JP2724549B2 - Production method of essential oil components by callus culture of sandalwood - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing essential oil components of sandalwood using sandalwood leaf tissue as a raw material. More specifically, the present invention relates to a method for producing essential oil components from callus cultured and grown on leaf tissue of sandalwood.

[0002]

2. Description of the Related Art Sandalwood is the scientific name Santalum, a semi-parasitic evergreen tree belonging to the sandalwood family with a height of about 3 to 4 m. It is used as a raw material for essential oils. The essential oil obtained from this is used as a component of a compounded fragrance, and as a component of flavoring, incense, and incense on decorative articles such as a fan due to its long-lasting aroma, and as a pharmaceutical.

[0003] Further, the extraction of essential oil components from sandalwood is generally carried out by chopping the core or root of an adult tree having a growth period of 30 years or more to obtain a coarse powder, which is then subjected to steam distillation. The yield is about 4.5-6.5% in total. The main components of the obtained essential oil are usually about 90% α-sanalol and β-santalol.

Conventionally, phenols and terpenes have been reacted as synthetic essential oils similar to the above to produce borneol (Borneo).
Many products have been developed, such as l) and phenol condensates, but their quality is inferior to sandalwood essential oils. Therefore, the essential oils obtained from adult sandalwood are still important. However, due to the low yields mentioned above, a considerable amount of log is required for its continuous harvesting. However, in recent years, there has been a problem that the massive logging of raw wood leads to environmental destruction.

[0005]

In view of the above-mentioned problems, various methods for mass production of sandalwood and a method for obtaining a large amount of essential oils thereof have been studied, but the established methods are not enough. not exist.

A method for producing a component contained in a plant by a tissue culture method is disclosed in Japanese Patent Application Laid-Open No. 1-181795. However, in this conventional method, the object is related to a method for producing crocin of a gardenia plant, not to sandalwood. It is. Therefore, the collection of the culture material is limited to a certain period, and the amount of collection is also limited. For this reason, this conventional method is not suitable for mass production of the essential oil component.

[0007] In particular, in the method of pure culture of plant tissue, when a strong aromatic component is contained in the plant, the culture and growth may be substantially inhibited. Therefore, the above-mentioned conventional method cannot be directly applied to sandalwood.

Therefore, an object of the present invention is to provide a production method capable of avoiding the above-mentioned problems and obtaining a stable and large amount of essential oil components contained in sandalwood.

[0009]

In order to achieve the above object, the present invention provides a method for producing an essential oil component by cultivating sandalwood callus as follows. That is, the process of inoculating the leaf tissue of sandalwood on a solid medium containing a carbon source to which a hormone according to a combination of 2,4-dichlorophenoxyacetic acid as auxin and kinetin as cytokinin is added and culturing callus, The method is characterized by comprising a step of subculturing the cultured callus on a solid medium in the same hormone-added section, and a step of extracting essential oil components of sandalwood from the grown callus.

The basic medium of each of the above-mentioned media is MS
A medium, white medium, LS medium, woody plant (WP) medium and the like can be used, but it is preferable to use a WP medium from the viewpoint of improving productivity. The basic medium may be a mixed medium of two or more kinds. The mixed medium preferably has a weight ratio of WP medium to Camborg-B5 medium of 1 to 1 from the viewpoint of improving the productivity of callus and α-santalol.

[0011] The carbon sources contained in the basic medium include carbohydrates such as sucrose, derivatives thereof, organic acids such as fatty acids, and primary alcohols such as ethanol. Hormones to be added to the medium include 2,4-dichlorophenoxyacetic acid.
Acids (hereinafter, referred to as "2,4-D") and kinetins (kinetins, hereinafter referred to as "kin") in combination with cytokinins are applied.

[0012] The amount of these hormones to be added is not particularly limited as long as the function can be effectively exerted in both the culture medium and the culture medium during the growth process.
2,4-D has a concentration of 10 ^{-5 to} 5 × 10 ^-7 mol, and ki
A combination in which n has a concentration of 10 ^{−5 to} 5 × 10 ⁻⁷ mol can be used.

Further, coconut milk (CAcasein hydrate) can be added to each medium to further improve callus productivity. The addition of coconut milk in an amount of 5 to 15% by weight and the addition of casein hydrolyzate in an amount of 0.05 to 0.5% by weight are preferable in that good productivity can be obtained. . Also,
To further improve the productivity, 10 to 100 μg of oligogalacturonic acid can be added.

The solidifying agent for each medium may be a common one such as agar or gelatin. Also,
0.9% agarose can also be used. The solidification of the medium can also be carried out by a general method such as pouring a solidifying agent in the form of an aqueous solution into a basic medium and treating the medium under heating and pressure in an autoclave.

The amount of the solidifying agent to be added is an amount sufficient to obtain a sufficient solidified state as a medium, and it differs depending on the kind. For example, in the case of gelatin, about 2% by weight is used.
Is an appropriate amount. The PH value of the medium is
It is preferably adjusted to 5.7 to 5.8 with an acid or an alkali.

It is preferable that the leaves of sandalwood are already sterilized with a bactericide such as ethanol in a state of having a petiole, and the leaves are, for example, 10 × 15.
It can be set as a section having a size of about mm.

The callus culturing process and the environmental conditions of the growing process can be carried out in a sterile room at 25 ± 2 ° C. in the dark and in a stationary state. The passage time can be determined appropriately according to the growth state of the callus, but it is preferable to carry out the passage every 6 weeks from the viewpoint of obtaining good growth.

Callus is induced and cultured in the leaf tissue of sandalwood planted on a solid medium containing a carbon source to which a hormone related to a combination of 2,4-D auxin and kin cytokinin is added.

In the callus thus produced, essential oil components of sandalwood are produced during the growth process.

[0020]

【Example】

(1) Induction of callus (Preparation of section) The leaves with the petiole of an adult sandalwood were washed with running water. Then, this was immersed in a solution of 10% anti-ormine containing 0.2% Tween 20 for 5 minutes, further immersed in 70% ethyl alcohol for several seconds, sterilized, and then washed 5 times with sterilized water. Next, the petiole and leaf margin were cut off at a width of several mm in a sterile dish, and then 10 × 1
The section was about 0 mm in size.

(Preparation of Medium) A basic medium was prepared by adding 3% by weight of sucrose to a WP medium. Add 2,4 to this basal medium
-D was added in a combination of 10 ⁻⁵ , 10 ⁻⁶ and 5 × 10 ⁻⁷ mol, and kin was added in a concentration of 10 ⁻⁵ , 10 ⁻⁶ and 5 × 10 ⁻⁷ mol. Next, the pH was adjusted to 5.7 to 5.8 using 0.1 N sodium hydroxide and 0.1 N hydrochloric acid.
To this was added 0.2% by weight of gelatin and dissolved in an autoclave.

33 ml of this lysate was dispensed into a 100 ml Meyer.
Each of them was dispensed and sterilized in an autoclave at 120 ° C. and 1.2 kg / cm ² for 15 minutes to obtain solid media.

(Induction operation) The leaf slices were inoculated on a solid medium of each of the above-mentioned hormone concentration-added sections, and they were cultured in a sterile room at 25 ± 2 ° C in the dark in the dark. .

The condition of the section of each leaf 7 weeks after planting is shown in Table 1 below.

[Table 1] In addition, in the table, when callus was induced, N. E. FIG.
Indicates that no callus was induced.

The induction of small white callus clusters on the back of the leaves was confirmed on the medium in the hormone-added group according to the combinations shown in Table 1. In addition, as a result of subculturing every 6 weeks thereafter for 1 year, callus was increased in the combined hormone-added group in which 2,4-D was 5 × 10 ⁻⁷ mol and kin was 5 × 10 ⁻⁷ mol. The growth was the best.

(Propagation operation) Next, this 2,4-D is 5
The callus induced in the combination hormone-added group containing × 10 ⁻⁷ mol and kin was 5 × 10 ⁻⁷ mol was compared with 5 × 10 ⁻⁵ and 10 ^{−5 of} 2,4-D obtained by the above-mentioned treatment method. , 10 ^-6 , 5
The cells were grown for one year on a solid medium in a group to which hormones were added in a combination of × 10 ⁻⁷ mol, kin at 10 ⁻⁵ , 10 ⁻⁶ , and 5 × 10 ⁻⁷ mol. The passage was performed every 6 weeks, and the growth conditions were the same as those described above.

The growth state is shown in Table 2 below.

[Table 2] The numerical values in Table 2 show the rate of weight increase every six weeks.

From the results in Table 2, it can be seen that the growth rate tends to decrease in the medium having a high 2,4-D concentration, but the growth rate is relatively high in the medium in the other combination hormone-added groups. Is confirmed.

[0029] Also among the highest growth was obtained in a medium with the addition group of combined hormonal with 10 ^-6 molar and 10 ^-6 moles of kin of 2, 4-D. The state of callus in this medium is shown in FIG. In the figure, 1 indicates a callus that has grown, 2 indicates a solid medium, and 3 indicates a container.

(Extraction of Essential Oil) In all the calli grown in Table 2, the essential oil-like odor of sandalwood was strongly felt. Of those shown in Table 2, 2,4-
The callus grown on the culture medium of the combination hormone-added group containing 5 × 10 ⁻⁵ mol of D and 10 ⁻⁶ mol of kin was extracted with n-hexane to obtain an extract.

(Identification of Extract) The obtained extract and the extract obtained by adding α-santalol to the extract were subjected to gas chromatography (GC) under the following conditions.
Analysis was performed. GC condition Model: HEWLETT PACKARD 5890A MS: JEOL JMS-SX102 ionization mood: EI, polarity: pos. column: NEVTRABOND-1 (0.25mmi.d. × 30m),
carrier gas: He column temp .: 130-250 ° C (rate 4 ° C / min
), Inj.temp .: 180 ° C det.temp .: 240 ± 10 ° C

The results of the GC analysis are shown in FIG. 2 for the extracted extract, and in FIG. 3 for the case where the α-santalol was added.

From the results of this analysis, as shown by the characteristics a and b in each figure, from the peak value of the same Retention Time of GC, the presence of the same compound as α-santalol was recognized in the above-mentioned extract. It can also be seen that a high yield is obtained.

(2) In preparing the medium of the above (1),
Further, the same culture and growth operations as in (1) above were performed using a solid medium containing 10% by weight of coconut milk, and as a result, the average was about 1.
A 6-fold amount of growth effect was confirmed.

From the callus thus grown, (1)
The yield of α-santalol in the extract obtained by the extraction operation of (1) was also improved in proportion to the increase in the growth rate.

(3) In preparing the medium of the above (1),
Furthermore, the same culture and growth procedures as in (1) above were performed by applying a solid medium containing 0.1% by weight of casein hydrolyzate. A 6-fold amount of growth effect was confirmed.

Further, from the grown calli, the above (1)
The yield of α-santalol in the extracted extract obtained by the extraction operation was also improved in proportion to the increase in the growth rate.

(4) In preparing the medium of the above (1), by using agarose which is a polysaccharide occupying about 70% of the agar component instead of agar, the callus proliferative property is improved. It was also confirmed that the productivity of α-santa roll could be improved.

[0039]

Since the present invention is constituted by the above-described steps, the following effects are exhibited. First, since the present invention was cultured on a solid medium to which a hormone related to the combination of 2,4-D as auxin and kinetin as cytokinin was added, callus was cultured on sandalwood leaf tissue. It became possible to produce the essential oil components of sandalwood during the growth process.
This has made it possible to artificially produce the essential oil component without using sandalwood logs.

In addition, since sandalwood leaves are used as a raw material for culture, since sandalwood is an evergreen tree, it can be produced throughout the year without any limitation at the time of production.

[Brief description of the drawings]

FIG. 1 is a partial cross-sectional perspective view showing a callus growing state.

FIG. 2 is a graph showing GC characteristics;

FIG. 3 is a graph showing GC characteristics;

Claims (4)

(57) [Claims] 1. A process of inoculating leaf tissue of sandalwood on a solid medium containing a carbon source to which a hormone related to a combination of 2,4-dichlorophenoxyacetic acid as auxin and kinetin as cytokinin is added, and culturing callus. And a step of subculturing the cultured callus on a solid medium in the same hormone-added section, and a step of extracting the essential oil component of sandalwood from the grown callus. A method for producing essential oil components by culturing. 2. The method according to claim 1, wherein 2,4-dichlorophenoxyacetic acid is 10 ^-5.
５5 × 10 ^-7 molar, with kinetin being 10 ^-5 -5
2. The method for producing an essential oil component of sandalwood callus culture according to claim 1, wherein the concentration is × 10 ⁻⁷ mol. 3. The method for producing an essential oil component according to claim 1 or 2, wherein coconut milk is further added to the solid medium in an amount of 5 to 15% by weight. 4. The essential oil obtained by cultivating sandalwood callus according to claim 1, wherein 0.05 to 0.5% by weight of casein hydrolyzate is further added to the solid medium. How to produce the ingredients.