WO2005063227A1

WO2005063227A1 - Process for producing triterpene composition

Info

Publication number: WO2005063227A1
Application number: PCT/JP2004/019428
Authority: WO
Inventors: Futoshi Matsuyama; Takashi Yoshida; Tsutomu Hatano; Shoko Taniguchi
Original assignee: Use-Techno Corporation
Priority date: 2003-12-26
Filing date: 2004-12-24
Publication date: 2005-07-14
Also published as: JPWO2005063227A1; KR20060123377A

Abstract

A process for producing a triterpene composition which comprises culturing a callus induced from banaba, loquat, perilla, or guava together with indoleacetic acid and a vegetable hormone other than the acid and obtaining from the cultured callus a triterpene composition which contains at least one member selected from the group consisting of corosolic acid, tormentic acid, 2α,19α-dihydroxy-3-oxours-12-en-28-oic acid, and maslinic acid.

Description

Specification

Method for producing triterpene composition

Technical field

The present invention relates to a method for producing a triterpene composition, a triterpene composition, and a callus for use in producing a triterpene composition.

Background art

[0002] Triterpenes such as corosolic acid, tolmentic acid, and maslinic acid are known to be useful substances that exhibit pharmacological activities such as blood sugar lowering action, and are extracted as components from natural plants. Examples of obtaining these triterpenes have been reported so far.

[0003] As a plant containing corosolic acid, for example, banaba (Lagerstroemia speciosa) is known (for example, Non-Patent Document 1), and as a plant containing tormentetic acid, for example, Tigerella polyphyla (Tiarella polyphyla) ) Is known (for example, Non-Patent Document 2), and as a plant containing maslinic acid, for example, Olive (Olea europasea) is known (for example, Patent Document 1).

Patent document 1: International publication 03Z057224 pamphlet

Non-Patent Document 1: "Pharmacology and Therapy", 1999, Vol. 27, No. 6, p. 1075— p. 1077 Non-Patent Document 2: Archives of Pharmacal Research, Korea, 2002 , 25 volumes, No. 1, p. 57— p. 60

Disclosure of the invention

Problems to be solved by the invention

[0004] However, the content of triterpenes in plants is not always high, but triterpenes are not always efficiently obtained. Furthermore, even when artificially cultivated, its growth requires at least several weeks or more, and the content of triterpene is easily affected by the weather and the soil of the cultivation area. was there.

[0005] In addition, triterpenes such as corosolic acid, tolmentic acid, 2α, 19α-dihydroxy-3-oxo-l-ul-12-en-28-oic acid and maslinic acid can be chemically synthesized at low cost. There is no known method.

[0006] Therefore, an object of the present invention is to provide at least one triterpene selected from the group consisting of corosolic acid, tolmentic acid, 2α, 19α-dihydroxy-3oxo-urose-12ene-28 euic acid and maslinic acid. Another object of the present invention is to provide a means for stably and efficiently supplying a triterpene composition containing the same.

Means for solving the problem

[0007] Callus culture is a technique for culturing calli induced by culturing parts of leaves, stems, roots, and the like cut out of a plant on a medium, and is used for basic research on biosynthesis of metabolites. May be

[0008] In the callus culture, the present inventors used banapa, vegetation, perilla, or guava as an original plant for inducing callus, and cultured the induced callus together with a specific plant hormone. The present inventors have found that specific triterpenes are produced with high efficiency in calli, and have completed the present invention.

[0009] That is, the method for producing a triterpene composition of the present invention is characterized in that a callus derived from banapa, vegetation, perilla or guano is cultured with indoleacetic acid and other plant hormones, This is a method for producing a triterpene composition containing at least one triterpene selected from the group consisting of corosolic acid, tolmentic acid, 2α, 19α-dihydroxy-3oxourose-12-en-28 euic acid and maslinic acid. .

[0010] In the method for producing a triterpene composition of the present invention, it is preferable to subculture callus. By subculturing the callus, a triterpene composition can be continuously obtained from the callus induced by Capana, such as banaba, without inducing another new virus.

[0011] Calli can also induce leaf, seed, stem, shoot apex, root or germ cell forces. By inducing such calligraphy, it becomes possible to obtain a triterpene composition efficiently in a shorter period of time.

[0012] The cultured callus contains maslinic acid, corosolic acid in an amount of 4 to 10 times the weight of maslinic acid, and tormentetic acid in an amount of 2 to 8 times the weight of maslinic acid. It is preferable to contain 1.5 to 4 times the amount of 2α, 19α-dihydroxy-3-oxo-1-urethane-12-28-ic acid! In particular, corosolic acid, which is highly useful in terms of blood sugar lowering action, is highly purified. It is important that the ratio of corosolic acid to maslinic acid is high, to make it easier to obtain at a high degree.

[0013] The present invention also provides a callus derived from banapa, vegetation, perilla, or guano and used for the above production method. The power of the present invention derived from vanapa, vegetation, perilla or guava is corosolic acid, tolmentic acid, 2α, 19α-dihydroxy-13-oxo-lour —12-en-28-oic acid And a large amount of triterpenes such as maslinic acid.

Therefore, it is obtained from the above production method and is selected from the group consisting of corosolic acid, tolmentic acid, 2α, 19α-dihydroxy-3-oxo-urose-12-en-28-oic acid and maslinic acid. A triterpene composition comprising at least one can be provided. This triterpen composition can be used as a raw material for health foods, medicines, cosmetics and the like.

[0015] The present invention further provides a triterpene of corosolic acid, tolmentic acid, 2a, 19α-dihydroxy-3-oxo-urus-12-en-28-oic acid or maslinic acid from this triterpene composition. Is provided by isolating and purifying the compound. By applying this production method, a high-purity triterpene that can be used for pharmaceuticals and the like can be obtained with high efficiency.

The invention's effect

According to the method for producing a triterpene composition of the present invention, corosolic acid, tolmentic acid, 2α, 19α-dihydroxy-3-oxo-urus-12-en-28-oic acid, and maslinic acid A triterpene composition containing at least one selected from the group consisting of carp can be stably supplied at a high efficiency.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments for carrying out the present invention will be described in detail.

In the method for producing a triterpene composition of the present invention, callus (undifferentiated cell mass derived from a plant cell) is induced from banapa, vegetation, perilla or guava.

[0019] Among them, it is preferable to induce banaba calli. Banaba (scientific name: Lager stroemia speciosa, Linn. Or Pers.) Is a plant belonging to the family Araceae, and is used as a street 、 and its leaves are used as a traditional herbal medicine in the Philippines.

[0020] Callus is induced by naturally growing power or artificially cultivated banana or vegetation. , Perilla or guava can be used. In order to efficiently obtain a triterpene composition in a short-term culture, it is more preferable to induce leaf, seed, stem, shoot apex, root or germ cell callus, and more preferably to induce leaf or seed force. In addition, calli may be induced from the prepared protoplasts with these site forces!

[0021] Callus induction is performed by, for example, sterilizing a site such as a leaf from which a force such as a banaba is cut out with sodium hypochlorite, cutting the portion to an appropriate size with a scalpel, and transplanting the force into a medium. Cultivation methods can be adopted.

[0022] The callus is preferably induced in a solid medium such as a Linsmeier-Skoog agar medium (hereinafter referred to as "LS agar medium") which can be induced in a solid medium or a liquid medium.

[0023] By culturing the induced callus in a medium supplemented with indoleacetic acid and other plant hormones and growing the same, a large amount of triterpene is produced in the callus. It is particularly important to use indoleacetic acid and other plant hormones in combination in cultivating calli derived from banapa, vegetation, perilla, or guava, and only plant hormones other than these are used. Compared to the callus, the efficiency of callus growth and triterpene production in the callus is significantly improved.

[0024] Examples of plant hormones other than indoleacetic acid used in combination with indoleacetic acid include potato-inetin, 1-naphthaleneacetic acid, 6-benzylazidenine, and the like, and potato-inetin is particularly preferred.

[0025] The callus is preferably cultured in a solid medium such as LS agar medium under aseptic conditions at 20-30 ° C in a dark place.

[0026] Cultivated callus power [0026] The triterpene composition can be obtained directly, or a part of the cultured callus is transplanted to another new solid medium or liquid medium and cultured again, that is, from the callus subjected to subculture. A triterpene composition can also be obtained. By repeating subculture, a triterpene composition can be stably obtained over a long period of time.

[0027] In the subculture, a part of the previously cultured callus may be used as it is, or a microplast may be prepared from a part of the cultured callus, and the microplast may be placed in a liquid medium. May be used for suspension culture. The conditions for subculture are as follows: Preferably, the conditions are the same as those described above. In addition, the culture period for one passage in the subculture is preferably 5 to 30 days. The subculture can be continued while renewing the generation as long as the production amount of the target triterpene in the callus is not significantly reduced.

[0028] Among the triterpenes produced in the callus cultured as described above, corosolic acid and tormentetic acid have great utility in terms of pharmacological activity and the like. Is preferably large. Specifically, maslinic acid, corosolic acid in an amount of 4 to 10 times the weight of maslinic acid, 2 to 6 times the amount of tolmentic acid, 1.5 to 4 times the amount of 2α, 19 α-dihydroxy-l-oxo-unoleth-l-l-en-28-oitic acid.

[0029] In particular, it is important that the ratio of corosolic acid to maslinic acid in callus is high in order to easily obtain corosolic acid having a large blood sugar level lowering action with high purity. Since corosolic acid and maslinic acid have very similar chemical structures, it is difficult to separate them.

[0030] As a method of quantifying the content of triterpene contained in the callus, a method of analyzing components extracted from the cultured callus using methanol or the like by liquid chromatography and quantifying from the area of the obtained chromatogram is used. Can be suitably adopted.

[0031] The extract obtained by extracting the cultured callus by using a known method is obtained by using corosolic acid, tonomenthetic acid, 2,19-dihydroxy-13-year-old quinone-unoleth-12-e. This is a triterpene composition containing a large amount of itic acid and maslinic acid. The triterpene composition can be used as a raw material for health foods or the like, or may be further purified to remove unnecessary components.

[0032] Extraction from the cultured callus may be performed using raw callus after the culture, or may be performed using a callus that has been dried and crushed with force!

As the extraction solvent, it is preferable to use a hydrophilic solvent such as water or an alcohol such as methanol or ethanol, and it is more preferable to use a heated water-alcohol mixed solvent. Specifically, a dry callus crushed material (raw material) is mixed with ethanol or an aqueous ethanol solution (ethanol content of 50 to 80% by weight) 5 to 20 times, preferably 8 to 10 times the weight of the raw material. Cook and heat at room temperature-90 ° C, preferably about 50-85 ° C for 30 minutes to 2 hours A method of refluxing and repeating this extraction 2-3 times can be suitably used.

[0034] From the triterpene composition obtained as described above, corosolic acid, tolmentic acid, 2α, 19α-dihydroxy-3-oxo-l-ul-12-en-28-ic acid or maslinic acid is isolated and purified. To obtain the respective triterpene. Isolation and purification of these triterpenes can be performed by methods such as silica gel column chromatography and recrystallization.

[0035] When the amount of the triterpene composition is large, the triterpene composition is suspended in water and distributed to ether or hexane to remove low-polar components, and then the aqueous layer is treated with diaion -20. It is preferable to sequentially elute with water, methanol and acetone using column chromatography or the like, and to separate and purify the methanol-eluted fraction by silica gel column chromatography or the like.

[0036] In addition, in order to increase the purity of the triterpene obtained by isolation and purification, column chromatography or recrystallization in which the hydroxyl group of the triterpene in the triterpene composition is acetylated or the carboxyl group is methyl esterified is performed. A method of obtaining the desired triterpene by carrying out purification by hydrolysis or the like, followed by hydrolysis, can also be suitably employed.

[0037] Corosolic acid, tolmentic acid, 2α, 19α-dihydroxy-3-oxourus-12-en-28 euic acid or maslinic acid obtained by isolation and purification as described above are At least 90.0% by weight or more,

More preferably, it contains 98.0% by weight or more, even more preferably 99.9% by weight or more.

[0038] The triterpene composition of the present invention is obtained by the above-described method, and is represented by corosolic acid represented by the following chemical formula (1), tolmentic acid represented by the following chemical formula (2), and represented by the following chemical formula (3) 2 OL, 19 A triterpene composition containing α-dihydroxy-3oxo-l-ul-12-28-ic acid and at least one maslinic acid represented by the following chemical formula (4). [Chemical 1]

Triterpene compositions containing these triterpenes are known to have pharmacological activities such as blood glucose level lowering activity and antitumor activity, respectively, and can be used as raw materials for health foods and medicines. In addition, among these triterpenes, the highly pharmacologically active corosori Triterpene compositions containing carboxylic acid and tolmentic acid are particularly useful. Example

Hereinafter, the present invention will be described in more detail with reference to Examples. The present invention is not limited to these Examples.

[0045] (Induction of callus)

Leaves cut from the banana were immersed in sodium hypochlorite for 15 minutes and sterilized. LS agar medium with ρ 酢酸 of 5.8 was prepared in an Erlenmeyer flask by adding 10 μΜ of indoleacetic acid and 10 μΜ of force inetine as plant hormones, and sterilized banana leaves were placed on the LS agar. The callus was induced by culturing in a dark place at ° C.

After culturing the induced calli for 30 days, a portion of the callus was transplanted to another LS agar medium and cultured, and it was confirmed that the transplanted callus proliferated again. That is, the subculture of the callus derived from Banano was able to be performed.

(Callus Culture and Triterpene Content in Callus)

The induced calli were cultured and grown on an LS agar medium supplemented with 10 M of indole acetic acid and 10 M of force inetine for 3 weeks, and the grown calli were extracted by cold immersion in methanol. The triterpene content of the cultured calli was determined from the area of the peak corresponding to each triterpene in the chromatogram (ultraviolet absorptiometer, detection wavelength: 220 nm) obtained by performing chromatographic analysis. In addition, the content of triterpene in the original plant (banapa leaf) before callus induction was determined by the same method. The results are shown in Table 1.

[Table 1]

Callus original plant

Triterpene

3 ri weight ratio 3 ri weight ratio

[mg / g dry wt] [mg / g dry wt]

Corosolin rooster g 10.7 6.7 1.1 .5 5.5 Tolmentic acid 6.0 3.8 Not detected

2 a, 1 9 Hi-hydroxy 3 _ 3.3 2.1 Not detected

Oxo-Urus 1 1 2—No 2

8-Oic acid

Ursolic acid 0.5 0.8 0.2 I .Q Oleanolic acid 0.4 0.3 0.1 0.5 Maslinic acid 1.6 1.0 0.2 1.0

[0048] The corosolic acid content in the calli increased about 10-fold relative to the original plant. In addition, it was found that tormentetic acid and 2a, 19α-dihydroxy-3-year-old xourous 12-28-ic acid, which were not detected in the original plant, were produced only by inducing callus.

[0049] On the other hand, LS agar medium to which only indole acetic acid or only force inetine was added as a plant hormone, or no plant hormone was added! / The force of trying to culture callus on LS agar medium Substantial callus growth was less vigorous.

Claims

The scope of the claims

[1] A callus derived from banapa, vegetation, perilla or guano is cultured with indoleacetic acid and other plant hormones, and from the cultured callus, corosolic acid, tormentetic acid, 2, 19 A method for producing a triterpene composition, which comprises obtaining a triterpene composition containing at least one selected from the group consisting of monodihydroxy-3-isoquinone-1-12-28-acetic acid and maslinic acid.

[2] The method for producing a triterpene composition according to claim 1, wherein the culture is a subculture of the callus.

[3] The triterpene composition according to claim 1 or 2, wherein the callus is a calla of a leaf, a seed, a stem, a shoot apex, a root, or an embryonic cell force of banapa, vegetation, perilla, or guava. Manufacturing method.

[4] The cultured callus is composed of maslinic acid, corosolic acid in a quantity of 4 to 10 times the weight of maslinic acid, 2 to 6 times the amount of tolmentic acid, 1.5 to 4 times the weight of the maslinic acid. 14. The method for producing a triterpene composition according to claim 13, wherein the composition further comprises 2α, 19α-dihydroxy-3-oxourous-12-28-oic acid.

[5] A method for producing a triterpene composition according to any one of claims 14 to 14, which is obtained by using corosolic acid, tolmentic acid, 2α, 19α-dihydroxy-3-oxo-l-urethane. 28 A triterpene composition comprising at least one selected from the group consisting of ic acid and maslinic acid.

[6] A callus derived from banapa, vegetation, perilla, or guano, for use in the method for producing a triterpene composition according to any one of claims 14 to 14.

[7] A callus derived from banapa, vegetation, perilla or guano is cultured with indoleacetic acid and other plant hormones, and from the cultured callus, a triterpene composition containing corosolic acid is obtained. A method for producing corosolic acid to obtain a triterpene composition cura corosolic acid.

[8] The method for producing corosolic acid according to claim 7, wherein the culture is subculture of callus.

[9] The production of corosolic acid according to claim 7 or 8, wherein the callus force is a callus induced by leaf, seed, stem, shoot apex, root or embryo cell force of banapa, vegetation, perilla or guava. Method.

[10] A callus derived from banapa, vegetation, perilla or guano is cultured with indoleacetic acid and other plant hormones, and a triterpene composition containing tormentetic acid is obtained from the cultured callus, A method for producing tolmentic acid, which obtains tolmentic acid from the triterpene composition.

[11] The method for producing tormentetic acid according to claim 10, wherein the culture is a subculture of callus.

[12] The tormentetic acid according to claim 10 or 11, wherein the callus is a callus induced by leaf, seed, stem, shoot apex, root or embryo cell of vanapa, vegetation, perilla or guava. Manufacturing method.

[13] A callus derived from banapa, vegetation, perilla or guano is cultured with indoleacetic acid and other plant hormones, and 2a, 19α-dihydroxy-3-oxo- A triterpene composition containing urus-12-en-28-oic acid is obtained, and from the triterpene composition, 2α, 19α-dihydroxy-3-oxourus-12-en-28-year-old acid is obtained. 19 A method for producing α-dihydroxy-l-3oxo-l-ul-l2-ene 28-oic acid.

14. The method according to claim 13, wherein the culture is a primary culture of callus.

—3-Oxo-Urus—Method for producing 12-en-28-oic acid.

15. The αα, 19 according to claim 13 or claim 14, wherein the callus power is a callus derived from leaves, seeds, stems, shoot tips, roots, or germ cells of banapa, vegetation, perilla, or guava. α-dihydroxy

—3-Oxo-Urus—Method for producing 12-en-28-oic acid.

[16] A callus derived from banapa, vegetation, perilla or guano is cultured with indole acetic acid and other plant hormones, and a triterpene composition containing maslinic acid is obtained from the cultured callus, A method for producing maslinic acid to obtain a triterpene composition masulinic acid.

17. The method for producing maslinic acid according to claim 16, wherein the culture is a subculture of callus.

[18] The production of maslinic acid according to claim 16 or 17, wherein the callus force is a callus induced by leaf, seed, stem, shoot apex, root, or embryo cell force of banapa, vegetation, perilla or guava. Method.