JPH01181795A - Production of crocin and gardenia ellis plant cell having crocin-producing ability - Google Patents

Abstract

PURPOSE:To industrially advantageously produce the title compound useful as a yellow dyestuff, by tissue-culturing the cells or tissues of the pericarps, peduncles or flower buds of a Gardenia Ellis plant fruit and collecting crocin from the resultant cultured product. CONSTITUTION:The cells or tissues of the pericarps, peduncles or flower buds of a Gardenia Ellis plant fruit are implanted on a solid medium and subjected to a stationary culture in a dark place at 25 deg.C to provide the calluses thereof. The calluses are subsequently cultured in a liquid medium and filtered to collect the cultured calluses. The calluses are dried with air to provide the dried calluses, which are extracted with methanol, etc. Crocin is obtained from the extracted solution.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to gardenia
The present invention relates to a method for producing crocin by plant tissue culture, and gardenia plant cells having crocin-producing ability used in this method.

[Conventional technology]

The following reports are examples of tissue culture of Gardenia plants. Planta, Medica, 41.186
(1981), Gardenia ja
sminoides f, gran-diflora)
Callus was induced by culturing seedlings and seedlings on Linsmeyer-Skoog agar medium containing 10-'M of 2,4-dichlorophenoxyacetic acid (2,4-D), and then the callus was Indole acetic acid (2×10-'
M), transferred to Murashige-Skoog agar medium containing kinetin (10-'M) and coconut milk.
It has been reported that gardenia cells producing iridoid glucosides were obtained by subculture every ~3 weeks.

Also, Plant 5science Letters 3
3+47 (1984), Gardenia (Gardenia)
Salicyl alcohol was glucosylated (glu-cosyl alcohol) using the callus of Ia jasminoides.
A method for obtaining salicin (sal 1cin) is disclosed.

However, including the above-mentioned literature, there has been no report to date of the production of crocin, a yellow pigment, through tissue culture of plants belonging to the genus Gardenia.

[Problem to be solved by the invention]

The present inventor recognized that crocin, which is a yellow pigment, cannot be obtained by the conventional tissue culture method of Gardenia plants, and under such circumstances, the present inventors developed a gardenia plant that can produce crocin. We created new cells by tissue culture and investigated a method for producing crocin using these cells.

[Means to solve the problem]

As a result, the inventors discovered that the above object could be achieved by employing the method described below, and completed the present invention.

That is, according to the first invention of the present application, gardenia genus (G.
Provided is a method for producing crocin, which comprises tissue culturing cells or tissues of the pericarp, fruit stalk, and flower bud of a fruit of the Ardenia Ellis plant, and collecting crocin from the resulting culture. Moreover, the second invention of the present application is a plant cell of the genus Gardenia used in the method for producing crocin of the first invention,
Crocin (crocin) obtained by tissue culture of cells or tissues of the pericarp, fruit stalk or flower bud of the fruit (Ia Ellis) plant.
cin) is a gardenia plant cell capable of producing cin.

In the tissue culture of the present invention, gardenia
nia Ellis) plants are used. Specifically, the gardenia plants include G, jas+ainoides
Ellis (Kolinkchinashi), G, jasmin
oides Ellis var, grandi-fl
ora Nakai (Gardenia), G, jasn+
1noides Ellisvar, ovalifo
lia Nakai, G, radi
-cans Thunb, (small gardenia), G, r
Adicans Thumb.

var, fasciculata Hort, G, radicans Thunb, var,
variegata Caar, (Futarikensaki) can be exemplified. Among these, gardenias and small gardenias are preferred.

In the present invention, tissue culture is carried out using the above-mentioned Gardenia plant, and in this case, the present inventor has found that by tissue culturing the cells or tissues from a specific part of the plant, the crocin-producing ability can be improved. We have completed the present invention by discovering that it is possible to obtain cells that have the following properties. That is, in the tissue culture of a gardenia plant according to the present invention, cells or tissues of a specific site selected from the pericarp, fruit stalk, and flower bud of the fruit of the plant are used as the cells or tissues to be cultured. Even if tissue culture is performed using cells or tissues from other parts of the gardenia, gardenia plant cells with high crocin-producing ability cannot be obtained. Cells or tissues from the site are used. This finding is a new finding by the present inventor. Furthermore, as mentioned above, there has been no report to date of obtaining cells capable of producing crocin through tissue culture of plants belonging to the genus Gardenia.

The flower buds of Gardenia plants used in the present invention refer to those in the state from two months before flowering to just before flowering, including flower pedicels, petals,
Contains flakes.

In the present invention, tissue culture is performed using cells or tissues of plants of the genus Gardenia selected from the above-mentioned specific parts.Specifically, the medium used for tissue culture in this case includes inorganic components, A medium is used which contains a carbon source and a plant hormone as essential components, to which vitamins are added, and if necessary, amino acids are added. Inorganic components of the medium include nitrogen, phosphorus, potassium, sodium,
Examples include inorganic salts containing elements such as calcium, magnesium, sulfur, iron, manganese, zinc, boron, molybdenum, chlorine, iodine, and cobalt, specifically potassium nitrate, sodium nitrate, ammonium nitrate, ammonium chloride, and potassium chloride. , calcium chloride, phosphate l
Potassium hydrogen, sodium dihydrogen phosphate, magnesium sulfate, magnesium chloride, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, copper sulfate, sodium molybdate, molybdenum oxide, potassium iodide, zinc sulfate, boric acid Examples include compounds such as cobalt chloride and cobalt chloride.

Examples of carbon sources for the medium include carbohydrates such as Sea 1m and their derivatives, organic acids such as fatty acids, and primary alcohols such as ethanol.

The plant hormone in the medium includes indole acetic acid (IA
A) Auxins such as naphthaleneacetic acid (NAA), p-chlorophenoxyisobutyric acid and 2,4-dichlorophenoxyacetic acid (2,4-D), and cytokinins such as kinetin, zeatin and benzyladenine (pendylaminopurine) Among these, it is particularly preferable to use a medium containing auxin and benzyladenine selected from naphthaleneacetic acid, indoleacetic acid, and indolebutyric acid because cells with high crocin-producing ability can be obtained.

The vitamins in the medium include biotin, thiamine (vitamin B), pyridoxine (vitamin B6), pyridoxal, pyridoxamine, calcium pantothenate,
Examples include ascorbic acid (vitamin C), inositol, nicotinic acid, nicotinamide, and riboflavin (vitamin B2).

Examples of amino acids in the medium include glycine, alanine, glutamic acid, cysteine, tyrosine, and lysine.

The liquid medium used in the present invention usually contains about 0.1 μi to about 110011 I of the inorganic component and about 1 μi of the carbon source.
g/! ~100g/Il, usually 0.01μM to 1000μH2 preferably 1μM to 1
00 μH range, and the above vitamins in the range of about 0.1 μH/
2~about 150■/! and 0 to about 10 of the above amino acids.
It is preferable to use it by including 00mg/ffi.

Specifically, media containing these components include conventionally known media used for plant tissue culture;
For example, Murashige Skoog ('62)
ge & Skoog], Linsmaier-Skoog (RM-1965) [Linsmaier
& Skoog] medium, white (”63) [
White] medium, Gamborg [Gaa+borg
] B-5 medium, Mitsui's To9 medium, etc., by adding the carbon source and plant hormones described above, and further adding the vitamins and amino acids described above as necessary.

Regarding the composition of the above-mentioned conventionally known culture medium, for example, see "New Plant Tissue Culture J P38" by Yakuen, Nakajima, and Furuya.
6-P391, Breakfast Shoten, 1979.

The medium that can be used in the present invention is a liquid medium, agar, Ge
It is a solid medium that usually contains 0.1 to 1% of 1lan Gum or the like.

In the present invention, cells capable of producing crocin can be obtained by continuing to subculture cells or tissues selected from a specific site of the above-mentioned gardenia plant using the above-mentioned medium for an appropriate period of time. In addition, by selecting cells that are thought to have a high crocin content in the subculture and subplanting them, cells with higher crocin-producing ability can be obtained.

Even if such culture is applied to cells or tissues other than the cells or tissues of the pericarp, fruit stalk, and flower bud of the fruit used in the present invention, such as leaves, roots, stems, and seeds, the cells or tissues with crocin-producing ability and those with crocin-producing ability You can't get high cells.

Light is not necessarily necessary for the tissue culture of Gardenia plants of the present invention, and culture can also be carried out in the dark.

The culture temperature is usually in the range of 15 to 30°C. Also,
The subculture period is usually about 30 to 45 days.

Rubiaceae gardenia (G.
Gardenia cells with crocin-producing ability obtained by culturing callus derived from the pericarp of Ardenia jasminodes (indication for identification; GaLIA4B)
S) was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology (hereinafter referred to as FERM P-9792).
) was deposited as.

The gardenia cells GaL 1A4B5 can be grown and preserved, for example, in a liquid or solid medium having the following medium composition.

Linsmeyer-Skoog medium 3-1ndoleacetic Ac1d 10-'
M6-Benzylaminepurine 10-
'MGellan Gu+++ 0.2X
(wt/vol) The pH of the medium is usually 5.0 before heat sterilization.
6, and the culture medium sterilization conditions are, for example, 121°C11
I can give an example of 5 minutes. The culture temperature is usually 25°C.

In the present invention, cells capable of producing crocin can be obtained by culturing a gardenia plant using the tissue culture method described above. Furthermore, within the cell, the above-mentioned Ga
If gardenia cells with high crocin-producing ability, such as LlA4B5, are grown and cultured in a similar medium, crocin can be efficiently obtained by separating the cultured cells from the culture mixture and performing extraction operations.

The gardenia plant cells having the ability to produce crocin according to the present invention usually contain 10% crocin based on the dry weight of the cells.
Contains -3■/g or more.

〔Example〕 Hereinafter, the present invention will be explained in more detail with reference to Examples.

Examples 1 to 3 A 2% antiformin solution or a 2% antiformin solution or Gardenia jasmin sterilized with 70% ethanol solution etc.
A part of the pericarp of ``Odes'' was placed on a bed and statically cultured in a dark place at 25°C to obtain each callus.

These calli were then inoculated every 14 days in a Linsmeyer-Skoog liquid medium under the same conditions as above, and cultured with rotation on a rotary shaker (amplitude 25 mm, 10
The growth rate of the callus was increased by 100 rpL, and stabilized gardenia callus was obtained.

After culturing, the callus was collected by filtration, air-dried at 40°C for one day and night, and its weight (dry weight) was measured.
The weight of cultured cells grown per cell was determined. Crocin was measured by extracting the obtained dry callus using methanol etc. and comparing it with a standard product using high performance liquid chromatography (HPLC). It was confirmed that crocin was produced, and the amount produced was determined.

HPLC conditions: column; TSKgel 0DS-120T (
4,6i,d,X 250nm) eluent; primary 5χMeOH
secondary 95℃MeOH 3.3%/Lllin linierflow
ate; 0.6m(/llll1nddetect
ion; 438nmTemp;
Room temperature TLC conditions: Mark Kieselgel 60F
254 ■Developing solvent; CHCl, -MeOH-1 (zO
(15:8.5:2) The development ratio of crocin in this case (R
f) Ha0.3■Developing solvent; EtOAc-i-PrO
H-HzO (65:25:10) In this case, Rf of crocin is 0.15 After development, P-anisaldehyde re
Heating after spraying agent.

The spectrum of HPLC analysis is shown in FIG.

Examples 4 to 6 Examples 1 to 3 are all the same as Examples 1 to 3 except that the part of the plant used is the flesh, peduncle, or young flower bud.
I did it in the same way.

Examples 7 to 9 Each of Examples 1 to 3 was conducted in the same manner as in Examples 1 to 3 except that the part of the plant used was the flower stalk.

Examples 10 to 12 In each of Examples 1 to 3, the same procedures as in Examples 1 to 3 were carried out except that the parts of the plant used were calyxes.

Comparative Examples 1 to 3 Each of Examples 1 to 3 was conducted in the same manner as in Examples 1 to 3 except that the plant parts used were leaves.

(Margins below this page) Table 1 The remainder of the ingredients listed in the table is water.M in the table is mol/I! The second table showing ratio) Miyabi is 2X in crocin production per dry weight of aI follicles.
It is shown that the amount of crocin produced ranges from 10-''/g to 4-cut Q-1-7g.

0 indicates that no peak of crocin was confirmed by HPLC.

〔Effect of the invention〕

According to the present invention, as a result of tissue culture using cells or tissues from specific parts of plants of the genus Gardenia selected from the pericarp, fruit stalks, and flower buds of plants of the genus Gardenia, the plants of the genus Gardenia have the ability to produce crocin. It becomes possible to efficiently obtain plant cells, and by culturing these gardenia plant cells with high crocin-producing ability, crocin can be efficiently produced.

[Brief explanation of the drawing]

FIG. 1 shows the spectrum of high performance liquid chromatography analysis of crocin. Applicant Mitsui Petrochemical Industries Co., Ltd. Agent Patent Attorney Yusuke Hiraki

Claims (2)

[Claims] (1) A method for producing crocin, which comprises tissue culturing the cells or tissues of the pericarp, fruit stalk, or flower bud of a gardenia (Gardenia Ellis) plant, and collecting crocin from the resulting culture. (2) Plant cells of the genus Gardenia having the ability to produce crocin, which are obtained by tissue culturing cells or tissues of the pericarp, fruit stalk, or flower bud of a gardenia genus (Gardenia Ellis) plant.