JPH0286773A - Production and separation of secondary metabolite of plant cell utilizing polymer adsorbing substance - Google Patents

Abstract

(57) [Abstract] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a method for producing and separating secondary metabolites of plant cells using a polymer adsorbent.

<Conventional technology> Since ancient times, plants have provided various important chemical substances for human life, such as pharmaceuticals and food additives.

Since the 1970s, with the development of plant cell tissue culture technology, various attempts have been made to separate and culture plant cells from the plant itself and use them as resources for various useful substances.

As a result, in the 1980s, it was possible to industrially produce Shikonin, a naphthoquinone-based plant pigment, for the first time.

Litho
It is a 1,4-naphthoquinone pigment that accumulates in the cork layer of the roots of Spermum erythrorhizon.

Shikonin was previously used as a dye for silk dyeing, but recently it has received the most attention as a pigment raw material for burns, skin regeneration medicine, and bio-cosmetics, and is an industrially more important substance than before. It is recognized that there is.

The key points to be solved by the present invention At present, industrially, shikonin is mainly used in liquid culture (s) of plant cells.
It is produced by ubmerged cullure).

After the cultured cells are collected, shikonin produced in the cells is separated using a solvent.

In such a process, shikonin is mainly distributed within the cells, so in order to effectively separate the target product, the cells must be destroyed, and then the shikonin leaked into the culture medium must be separated. This includes factors that increase costs in the process, such as processing a large amount of solvent.

<Objective of the present invention> The present invention was made in order to improve the above-mentioned drawbacks, and an object of the present invention is to provide a method for producing a target product more effectively.

In addition, only the target product adsorbed from the culture to the polymer substance can be easily separated and recycled without damaging the cells.
The purpose is to provide a method that has process advantages such as extraction with a solvent.

Furthermore, it is an object of the present invention to provide a method that can shorten the culture time during a batch process.

Another object of the present invention is to provide a method that enables the separation and production of target products from cells once cultured by reusing adsorbed substances in a semi-continuous or continuous manner.

Another object of the present invention is to provide a method that can more effectively reduce the production cost of secondary metabolites of plant cells, including naphtokins.

<Configuration of the present invention> The detailed explanation of this invention is as follows.

<B> Cell culture Using 5HND medium (SHND-medium), grow rinse vermum iris lorizon cells.

KNO32500mg/L Inositol
1000mg/LMgSO47) (20400Thi
amine IIcI 5Mn5O4H2010N
1cotinic acid 51nsO47fI2
0 1 Pyridoxine O,5
CIISO45H200,2CPA 2
C'aC122Hx0 200 Kinetin
e O, IKl l 5u
close 30.000COC126H200,
1pH5,8N)+4H2PO4300Cu1ture temp,
25 CthP045 1100rp in r
otary shakerNa2Mo042H200,
l Fe50+71120 15 Na2-EDTA 20 <Example> The conditions and method for producing shikonin are as follows.

After washing the cells grown by the method of <A> above,
M-9 medium containing polymer adsorbent (Ro-9med
ium) to adsorb and separate shikonin.

The preparation and production conditions of M9 medium are as follows.

Ca(NO3)24t120 694mg/L 3-
IAA 1.75mg/LKNO3805ucr
osc 30.000CuSfl+7t120
0.3 bugsO, +71h0 750 pits,
sNa! ! SO41480culture temp,
25 C2n5O47t120 3 1100r
p in rotary 5hakerKCl
65 112803 4.5 N a 112 P 042 Il 20 19F
eSO471 (201J7 Na2EDTA 1.83 <c> Quantification of shikonin and fixed shikonin are carried out by quantification of shikonin in cultured cells or adsorbed to a polymeric substance in chloroform (chl).
oroform), chloroform was removed using a vacuum evaporator, 2.5% potassium hydroxide (KO2) was added, color was developed, and absorbance (OD6B) was extracted using a spectrophotometer.
) was comparatively quantified using a standard curve using 7111+.

(References: Mizukami, Il, et al.
, (1977), Phytochemistry, 1
6:1183-1186).

For fixation of shikonin, the extracted sample was immobilized on silica gel (sili
Make the chloroform mobile by dripping it onto the plate (cagel),
Thin layer chromatography method
chromatography).

Here, the standard sample is acetylshikonin (acetylsh
iconin) was used.

According to this method, the Rf value on the chromatograph of the sample produced was at the same location as the Rf value of the standard sample acetylshikonin, and four slight traces were found that were slightly different from the Rf value. These were found to be shikonin derivatives.

This is mass spectrometry.
It was also confirmed again by the analytical method (rometry).

(Example 1) Polyurethane foam as an adsorbent
(Table 1) Cut polyurethane foam (cushion sponge), which can be easily obtained from cloth, into a suitable size with a diameter of 1 cm or less, and add it together with the cells grown in 5HND medium. When cultured in M-9 medium in a batchwise semi-continuous manner or in a continuous manner, most of the shikonin produced as the cells proliferate is absorbed into the polyurethane.

The results shown in Table 1 were obtained by extraction with chloroform adsorbed from the polyurethane foam separated from the medium.

Table 1 (Example 2) Silicone rubber (sili) as an adsorbent
(Table 2) When using silicone rubber (product name: Masterflex) (Table 2)
rflex), America, ColeP-7- (ColeP
ermer) product) to an appropriate size, and
When cells grown in HND medium are cultured in M-9 medium in a batch, semi-continuous or continuous manner, most of the shikonin produced as the cells grow is adsorbed to the silicone rubber.

Shikonin adsorbed on silicone rubber was extracted with chloroform and the results shown in Table 2 were obtained.

A. Wet weight ffi (g/40m1) B. Shikonin released into the sea urchin culture medium in the cells; Shikonin adsorbed to polyurethane foam *Shikonin g/40m1, IQ6 polyurethane foam stool table-2 A: ゛2 body weight Fleas (g/40m1) A: Shikonin released into the intracellular sea urchin culture medium: Shikonin adsorbed to silicone rubber Nikita Shikonin: g/40m 1.1% silicone rubber used for use Effects of the present invention> This invention During the culture of plant cells, an appropriate polymeric adsorbent is added to induce the flow of naphthoquinones containing the target product shikonin out of the cells. Product inhibition (p
It is intended to reduce the product 1nhibition).

Furthermore, this invention is a bioreactor that uses Lithospermum iris lorhizon (Lithospermum iris, which belongs to the family Murasaceae).
When producing plant secondary metabolites such as naphthoquinones, including shikonin, by culturing large amounts of plant cells such as A. Needless to say, there are many ways to improve the ability of plant cells to produce secondary metabolites.

As a result, the desired product can be produced more effectively.

Furthermore, in addition to the process advantages of easily separating, recycling, and extracting with a solvent only the target product adsorbed from the culture to the polymeric substance, without giving a variety to the tidal volume, it also has the advantage of It has the characteristics that it can be expected to have multipurpose effects, such as being able to shorten culture time.

Another great advantage is that by reusing adsorbed substances, it is possible to separate and produce the target product from cells once cultured in a semi-continuous or continuous manner.

Furthermore, the present invention provides such process advantages, thereby effectively reducing the production cost of secondary metabolites of plant cells, including naphtoquine necks.

Applicant: Korea Institute of Science and Technology, Director Jeon Mok Jae

Claims (4)

[Claims] (1) In the production of secondary metabolites by culturing plant cells, one or more of polyurethane, silicone rubber, and related hydrophobic polymeric substances are added to the bioreactor and cultured in the plant cells. A method of increasing the productivity of secondary metabolites by inducing or promoting their flow out of cells. (2) Part or all of the secondary metabolites produced according to claim (1) are adsorbed onto the polymeric substance according to claim (1), and a batch system, semi-continuous system, or continuous system is used. How to separate and produce. (3) Claim (1) characterized in that the plant is a plant of the Boraginaceal family.
) or the method according to paragraph (2). (4) Any one of claims (1), (2), and (3), characterized in that the secondary metabolite of the plant is a naphthoquinone compound containing shikonin. One method.