JPH025856A - Production of vincamin and epivincamin by cell culture of vinca rosea - Google Patents

Abstract

PURPOSE: To selectively produce vincamine and epivincamine in high yield by culturing a new cell-cultured strain originated from Vinca minor under aero bic condition.

CONSTITUTION: A cell-cultured strain FICE-V3 (DSM4365) originated from the plant body of Vinca minor is cultured in a liquid medium containing assimilable carbon source and nitrogen source as well as inorganic salts under aerobic condition in dark place at pH 5.1-6.8 and 1.8-30°C for 8-16 days and the objective vincamine and epivincamine are separated from the cultured cell and/ or cultured broth.

COPYRIGHT: (C)1990,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing vincamine and epivincamine.

For the past several years, research has been underway to produce tonka alkaloid by fermentation in an artificial medium. French patent application FR-A2300131 discloses a method for producing a mixture of unidentified vinca alkaloids. According to this, a complex mixture of alkaloids is produced by fermentation in cell suspension culture for 20 days or more, preferably under continuous light irradiation.
It has been shown that it can be produced with a yield of 5/1.

In the present invention, surprisingly, a new cell culture strain derived from Periwinkle (L.camlnor L.) can produce vincamine and its isomer Ebivicamine in good yield without producing other alkaloids. It was found that it can be produced. Therefore, the present invention aims to cultivate the culture strain FI CE-V3 under aerobic conditions in a liquid medium containing an assimilable carbon source, an assimilable nitrogen source, and inorganic salts.
(DSM 4365) [111] Provides a method for producing vincamine and shrimp vincamine, which comprises culturing.

According to the present invention, approximately 1 g/j in liquid medium, regardless of whether or not the culture is irradiated with light! Or higher yield of vinkamin and shrimp vinkamin 1! ! J
can be built. The most important features of the present invention are that the yield is high, that it is a simple process that can be carried out even in a normal industrial-scale M generation tank, and that only two isomers, vincamine and shrimp vincamine, are produced. This method is more advantageous than the method of extracting alkaloids from the Periwinkle plant and the semi-synthetic production method of the French patent application F r<-8-2300131.

Vincamine and shrimp vincamine thus obtained are recovered from the cells and/or culture broth. Each of these can be separated. Incidentally, vincamine and vincamine are stereoisomers that differ in the configuration at the 16th position.

In the literature, it is known that vincamine can be converted to shrimp vincamine in an acidic solution with a high yield (G, 5zo).
Tetrahedron
1ett.

p. 191, 1973). Therefore, vincamine produced according to the present invention can be converted to shrimp vincamine according to this method.

The new culture strain producing vincamine and shrimp vincamine of the present invention is referred to as the FICE-V3 strain. 19 under the international Budapest Treaty on the Deposit of Microorganisms.
12J, 1981: German Microbiological Depository on J23rd
eutche s ammlung VOnM i
kroorganismen, DSM) with the deposit number DSM4365.

The morphological characteristics of FICE-V3 cells are as follows.

Cultures grown on solid media (callus cultures) are colorless and easily disaggregated cell populations. Cells are 50~
It has a 100-circular or elliptical shape. Under the culture conditions tested, callus cultures do not exhibit organogenesis or any differentiation process. When exposed to 2000 lux of light, the callus culture becomes green due to the biosynthesis of Chlorofil. However, light exposure has no effect on alkaloid biosynthesis.

When grown in liquid medium, undifferentiated cultures have 5 to 50
Grows as small clumps of cells.

This m cell has the same shape and size as that grown on a solid medium.

Other characteristics are the two alkali hydrides of vincamine and shrimp vincamine (hereinafter simply referred to as ``crude vincamine J'').
It is to produce.

This new 1 m product is derived from a plant of a periwinkle (V 1nca m1nor) collected near Lecco [Como, Italy] +I. Sterilize the plant parts (capsules, shoots, and roots) by washing them with 70% ethanol for 5 minutes, 2% epithorium hypochlorite for 2 minutes, and 0.5% mercuric chloride for 45 seconds. did. After each wash, rinse with sterile distilled water.

The leaves, shoots and roots were then cut into small pieces and treated with 1 mg/d of naphthalene II (NAA) and 7 g/! Gamborgh B5 medium supplemented with agar (0°, Gamborget
al, EXD, Ce1l Res, 50゜p
151 (1968)) and in a dark place for 28 hours.
It was kept at ℃. After 10-15 days, undifferentiated tissue (callus) began to grow, and after 20-30 days it was transferred onto agar slants in the same medium. Typically, approximately 20 days in the dark at 28°C are required to obtain a fully grown culture. After two to three transfers, stable cultures were obtained and used as inoculum in shake flasks.

After homogenizing about 1-2 g of fresh callus with a glass rod, 50#1e (7) was added with 1 m9/It of NAA.
Transferred to a 300 m Erlenmeyer flask containing liquid Gamborg's medium. 28 hours in the dark on a rotary shaker (120 rpm)
After 7 days of incubation at ℃, the culture had a dry weight of 10 m
y/ln1. Then, the 5th proliferative culture,
45 with 309/It sucrose and 1/1 NAA
300 containing Gamborg B5 medium (G30 medium) of d.
Inoculate an Erlenmeyer flask with a volume of In1. These cultures were grown for 1 hour in the dark at 23°C on a 12Orpm rotary shaker.
Culture for 0-14 days.

In the method of the present invention, FICE-V3111111
is cultured in liquid medium. Flasks or fermenters of glass or other commonly used materials such as stainless steel can be used. The liquid medium may include a nutrient solution containing an assimilable organic carbon source, an assimilable organic or inorganic nitrogen source, inorganic salts, and optionally plant hormones and/or vitamins. Assimilable organic carbon sources can include carbohydrates such as sucrose, buto-sucrose, fructose, starch, dextrin, glycerin, mannitol and mannose.

Organic or inorganic nitrogen sources that can be assimilated include amino acids and their mixtures, peptides and proteins or their hydrolysates, casein hydrolysates, and grains such as corn [cocose and wheat distillation residue] for alcohol synthesis. Examples include water-soluble fractions of enzymes, inorganic nitrates, and inorganic ammonium salts.

The process of the invention typically involves plating by suspension culture, for example in a flask with stirring or in an aerated fermenter.
H is in the range of 4.4 to 7.0, preferably at pH 6.5,
The temperature range is 18-36°C, preferably 23°C. - Preferred culture conditions within the membrane are dark, 5.1-6.
8(7) I)H range, 18-30'C(7)m degree range and duration of 8-16 days. Production of crude vincamine begins after 2-3 days of growth and reaches its peak after 10-14 days.

Crude pinkamine can be extracted from both cells and culture broth. Crude vincamine can be extracted from the filtered cells, typically using a water-miscible organic solvent such as methanol, ethanol or acetone. Culture broth that has been made alkaline to pH 8-9 after removing cells is usually treated with chloroform, methylene dichloride,
Crude vincamine can be extracted with a water-immiscible solvent such as ethernidel.

Vincamine or epivincamine thus produced can be made into a pharmaceutical composition together with a pharmaceutically acceptable carrier or diluted crude agent.

The present invention will be explained in more detail below using Example F1. However, the following description does not limit the present invention in any way.

Example 1 50d 03 with I)H adjusted to 6.5 with dilute ammonia
This was carried out in a 300 d Erlenmeyer flask containing 0 nutrient medium, and this was carried out using a rotating shaker (rotation speed: 120 rpm, eccentric distance).
! 18 cm). The best culture temperature is 2
The temperature was 3°C. The flask was inoculated with cells obtained from a 14 day old FIC Pei 3 culture in 85 agar medium.

After 14 days, high performance liquid chromatography (HPLC) measurements revealed that approximately 750 rng/j of vincamine and 340 rng/j of vincamine were detected.
■／j! It was found that shrimp binkamine was produced. 101 cell suspension cultures from 200 flasks were centrifuged and the cells and supernatant were extracted separately.

The supernatant was adjusted to pH 9 with sodium carbonate and extracted twice with 101 methylene chloride.

The organic extract was then extracted with 2% aqueous tartaric acid.

The resulting acidic solution was adjusted to pH 9/10 and then extracted with methylene chloride.

The centrifuged cells were homogenized with a 50% ahitone aqueous solution containing 2% tartaric acid and filtered. The P solution was compressed under reduced pressure, and the resulting aqueous solution was made alkaline to 1)) -19 to 10, and then extracted with methylene chloride.

The organic extracts of the cells and supernatant were combined and evaporated to dryness, yielding 8.8 g of crude vincamine (N-1, consisting of 6.0 g of vincamine and 2.83 g of shrimp vincamine as determined by plc). The two alkaloids were separated by silica gel column chromatography. At this time, methylene chloride containing gradually increased amount of ethanol was used as the eluent. 5.69 g of pure vincamine and 2.5 g of shrimp vincamine were recovered from the column.

Example 2 The method of the invention was carried out in a flask by a two-stage fermentation method. First, Murashige medium (T, Murashiage medium) containing 209/l glucose and 1/l NAA was used.
al, Physiol, Plant, Magazine
Volume 15, page 413.

(1962) under the same conditions as in Example 1. After 8 days, the dry Φ mother had reached 10-13 μg/l, the culture was centrifuged aseptically, and the cells were transferred to production medium G30.
resuspended in Fermentation for 6-8 days produced 1050 mg/l of crude vincamine. Hplc analysis showed that vincamine was 73% of the total alkaloids and shrimp vincamine was 25%. Using 100 Erlenmeyer flasks as described in Example 1, 3.6 g of pure vincamine and 1.09 g of shrimp vincamine were isolated. Almost the same yield was obtained when fructose or maltose was used in the medium instead of glucose.

Example 3 It was carried out in an IJ culture tank containing 61 g of G30 nutrient medium. Cultivation of the periwinkle culture strain FICE-v3 was carried out in accordance with Example 1 in a 300 d Erlenmeyer flask containing 50 rd of 85 medium. 1-10 days later (drying
12rftg/d), the culture was inoculated into a 20007 volume round bottom flask containing 450me of 85 medium and incubated in the dark for 2 hours.
Culture was carried out at 8°C on a rotating shaker with a rotational speed of 1100 rpm. After 7-10 days fully grown culture (dried lff1
10ay/d), 6j! (7) M10 containing G30 medium
It was used as an inoculum for the R1 culture tank. 1100rp
Growth was carried out in the dark at 27° C. with an initial stirring rate of 0.5 vol/vol/min and an aeration rate of 0.5 vol/vol/min. Dissolved oxygen concentration is 20
% decrease, the stirring speed was increased by 20 rpm.

After 10-14 days the culture had a dry part of 20 η/d and an alkaloid content of 7 G expressed as crude vincamine.
There was OIng/β. This alkaloid was extracted and purified as described in Example 1 to yield 3.39/l of vincamine and oJg/j2 of epivincamine.

Claims (1)

[Scope of Claims] (1) Culture strain FI under aerobic conditions in a liquid medium containing an assimilated carbon source, an assimilated nitrogen source, and inorganic salts.
A method for producing vincamine and epivincamine, which comprises culturing CE-V3 (DSM4365) cells. (2) Cells were grown in the dark at a pH of 5.1-6.8, 18-30
The method according to claim 1, characterized in that the culture is carried out at a temperature of 8 to 16 days. (3) The method according to claim 1 or 2, characterized in that the produced vincamine and epivincamine are recovered from cells and/or culture broth. (4) Claims 1 to 3 characterized in that the produced vincamine and epivincamine are separated from each other.
The method described in any of the above. (5) The method according to any one of claims 1 to 4, characterized in that the produced vincamine is converted to epivincamine. (6) The method according to any one of claims 1 to 5, which is carried out in a fermenter. (7) A method for producing vincamine or epivincamine as described in substantially any of the Examples. (8) Claim 1 as a pharmaceutically acceptable carrier or diluent and an active ingredient.
A pharmaceutical composition comprising vincamine or epivincamine produced by the method according to claim 6. (9) Cell culture line FICE-V3 (DSM4365).