JPH0484894A - Production of rutin - Google Patents

Abstract

PURPOSE:To inexpensively obtain a large amount of rutin industrially and advantageously by culturing callus derived from an organ and/or tissue of a plant containing rutin. CONSTITUTION:Bud of Sophora japonica L. is sterilized with ethyl alcohol and cut to give a small piece. Then the small piece is inoculated into a medium comprising a carbon source such as sucrose, an inorganic component such as Ca, an organic component such as biotin and indole-3-acetic acid, etc., irradiated with light rays at 18-32 deg.C at 190-500nm wavelength at 100-10,000lux intensity for several days to several weeks and sterilely subjected to callus induction culture to give callus. Then the callus is inoculated into a liquid medium having reduced nutrients of the above-mentioned induction medium, irradiated in the same way with 20-300l/minute based on 1m<3> medium aeration and subjected to rutin biosynthesis cell culture for plural days to several weeks to give a culture mixture. Then the culture mixture is filtered, cells are dried, ground, impregnated with water and extracted to produce rutin.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for producing rutin, and more particularly, to an efficient method for producing rutin in large quantities from the organs and/or tissues of plants containing rutin. It relates to a method of manufacturing well.

[Prior Art] Rutin, which is a plant pigment, is a typical compound of vitamin P and has an antioxidant effect. Its uses include coloring agents for foods and beverages, blood vessel reinforcement and hemostatic agents, and antioxidants.

Rutin is widely distributed in natural plants, such as apricot of the legume family, buckwheat of the polygonaceae family, hemruda of the rutaceae family, stevia of the asteraceae family, azuki bean, tobacco, tomato, red bean paste, and lemon. About 10 to 20% (based on dry weight), about 4% in buckwheat leaves (based on dry weight), about 3% in whole azuki plants (based on dry weight)
Some products also contain a large amount of rutin. the current,
Rutin is produced by extracting rutin from these rutin-containing plants.

The manufacturing method involves drying these plants, turning them into powder, immersing them in about 10 times the volume of hot alcohol, extracting them for 24 hours with occasional stirring, and then treating the extract with boiling water to immediately extract the obtained product. It is produced by extracting rutin with ethyl alcohol in twice the volume of the collected tear fluid.

In addition, when using rutin as a medicine, etc., the rutin crystals obtained by extraction are recrystallized by dissolving them in 5 times the amount of hot methyl alcohol, and then recrystallized by treating them with 200 times the amount of hot water. Refined.

Producing rutin through chemical synthesis is difficult and expensive due to its chemical structure, so it is not currently possible to produce rutin through chemical synthesis. In this case, from the viewpoint of safety, it is preferable to use rutin derived from natural products rather than rutin produced by chemical synthesis.

[Problems to be solved by the invention] However, the production method of extracting rutin from natural plants has poor extraction efficiency, and it is difficult to use natural cultivation methods, which are susceptible to various natural environment constraints such as season, weather, latitude, and topography. Therefore, it is difficult to stably supply rutin. Furthermore, cultivating large amounts of rutin requires a large amount of land and a large amount of labor costs, which causes the production price of rutin to become expensive.6 The present invention solves these problems in the past. rutin, a naturally derived product, in a planned manner and on an industrial scale.
The object of the present invention is to provide a method for producing rutin that can be supplied inexpensively and in large quantities.

[Means for Solving the Problems] As a result of intensive studies, the present inventors were able to find a method for producing rutin that solves the above problems.

That is, the present invention involves inducing callus from rutin-containing plant organs and/or tissues, then further culturing the induced callus to biosynthesize rutin, and collecting rutin from the cultured cells. The present invention provides a method for producing rutin.

The present invention will be explained in more detail below.

The plant and its organs and/or tissue parts used in the present invention may be any M products and parts as long as they contain rutin, but are preferably rutin buds,
Rutin can be produced more efficiently by using plant organs and/or tissues containing a large amount of rutin, such as buckwheat leaves.

In the method for producing rutin of the present invention, first, cut out the organs and/or tissues of a plant containing rutin, and
% ethyl alcohol, sodium hypochlorite, etc., sterilize it, wash it with sterile distilled water, and then cut it into small pieces.
Aseptically place a solid medium or a liquid medium, or in the case of a liquid medium, place a filter paper, etc. on top and culture to induce callus (callus induction culture).The medium used at this time is sucrose, etc. carbon sources, inorganic components such as nitrogen, phosphorus, potassium, calcium, magnesium, zinc, copper, sodium, iron, manganese, and molybdenum, various vitamins, and organic components such as myo-inositol, nicotinic acid, biotin, folic acid, and thiamate. From among the components, appropriate carbon sources, inorganic components, and organic components are selected and added to the medium. Furthermore, the medium contains a plant growth regulating substance, namely Mel.
I hormone, 2.4-D, α-naphthalene acetic acid,
An appropriate one is selected from among auxins such as indole triacetic acid, and cytokinins such as kinetin, 6-benzyladenine, and zeatin, and is added in a small amount.

The types and concentrations of carbon sources, inorganic components, organic components, and plant hormones in these media can be appropriately selected depending on the type of plant placed on the medium and the site of its organs and/or tissues.

For callus induction culture, the culture temperature is 18-32℃, the wavelength is 190-500r+w+, and the intensity is 1000-10.0.
The test is carried out in a dark place for several days to several weeks without irradiation with 00 lux of light continuously or intermittently every day, or without irradiation with light. The range of temperature and light values at this time is
This is because appropriate values are selected depending on the type of plant and its organs and/or tissues. Callus induction culture induces callus, which is a cell mass consisting of amorphous undifferentiated cells, from small plant fragments. This callus is
Although subculture can be performed, it is preferable to perform subculture using the medium and culture conditions used for callus induction culture.

Next, the induced callus is further cultured in order to biosynthesize rutin (rutin biosynthetic cell culture).

The medium used at this time may be the same as the medium used for callus induction culture, but preferably nitrogen, phosphorus,
Rutin production will be more efficient if a medium containing reduced amounts of plant growth nutrients such as potassium is used. For rutin biosynthetic cell culture, cells are cultured for several days to several weeks using a culture tank, for example, a closed metal culture tank, and rutin is biosynthesized from callus. In addition, in order to supply oxygen necessary for cell growth and rutin biosynthesis, a liquid medium 11
This is done while bubbling 20 to 300 l of sterile air per minute into the liquid medium. At this time, it is preferable to supply oxygen evenly and sufficiently to the cells by flowing aeration air along an air passage provided in the culture tank and further stirring the liquid medium and cells.

Regarding light and temperature in rutin biosynthesis cell culture, the optimal conditions differ depending on the plant from which the cells are derived and its organs and/or tissues, as in the case of callus induction culture, but the culture temperature is 18-32℃ and the wavelength is 190℃. ~500nI1
1 and a light or dark place with an intensity of 1,000 to 10,000 lux is preferred.

During rutin biosynthetic cell culture for several days to several weeks, some of the produced rutin elutes out of the cells, but the majority is water-insoluble and remains within the cells, so it is difficult to remove the cells from the liquid medium. The rutin in the cells is extracted by an appropriate method. For example, rutin can be extracted by drying cultured cells separated by filtration from a liquid medium, pulverizing them, immersing them in water, and stirring occasionally. Of course, rutin can be extracted using ethyl alcohol, which is a conventional extraction solvent from natural plants, but the production cost is high.

Although a solid medium can be used as the medium for culturing rutin biosynthetic cells, the components added to the medium can only be effectively used in the surface layer of the solid medium, which is not practical.

[Function] By callus induction culture for several days to several weeks, callus is induced from small pieces of plants placed in a medium, cells proliferate, and callus with a diameter of several II to several centimeters develops. When this callus is used to culture rutin biosynthetic cells in a liquid medium for several days to several weeks, rutin is biosynthesized by metabolic action within the cells. The amount of rutin produced is
It can be confirmed by measurement using high performance liquid chromatography, etc., and the rutin content is usually higher than that in natural plants, with a content of 5 to 30%.
% (based on dry cell weight).

The produced rutin can be extracted from dry and crushed cultured cells.
It can be extracted using water or the like as a solvent.

At this time, it is difficult to extract rutin from natural plants because they have hard tissues such as the epidermis of leaves and stems, but in the cultured cells of the present invention, extraction is easy because it is extracted only from the cells. can be done.

[Example] The present invention will be explained below by way of examples.

K1■'' Buckwheat leaves were thoroughly washed with running water, further washed with distilled water, and then cut into pieces approximately 5 cm in size. Next, in a clean bench, this small piece was immersed in 70% ethyl alcohol for 5 seconds, 8% sodium hypochlorite solution for 10 minutes to sterilize it, and then immersed in sterile distilled water 4 times to thoroughly remove the sterilizer. Washed and removed. The buckwheat leaves were cut into small quadrilateral pieces of about 1 cm on each side using a sterile scalpel, and then added to the Murashige-Skoog (MS) medium shown in Table 1 with α-naphthalene vinegar MO, 8 pp+* and 6 ppm as N-hormones. Benzyl adenine t, opps was added, and the mixture was aseptically placed on a solid medium to which agar had been added in an Erlenmeyer flask.

When the small pieces placed in the medium were cultured for 2 weeks at a culture temperature of 25° C. and under light irradiation of 2000 lux, callus generated around the cut end of the leaf grew to a diameter of several cm. The callus was pale yellow.

This callus was divided into small pieces, and 400ml of ammonium nitrate (NH, NO,
g/l, add potassium dihydrogen phosphate (KH2PO,) to 1
0C) + g/f, and put a liquid medium without adding agar into a culture tank.
Cell culture was performed. Air aeration rate to the liquid medium 801/min, z ff, rotation speed of stirring blade 5 Q rpm, temperature 2
As a result of culturing at 5° C. for 2 weeks, countless yellow cultured cells were produced.

Next, the cultured cells were separated from the liquid medium using a cloth separator, dried in an oven, crushed using a coarse pulverizer, and immersed in water for 24 hours to remove rutin. After extraction, the cultured cells were separated using an F cloth separator to obtain rutin.

The components of the crushed cultured cells at this time were measured using a high performance liquid chromatograph, and the results are shown in FIG. 1 under six measurement conditions as follows.

Column, prebacked column 4xxφX250+++
+L Packing material: Licrofer-100RP-18 (5μ) manufactured by Merck Flow rate: 0.8 shoes l/min Detector: UV300nm Model used: Hitachi L-6200 Eluent: Methyl alcohol As can be seen from Figure 1, culture The cells contained as much as 24% (dry weight basis) rutin.

Warise Yu The apricot buds were washed with water and then rinsed with distilled water.

The sample was sterilized by being immersed in 70% ethyl alcohol for 5 seconds and 3% sodium hypochlorite solution for 10 minutes in a clean bench, and then immersed in sterile distilled water 4 times to thoroughly wash and remove the sterilizer. Next, using a sterile scalpel, remove the calyx from the buds of Japanese amberjack and add to the Linsmeyer-Skoog (LS> medium shown in Table 2) 0.2 pp'm of indole triacetic acid and 1.5 ppm of kinetin as plant hormones.
were added and placed aseptically on a solid medium supplemented with agar in an Erlenmeyer flask.

The buds of Japanese amberjack placed in the culture medium were cultured at a temperature of 28℃, 3
When cultured for 3 weeks under light irradiation of 000 lux, fresh calli grew to a diameter of 1c+y.

The callus was pale yellow.

Of the components of the medium used for the above-mentioned callus induction, potassium nitrate (KNO,) was added at 900 ty/N, and ammonium nitrate (NH, NO,) was added at 400 x g, y'
1, add calcium chloride (CaCI24HzO) to 340
xy/l, magnesium sulfate (?IgSO, 71(
20) to 200 zg/l, and a liquid medium without adding agar was placed in a culture tank for cell culture. Under 2000 lux light irradiation,
Air was fed into the liquid medium at Loot'/min·13, and the cells were cultured for 10 days while refluxing the cells in the liquid medium. Countless yellow cultured cells were produced.

Rutin was extracted from cultured cells in the same manner as in Example 1, and measured using a high performance liquid chromatograph.
% (on a dry weight basis) of rutin.

[Effect of the invention] From the axis and/or tissue of a plant containing rutin,
By inducing callus, then further culturing the induced callus to biosynthesize rutin, and collecting rutin from the cultured cells, rutin derived from natural products can be produced by an industrial method.
It is now possible to manufacture on a large scale, systematically, and at low cost, which is extremely useful in practice.

[Brief explanation of the drawing]

FIG. 1 is a high performance liquid chromatogram of rutin obtained in Example 1.

Claims (1)

[Claims] from plant organs and/or tissues containing rutin;
A method for producing rutin, which comprises inducing callus, then further culturing the induced callus to biosynthesize rutin, and collecting rutin from the cultured cells.