 l medium G30 contains used. The production phase took place at 27 ° C in the dark with an initial stirring of 100 rpm and a ventilation volume of 0.5 vol / vol / min. If the Concentration of dissolved oxygen decreased by 20% stirring increased by 20 rpm.

After 10 to 14 days, the culture has a dry weight of 20 mg / ml and an alkaloid content of 760 mg / l squeezes as raw vincamine. The alkaloids were, as in Example 1 described, extracted and purified. The Yield was 3.3 g / l vincamine and 0.9 g / l epivamine.

Claims (8)

1. A process for the preparation of vincamine and epivin camin, characterized in that cells of the culture FICE-V3 (DSM 4365) are grown under aerobic conditions in a liquid medium which contains an assimilable carbon source, an assimilable nitrogen source and mineral salts. 2. The method according to claim 1, characterized records that the cells in the dark a pH of 5.1 to 6.8 at a temperature of 18 to 30 ° C and be bred from 8 to 16 days. 3. The method according to claim 1 or 2, characterized ge indicates that the vincamin thus formed and epivamine from the cells and / or the culture broth be isolated. 4. The method according to any one of the preceding claims, characterized in that the so formed ten Vincamin and Epivincamin be separated from each other.   5. The method according to any one of the preceding claims, characterized in that the gebil dete Vincamin is converted into Epivincamin. 6. The method according to any one of the preceding claims, characterized in that it is in one Fermentor is carried out. 7. Pharmaceutical preparation, characterized thereby records that it is a pharmaceutically acceptable Carrier or diluent and as active ingredient Partly Vincamin or Epivincamin, which after a procedure of the preceding claims. 8. Cell culture FICE-V3 (DSM 4365).