JPH0559713B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a method for producing alkylhydroquinone-β-D-glucoside (hereinafter referred to as alkylhydroquinone glucoside) by tissue culture of plants. [Prior Art] Alkylhydroquinone glucosides are known to have remarkable effects as skin whitening agents (Japanese Patent Application Laid-Open No. 164815/1983), but conventionally, they have been produced by synthetic methods. Regarding homoarbutin, a specific alkylhydroquinone glucoside, a method for extracting it from Pirola incarnata Fisch. has also been reported {Pharm.Bull. (Tokyo)
4 281-283 (1956)}. [Problems to be solved by the invention] Synthetic methods usually consist of three steps. Namely, (1) acetylation of glucose in the presence of pyridine and ethyl acetate, (2) condensation to alkylhydroquinone using phosphorus oxychloride or P-toluenesulfonic acid as a catalyst, and (3) acetylation of glucose in the presence of pyridine and ethyl acetate. Deacetylation using methoxide requires the above three steps and is extremely complicated. Furthermore, there is no general method for extracting alkylhydroquinone glucoside from plants, and the reported content of homoarbutin is low (0.9% dry weight), making it difficult to obtain in large quantities. [Means for Solving the Problems] In view of the above circumstances, the present inventors have conducted extensive research and found that when alkyl hydroquinone is added to the tissue culture medium of periwinkle callus, an extremely large amount of alkyl hydroquinone is added to the culture medium. It was recognized that hydroquinone glucoside is accumulated, and the present invention was completed based on this knowledge. That is, the present invention is characterized in that alkylhydroquinone is added to and cultured in a tissue culture medium of Catharanthus roseus L., and alkylhydroquinone-β-D-glucoside represented by the following general formula is separated and collected from the culture. This is a method for producing alkylhydroquinone-β-D-glucoside by plant tissue culture. (In the formula, R represents an alkyl group having 1 to 4 carbon atoms.) The present invention will be explained in detail below. First, roots, hypocotyls, cotyledons, etc. of periwinkle sprouts (seedlings), or roots of mature periwinkle,
Callus can be induced by using cell groups or tissue pieces of stems, leaves, petioles, flowers, pollen, etc. as starting materials, sterilizing them in a conventional manner, and culturing them in a medium supplemented with auxin or cytokinin. At that time, the culture medium used is Murashige and Skoog.
Linsmeier-Skoog medium, White
Medium, Gamborg medium, Nitsch medium, Heller medium, Schenk-Hildebranat medium, Nitsch-Nitsch medium,
Kohlenbach and Schmidt
Schmidt) medium, etc. may be used. Normally, callus formation is performed in a solid medium containing agar, but callus can also be induced in a liquid medium that does not contain agar. In addition, auxin is generally required for callus induction, including 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA), and 2,4,5-trichlorophenoxyacetic acid. (2,4,5-T), indole acetic acid (IAA)
You may add any of the following. Furthermore, any of the cytokinins such as zeatin, 6-benzyladenine, kinetin, ribosylzeatin, and isopentenyladenine may be added. The concentration of auxin added is 10 ^-7 M to ^{10 -5} M.
The concentration of cytokinin also ranges from 10 ^-8 M to
It is in the range of 10 ^-4 M. The callus thus induced is transferred to liquid culture without adding agar to the above medium and subjected to a shaking medium. Of course, callus can divide and grow even in a medium containing agar, and liquid culture is not limited to shaking culture, but can also be carried out with aeration agitation or an airlift type jar infermenter. Liquid culture can be carried out by conventional known methods. During culturing, light may or may not be irradiated. The culture temperature is between 20°C and 30°C, with a temperature of about 27°C being preferable. Cultured cells are periodically transferred to new medium and subcultured. To obtain alkylhydroquinone glucosides, alkylhydroquinone is added to this medium. Even if alkylhydroquinone is added one week after callus is induced, alkylhydroquinone glucoside will be produced, but it is better to subplant the callus more than 100 times until the traits of the callus are stabilized. Alkylhydroquinone may be added at any time after transplanting newly cultured cells, but preferably at the end of the cell division phase. The glucoside can be produced at any concentration of alkylhydroquinone of 10 mM or less, but a range of 2 to 5 mM is particularly desirable. After adding the alkylhydroquinone, the culture is continued for one to several days, and then the culture is collected. Extraction of alkylhydroquinone from the collected culture can be performed by a conventional known method. [Examples] Hereinafter, the present invention will be explained in more detail with reference to Examples, but these are not intended to limit the present invention. Example 1 2.2×10 ^-6 M of 2,4-D as auxin
Murashige and Skoog liquid medium containing (manufactured by KC)
360ml each was dispensed into 1 Erlenmeyer flask and sterilized using 10 autoclave irradiation flasks.
The cultured cells used were callus derived from the stems of Catharanthus periwinkle by a conventional method and subcultured for over 3 years. 40 ml of cell suspension cultured for 7 days (fresh weight: 4
(containing cultured cells of g) in a sterilized medium,
Shaking culture was performed at 110 rpm using a rotary shaking culture device (manufactured by Iwashiya Kagaku) without light irradiation.
The culture temperature was 27°C. Seven days after implantation, 149 mg of methylhydroquinone (manufactured by Wako Pure Chemical Industries, Ltd.) was made into a 10 ml aqueous solution and added aseptically to the cell suspension (3 mM). Such treatment was performed on five flasks, and as a control, five flasks were cultured under the same conditions except that methylhydroquinone was not added. On the 9th day, the culture solution was suction filtered through Toyo Roshi No. 2, and the culture was thoroughly washed with pure water. Each culture was transferred to a 300 ml eggplant-shaped flask, 200 ml of methanol was added thereto, homogenized for 5 minutes using a Hiscotron (manufactured by Nichion Medical Instruments Manufacturing Co., Ltd.), and hot methanol extraction was performed at 65° C. for 2 hours on a hot water bath. The supernatant was stored by centrifugation, and the precipitate was extracted with hot methanol again under the same conditions. 400 ml of the extract obtained from the two extractions was concentrated to dryness using an evaporator, and the solid was dissolved in 30 ml of a mixture of chloroform, methanol, and water (30:10:1).
Dissolve this in a silica gel column (Wakogel
C-300 (manufactured by Wako Pure Chemical Industries). Silica gel is 200
g was suspended in the above-mentioned mixed solution, which had been previously packed in a column and equilibrated. Elution was performed with the above mixture, and a portion of each 10 ml fraction was collected by TLC (Kieselgel60F254,
(manufactured by Merck) (the developing solution was the above mixed solution).
Arbutin was developed simultaneously as a control. Fractions in which a spot with an Rf value slightly higher than that of arbutin was observed were determined to be a fraction containing methylhydroquinone glucoside, and these fractions were collected and concentrated to dryness. After further dissolving with a small amount of methanol, chloroform was successively added dropwise to effect recrystallization. Table 1 shows the average value and standard deviation of the amount of methylhydroquinone glucoside produced in the five flasks. In the control group, when the elution fraction from the silica gel column was analyzed by TLC, no spots were detected at the expected Rf value, and the methanol extract from the culture was directly analyzed by HPLC (the column was manufactured by Hanui Chemical Co., Ltd.). When Cosmosil 5C ₁₈ (eluent was 5% methanol adjusted to pH 2.1 with phosphoric acid), no peak was observed at the expected retention time. In order to confirm that the recrystallized product obtained in the experimental group (methyl hydroquinone addition) was methyl hydroquinone glucoside, its weight was measured and subjected to mass spectrometry and NMR. Mass spectrum m/z = 286 C-13 NMR spectrum δ value (solvent CD ₃ OD) Signal derived from benzene ring 152.0, 151.6, 120.8, 116.2, 103.4ppm Signal derived from glucose 103.4, 77.8, 74.8, 71.2, 62.4ppm Signal derived from methyl group 16.4, 16.6ppm As is clear from the above data, the recrystallized product is methyl hydroquinone glucoside. Furthermore, since two signals derived from the methyl group appeared, it was found that the following two isomers were produced.

【formula】

[Formula] Example 2 A culture medium similar to that in Example 1 was prepared, and the medium was dispensed into three aeration-stirring 5-jar fermenters manufactured by Marubishi in 2.7 portions and sterilized in an autoclave. Cultured cells of the same strain as in Example 1 were cultured in 300 ml of medium in one flask for 10 days, and then the entire cell suspension was aseptically added to a jar fermentor. The agitation speed for culturing the jar fermentor
Performed at 60rpm, airflow rate 0.5~3.0ppm, temperature 26℃, PH
was not controlled. After culturing for 10 days, 1490 mg of methylhydroquinone (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 100 ml of water and added aseptically (4 mM). The amount of aeration was adjusted at appropriate times so that the dissolved oxygen did not reach zero, and the culture was continued for two more days, and the culture was collected by centrifugation. Extraction and purification were performed in the same manner as in Example 1,
We looked at the production amount of methylhydroquinone glucoside. The results are shown in Table 1. It is clear from the results of Examples 1 and 2 that the content of alkylhydroquinone glucoside is much higher than the level of extraction from A. japonica that has been known so far.

[Table] Example 3 In the same manner as in Example 2, three periwinkles were cultured in three 5-jar fermenters. After culturing for 12 days, 1992 mg of tert-butylhydroquinone (manufactured by Tokyo Kasei) was dissolved in 10 ml of ethanol and added m/z (4 mM) aseptically. The amount of aeration was adjusted at appropriate times so that dissolved oxygen did not reach zero, and the culture was continued for another 4 days, and the culture was collected by centrifugation. Extraction and purification were performed in the same manner as in Example 1, and the production amount of methylhydroquinone glucoside was observed. The results are shown in Table 2.

【table】

Claims (1)

[Claims] 1. Catharanthus roseus L.
Alkylhydroquinone produced by tissue culture of plants, characterized in that alkylhydroquinone is added and cultured in a tissue culture medium of callus, and alkylhydroquinone-β-D-glucoside represented by the following general formula is separated and collected from the culture. -Method for producing β-D-glucoside. (In the formula, R represents an alkyl group having 1 to 4 carbon atoms.)