GB2214913A - Production of vincamine and epivincamine by cell cultures of vinca minor - Google Patents

Description

1 1 t 1 1 2"' 11, 9 13 1 2 PP 4309 (FC 363) "PRODUCTION OF VINCAMINE AND

EPIVINCAMINE BY CELL CULTURES OF VINCA MINOW' The invention relates to the production of vincamine and epivincamine.

The production of Vinca alkaloids by a fermentation process in an artificial medium has been studied for several years. A process for preparing a mixture of undefined Vinca alkaloids is disclosed in FR-A2300131. This describes the production of complex mixtures of alkaloids after more than 20 days of fermentation in cell suspension cultures and preferably under continuous illumination, with yields of about 20-45 mg/1.

It has been surprisingly found according to the present invention that a new cell culture derived from Vinca minor L. is capable of producing vincamine and its isomer epivincamine, without any further alkaloids, in good yields. Accordingly, the present invention provides a process for the production of vincamine and epivincamine, which process comprises 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.

It is thus possible to obtain yields of vincamine and epivincamine of about lg/1 or more in liquid culture - 2 regardless of whether or not the culture is illuminated. The high yield of production, the simplicity of the process which may be carried out in the usual industrial fermenters, and the fact that the only two isomers, vincamine and epivincamine, are formed, are the most important advantages of the present invention and represent an improvement over the extraction of the alkaloids from the Vinca minor plants and the semisynthetic production process of FR-A-2300131.

The vincamine and epivincamine thus-produced can be recovered from the cells and/or the culture broth. They can be separated from each other. Further, vincamine and epivincamine differ from one another in their stereochemistry at the 16-position. It is known from the literature that transformation of vincamine into epivincamine may be easily carried out with high yields in acidic solution (G.-Szantay et al, Tetrahedron Lett. (1973) p. 191). The vincamine produced in accordance with the present invention may therefore be converted into epivincamine.

The new culture producing vincamine and epivincamine is designated as strain FICE-V3. It was deposited on 23 December 1987 at the Deutsche Sammlung von Mikroorganismen (DSM) under accession number DSM 4365. The morphological characteristics of FICE-V3 cells are as follows:

Cultures grown on solid medium (callus cultures) are colourless, easily disaggregateable, masses of cells. The 1; I.

1 cells have a round to elliptical shape having a 50-100pm diameter. Under the culture condition examined, the callus cultures do not show organogenesis or any differentiation process. if exposed to 2000 lux of light, callus cultures become green due to chlorophyll biosynthesis. However, light exposure does not affect alkaloid biosynthesis.

When grown in liquid media, undifferentiated cultures grow as small aggregates of 5 to 50 cells. The cells have the same shape and dimension as those grown on solid media.

Another property is the production of the two alkaloids vincamine and epivincamine essentially pure, which hereinafter will be simply called "crude vincamine".

The new culture has been derived from a plant of Vinca minor collected near Lecco (Como - Italy). Parts of the plant (leaves, shoots and roots) were sterilized by washing with 70% ethanol for 5 minutes, 2% sodium hypochloride for 2 minutes and 0.5% mercuric chloride for 45 seconds. After each wash they were rinsed with sterile distilled water.

The leaves, shoots and roots were then cut into pieces and fixed aseptically in Gamborg B5 medium (O.L. Gamborg 2t al. Exp. Cell Res. 50, p. 151 (1968)) supplemented with 1 mg/ml of naphthalene acetic acid (NAA) and 7 g/l of agar and maintained in the dark at 280C. After 10-15 days an undifferentiated tissue (callus) started growing and after 20-30 days it was transferred onto agar slants of the same medium. usually it takes about 20 days at 280C in the dark - 4 to have completely grown cultures. After 2 or 3 transfers, stabilized cultures were obtained and used as inoculum in shaken flasks.

About 1-2 g fresh weight of callus can therefore be homogenized with a glass rod and transferred into a 300 ml Erlenmeyer flask containing 50 ml of liquid Gamborg medium supplemented with 1 mg/1 of NAA. After 7 days incubation at 280C in the dark on a rotatory shaker at 120-rpm, the dry weight of the culture is about 10 mg/ml and 5 ml of vegetative culture is inoculated into 300 ml Erlenmeyer flask containing 45 ml of Gamborg B5 medium with 30 g/1 of sucrose and 1 mg/1 of NAA (G30 medium). These cultures are incubated at 230C in the dark on a rotatory shaker at 120 rpm for 10-14 days.

According to the process of the present invention, FICE-V3 cells are cultured in a liquid medium. Flasks or fermenters of glass or other generally used materials, such as stainless steel, may be employed. The liquid medium may be a nutrient solution containing an assimilable organic source of carbon, an assimilable organic or inorganic source of nitrogen, inorganic salts, and, optionally, plant hormones and/or vitamins. The assimilable organic carbon source may consist of carbohydrates such as sucrose, glucose, fructose, starch, dextrin, glycerol, mannitol and mannose. The assimilable organic or inorganic source of nitrogen may consist of amino acids or their mixtures, 1 W - 5 peptides and proteins or hydrolyzates thereof, caseine hydrolyzate, water-soluble fractions of cereals such as the residues of the distillation of maize or wheat in alcohol production or yeast and also inorganic nitrates and inorganic salts of ammonium.

The present process typically is carried out in suspension culture, for example in a flask under stirring or in aerated fermenters at a pH ranging from 4.4 to 7.0, preferably 6.5 and at a temperature ranging from 18 to 36C, preferably 23C. General preferred culture conditions are in the dark at a pH of from 5.1 to 6.8, at a temperature of from 18 to 300C and for from 8 to 16 days. The production of crude vincamine begins after 2-3 days of growth and reaches its maximum after 10-14 days.

The extraction of crude vincamine can be carried out both from the cells and from the culture broth. From the filtered cells the crude vincamine is typically extracted with a water miscible organic solvent such as methanol, ethanol or acetone. From the culture broth, separated from the cells and made alkaline at pH 8-9, crude vincamine is generally extracted with a water immiscible solvent such as chloroform, methylene dichloride or ethyl ether.

The vincamine or epivincamine thus-produced may be incorporated in a pharmceutical composition with a pharmaceutically acceptable carrier or diluent.

The following Examples serve to illustrate, without 1 limiting, the invention. EXAMPLE 1 The process was carried out in 300 ml Erlenmeyer flasks containing 50 ml of nutrient medium G30, whose pH was adjusted to 6.5 with dilute ammonia. The flasks were shaken by a rotatory shaker (120 rpm; eccentric throw: 8 cm). The optimal incubation temperature was 230C. The flasks were inoculated with cells which were obtained from 14 day old FICE-V3 cultures in B5 agar medium.

After 14 days the production was about 750 mg/l of vincamine and 340 mg/l of epivincamine determined by Hplc. Ten liters of cell suspension cultures obtained from 200 flasks were centrifuged and cells and the supernatant were extracted separately. The supernatant was brought to pH 9 with sodium carbonate and extracted twice with 10 litres of methylene chloride. The organic extracts were in turn extracted with 2% aqueous tartaric acid solution. The acidic solution was brought to pH 9-10 and extracted with methylene chloride.

The centrifuged cells were homogenized with a 50% aqueous acetone solution containing 2% of tartaric acid and filtered. The filtrate was concentrated in vacuo and the resulting aqueous solution, made alkaline to pH 9-10 was extracted with methylene chloride.

The organic extracts of cells and supernatant were t 4.

- 7 combined and evaporated to dryness. A quantity of 8.8 g of crude vincamine (consisting of 6.0 g of vincamine and 2.8 g of epivincamine determined by Hplc) were obtained. The two alkaloids were separated by column chromatography on silica gel in methylene chloride with increasing amounts of ethanol. From the column 5.6 g of pure vincamine and 2.5. g of epivincamine were recovered. EXAMPLE 2 The process was carried out in flasks by a two-stage fermentation. At first the growth was performed on Murashige medium (T. Murashige et al, Physiol. Plant. 15, (1962) p 473), with 20 g/1 of glucose, 1 mg/1 of NAA and in conditions as in Example 1. After 8 days dry weight reached 10-13 mg/ml, the cultures were centrifuged aseptically and the cells resuspended in the production medium G30. After 6-8 days of fermentation, the production of crude vincamine was 1050 mg/1. By Hplc analysis vincamine corresponded to 73% and epivincamine to 25% of the total alkaloids. By operating 100 Erlenmeyer flasks as reported in Example 1, 3. 6 9 of pure vincamine and 1.0 g of epivincamine were separated. Almost the same yields were obtained when glucose or maltose was used in the medium instead of glucose. EXAMPLE 3 The process was carried out in 10 litre fermenters containing 6 litres of nutrient medium G30. A culture of Vinca minor FICE-V3 was performed according to Example 1 in 300 ml Erlenmeyer flask with 50 ml of medium BS. After 7-10 days (dry weight 12 mg/ml), the culture was used to inoculate a 2000 ml spherical flask containing 450 ml of medium BS. This culture was incubated on a rotatory shaker at 100 rpm in the dark at 280C. After 7-10 days the fully grown culture (dry weight was 10 mg/ml) was used as inoculum for a 10 litre fermenter containing 6 litres of medium G30. The production phase was carried on at 270C in the dark with an initial stirring of 100 rpm and an aeration rate of 0.5 vol/vol/minute. If the dissolved oxygen concentration decreased by 20%, stirring was increased by 20 rpm.

After 10-14 days the culture had a dry weight of 20 mg/ml and an alkaloid content of 760 mg/1 expressed as crude vincamine. The alkaloids were extracted and purified as reported in Example 1. The yield was of 3.3 g/1 of vincamine and 0.9 g/1 of epivincamine.

Claims (9)

1. A process for the production of vincamine and epivincamine, which process comprises culturing cells of the culture FICE-V3 (DSM 4365) under aerobic conditions in a liquid medium containing a s ource of assimilable carbon, a source of assimilable nitrogen and mineral salts.

2. A process according to claim 1, wherein the cells are cultured in the dark at a pH of from 5.1 to 6.8, at a temperature from 18 to 30C and for from 8 to 16 days.

3. A process according to claim 1 or 2, wherein the vincamine and epivincamine thus-produced is recovered from the cells and/or the culture broth.

4. A process according to any one of the preceding claims, wherein the vincamine and epivincamine thus-produced are separated from each other.

5. A process according to any one of the preceding claims, wherein the vincamine thus-produced is converted into epivincamine.

6. A process according to any one of the preceding claims, which is carried out in a fermenter.

7. A process for the production of vincamine and epivincamine, said process being substantialy as hereinbefore described in any one of the Examples.

B. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and, as - 10 active ingredient, vincamine or epivincamine which has been produced by a process as claimed in any one of the preceding claims.

9. A cell culture FICE-V3 (DSM 4365).

Published 1989 at The Patent Office, State House, 66,171 High HolbornLondonWCIR4T?.FUrther copies maybe obtainedfrom The Patent Office. Wes Branch, St Mary Cray Orpington, Rent BR5 3RD. Printed by Multiplex techniques ltd, St Mary Cray, Rent, Con. 1/87