KR102236425B1 - Method for producing callus mass-producing Tiarellic acid from Tiarella polyphylla and callus produced by the same method - Google Patents

Abstract

The present invention relates to a method for producing a callus for mass production of tiarelic acid derived from a panting plant and to a callus for mass production of tiarelic acid produced by the method, which is contained in a small amount in the root of the panting plant of the present invention. Tiarelic acid is an excellent natural new drug candidate that has proven its excellent asthma effect, inhibition of LTC4 production, inhibition of IgE, IL-4, -5, -13 production in asthmatic animals, and improvement of airway hypersensitivity. Since it is possible to supply tiarelic acid without depletion of plant resources and environmental destruction through infinite growth and production through cultivation of whole, plant tissue or callus-derived plant cells, it is very useful for related industries.

Description

[Method for producing callus mass-producing Tiarellic acid from Tiarella polyphylla and callus produced by the same method}

The present invention relates to a method for producing a callus for mass production of tiarelic acid derived from a panting plant and to a callus prepared by the method.

Tiarella polyphylla ) is a plant of the genus Saxifragaceae, a perennial herb distributed in Korea, China, and Japan, and is the only plant native to Ulleungdo in Korea. In the Ulleungdo area, the extract of pantweed was called "asthma herb" and was used for coughing and asthma, but it is not a widely known plant due to its limited distribution of habitat. As a study on the components and activity of panting grass, oleanolic acid glycosides showing anti-complement activity have been isolated and reported (Park SH et al., Arch. Pharm. Res., 22(4), pp428-431, 1999), components such as tiarelic acid, corosolic acid, and tormentic acid have been isolated as triterpenoid compounds of the lupan skeleton (Park SH et al., Arch.Pharm.Res., 25(1), pp57-60, 2002), MMP-1 expression induced by ultraviolet irradiation in tissue cultured fibroblasts and inhibitory activity of type 1 procollagen (Moon HI et al. , J. Ethnopharmacol., 98, pp185-189, 2005) have been reported.

In many cases, securing a large amount of materials is the most important key in the process of discovering excellent natural active materials of plants and transferring them to commercialization. For example, woody plants with slow growth, specific organs of woody plants with limited sample acquisition (e.g. flowers or roots), herbaceous plants with a small body (biomass), materials that are difficult to secure or germinate, and whose active ingredients are different during cultivation. In the case, finally, when it is difficult to secure a large amount of material, such as when the activity is very excellent, but the body content is very small, a fundamental method to solve this problem is needed. As a fundamental solution to this, plant tissue and cell culture technology is drawing attention as the only method. Mass production of natural active materials based on plant tissue and cell culture does not directly collect and utilize plant resources living in nature, but infinite proliferation through cultivation of whole plants, plant tissues (negative roots, hairy roots) or callus-derived plant cells in a bioreactor. Because it is produced, it can be free from depletion of plant resources and environmental destruction, and can be used as a more active means of preserving the natural environment.

On the other hand, Korean Patent No. 1163215 discloses'a composition for the prevention and treatment of cancer diseases containing an extract or a triterpene compound isolated therefrom as an active ingredient', and Korean Patent No. 0693400 discloses' Although a method for producing corosolic acid by plant cell suspension culture is disclosed, as in the present invention,'a method for producing a callus for mass production of tiarelic acid derived from a panting plant and a callus produced by the method' Nothing has been revealed about.

The present invention was derived from the above requirements, and the present invention is to mass-produce tiarelic acid, which is a natural component mainly contained in the root of the panting plant, by the plant cell culture method. To induce excellent callus cells from the leaves, stems, and roots of plants grown by germinating seeds in aseptic phase, callus of white or pale yellow using a solid medium for cultivation of callus callus cells containing auxin and cytokinin in an appropriate concentration After passing through the subculture process to induce and homogenize the cells, the callus showing the highest content was finally selected by performing the tiarelic acid analysis, and the callus was continuously maintained and cultured without degeneration, resulting in mass production of tiarelic acid. By confirming the points that can be used usefully, the present invention was completed.

In order to achieve the object of the present invention, the present invention

(a) Tiarella polyphylla ) germinating in a germination medium after sterilizing the seeds of the plant to induce stems, leaves and roots;

(b) inducing and proliferating callus derived from stem, leaf and root by culturing the induced stem, leaf and root fragments in a callus induction medium added with a growth regulator;

(c) subculturing the proliferated stem, leaf, and root-derived callus in a callus growth medium to proliferate each; And

(d) Tiarelic derived from panting plants comprising the step of selecting callus having excellent tiarelic acid productivity by measuring the content of tiarelic acid for calli derived from stems, leaves and roots grown by subculture. It provides a method of manufacturing callus for mass production of Tiarellic acid.

In addition, the present invention is prepared by the method described above ( Tiarella polyphylla ) It provides callus for mass production of tiarelic acid derived from plants.

In addition, the present invention is gasping grass ( Tiarella polyphylla ) It provides callus (KCTC 18650P) for mass production of tiarelic acid derived from plants.

In addition, the present invention is gasping grass ( Tiarella polyphylla ) provides a method for producing tiarelic acid comprising the step of producing tiarelic acid by culturing callus for mass production of tiarelic acid derived from a plant.

In addition, the present invention contains TDZ (Tidiazuron) and picloram (picloram) MS basal medium added as an active ingredient, as an active ingredient ( Tiarella polyphylla ) provides a medium composition for induction and propagation of callus for mass production of tiarelic acid derived from plants.

Tiarelic acid, which is contained in a small amount in the roots of the panting grass of the present invention, has excellent asthma effect, suppresses LTC4 production, inhibits IgE, IL-4, -5, -13 production in asthmatic animals, and has proven effective in improving airway hypersensitivity. As a candidate for a new drug, the present invention enables the supply of tiarelic acid without depletion of plant resources and environmental destruction through infinite growth and production through cultivation of whole plants, plant tissues or callus-derived plant cells in a bioreactor. useful.

Figure 1 shows the chemical structure of tiarelic acid (Tiarellic acid, 3,23-dihydroxy-20(29)-lupen-27-oic acid).
Figure 2 is a pant ( Tiarella polyphylla D) In vitro germination and growth of seeds are shown.
3 shows a process of inducing callus in a callus-inducing medium to which TDZ (Tidiazuron) and picloram for each concentration were added. The composition of the medium is as follows; TP1 (TDZ 0.1 mg/L + Picloram 0.2 mg/L), TP2 (TDZ 0.5 mg/L + Picloram 0.2 mg/L), TP3 (TDZ 1.0 mg/L + Picloram 0.2 mg/L) , TP4 (TDZ 0.1 mg/L + Picloram 1.0 mg/L), TP5 (TDZ 0.5 mg/L + Picloram 1.0 mg/L), TP6 (TDZ 1.0 mg/L + Picloram 1.0 mg/L ), TP7 (TDZ 0.1 mg/L + Picloram 2.0 mg/L), TP8 (TDZ 0.5 mg/L + Picloram 2.0 mg/L), and TP9 (TDZ 1.0 mg/L + Picloram 2.0 mg /L)
4 shows the results of induction of callus in a callus induction medium to which TDZ (Tidiazuron) and picloram are added.
5 shows the HPLC analysis peaks of tiarelic acid standard.

In order to achieve the object of the present invention, the present invention

(a) Tiarella polyphylla D.) sterilizing the seeds of the plant and then germinating in a germination medium to induce stems, leaves and roots;

(b) inducing and proliferating callus derived from stem, leaf and root by culturing the induced stem, leaf and root fragments in a callus induction medium added with a growth regulator;

(c) subculturing the proliferated stem, leaf, and root-derived callus in a callus growth medium to proliferate each; And

(d) Tiarelic derived from panting plants comprising the step of selecting callus having excellent tiarelic acid productivity by measuring the content of tiarelic acid for calli derived from stems, leaves and roots grown by subculture. Provides a method of manufacturing callus for mass production of Tiarellic acid,

Preferably, the method according to an embodiment of the present invention

(a) Tiarella polyphylla D.) sterilizing the seeds of the plant and germinating in MS medium containing 0.5-1.5 mg/l gibberellin to induce stems, leaves and roots;

(b) The induced stem, leaf and root fragments were cultured in MS medium supplemented with 0.05 to 0.15 mg/l TDZ and 0.15 to 0.25 mg/l picloram to induce callus derived from stem, leaf and root, respectively. And proliferating;

(c) subculturing the proliferated stem, leaf, and root-derived callus in a callus growth medium to proliferate each; And

(d) Tiarelic derived from panting plants comprising the step of selecting callus having excellent tiarelic acid productivity by measuring the content of tiarelic acid for calli derived from stems, leaves and roots grown by subculture. To provide a method of manufacturing callus for mass production of Tiarellic acid, but is not limited thereto.

In the method according to an embodiment of the present invention, the germination medium in step (a) is MS medium containing 0.5 to 1.5 mg/l gibberellin, preferably MS medium containing 1 mg/l gibberellin, , Is not limited thereto.

In the method according to an embodiment of the present invention, the induction medium in step (b) may be MS medium to which 0.5 to 1.5 mg/l TDZ (Tidiazuron) and 0.15 to 0.25 mg/l picloram are added. And, preferably, 1 mg/l TDZ and 0.2 mg/l picloram, but are not limited thereto.

In the method according to an embodiment of the present invention, the callus induction medium in step (b) may be an MS medium to which TDZ (Tidiazuron) and picloram are added, and the added TDZ and picloram content is As described above.

In the method according to an embodiment of the present invention, the callus growth medium of step (c) may be an MS medium to which Tidiazuron (TDZ) and picloram are added, but is not limited thereto.

In the method according to an embodiment of the present invention, the callus having excellent tiarelic acid productivity in step (d) may be derived from leaves, roots, or stems, and preferably may be derived from leaves, but is not limited thereto.

The present invention induces excellent callus cells from the leaves, stems, and roots of the plant by germinating the plant seeds in aseptic in order to mass-produce tiarelic acid, which is a natural component mainly contained in the roots of the panting plant. To induce white or pale yellow callus cells using a solid medium for cultivating panting callus cells containing auxin and cytokinin in an appropriate concentration, go through a subculture process to induce a homogenized state, and then tiarelic acid. Analysis was performed to confirm that the callus showing the highest content was finally selected, and that the callus was continuously maintained and cultured without deterioration.

In addition, the present invention is prepared by the method described above ( Tiarella polyphylla D.) Provides callus for mass production of tiarelic acid derived from plants.

The callus according to an embodiment of the present invention has a deposit number of KCTC 18650P, and was deposited with the Korea Research Institute of Bioscience and Biotechnology on January 08, 2018.

In addition, the present invention is the gasping grass ( Tiarella polyphylla D.) It provides callus (KCTC 18650P) for mass production of tiarelic acid derived from plants.

In addition, the present invention is the gasping grass ( Tiarella polyphylla D.) Provides a method for producing tiarelic acid comprising the step of culturing callus for mass production of tiarelic acid derived from a plant to produce tiarelic acid. The method of culturing the callus for mass production of tiarelic acid selected through the present invention is a method of culturing callus generally used in the art, and is not limited to a specific method.

In addition, the present invention contains TDZ (Tidiazuron) and picloram (picloram) MS basal medium added as an active ingredient, as an active ingredient ( Tiarella polyphylla D.) It provides a medium composition for induction and propagation of callus for mass production of tiarelic acid derived from plants. The medium composition for inducing and proliferating callus for mass production of tiarelic acid derived from the panting plant of the present invention contains 0.1 mg/l TDZ and 0.2 mg/l picloram-added MS base medium as an active ingredient, It is effective in inducing and proliferating callus derived from panting plants that mass-produce tiarelic acid.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Example 1.Puffing ( Tiarella polyphylla D.) Seed germination and in-flight culture

The panting seeds were surface sterilized with 70% ethanol and washed once with sterile water. The washed seeds were sterilized with 10% sodium hypochloride, washed three times with sterile water, and dried on sterile paper. The dried seeds induce germination in a germination medium in which 1 mg/L of GA (gibberellin) was added to MS basal medium (MS salt, 80-120 mg/L myo-inositol, vitamin + 3% sucrose + 0.8% agar). Culture conditions: 25 ℃, long-day photoperiod (16 h ^{light / 8 h dark, 45μmol photons m -2} s ^-1 white light). About 2 months after inducing germination, it developed into a complete plant, and 6 months later, it grew to the required level of subculture. Due to the nature of the plant, it shows very slow growth, which is similar to the results in general soil.

Example 2. From in-flight cultured plants Callus Judo

The leaves, stems and roots of the in-flight cultured plants obtained as a result of the previous experiment were sufficiently secured. The obtained plant was combined with each concentration of TDZ (Tidiazuron), a cytokinin, and picloram, auxin, in MS basal medium and MS basal medium, and sectioned each of 30 leaves, stems, and roots. Cut the teeth and induce callus in a culture chamber at 25° C. in a dark state (FIG. 3). The composition of the medium is as follows; TP1 (TDZ 0.1 mg/L + Picloram 0.2 mg/L), TP2 (TDZ 0.5 mg/L + Picloram 0.2 mg/L), TP3 (TDZ 1.0 mg/L + Picloram 0.2 mg/L) , TP4 (TDZ 0.1 mg/L + Picloram 1.0 mg/L), TP5 (TDZ 0.5 mg/L + Picloram 1.0 mg/L), TP6 (TDZ 1.0 mg/L + Picloram 1.0 mg/L ), TP7 (TDZ 0.1 mg/L + Picloram 2.0 mg/L), TP8 (TDZ 0.5 mg/L + Picloram 2.0 mg/L) and TP9 (TDZ 1.0 mg/L + Picloram 2.0 mg/ L)

Callus began to be generated about 8 weeks after the leaf tissue was implanted. As the callus was browned, a new callus was generated on some surfaces. Callus was continuously induced and proliferated by subculture in the same medium every 4 weeks. The induction rate of each medium was investigated based on a new callus with a yellow color and relatively fast proliferation. It can be determined that the proliferated callus is homogenized because the callus was induced again in a part after browning in the first induced callus. The results are shown in FIG. 4.

As a result of the analysis, as a result of the induction of panting callus, the callus induced from the leaves showed a pattern of inducing browning as a whole after the initial induction, and then inducing the callus again.The initial induction rate was high in the overall medium combination, but the callus was browned. Afterwards, the induction rate in the process of forming a new callus differed by medium composition, and the best induction medium combination was TDZ 1.0 mg/L + Picloram 0.2 mg/L, followed by TDZ 1.0 mg/L. The induction rate was high in the order of + picloram 2.0 mg/L, TDZ 0.1 mg/L + picloram 2.0 mg/L. The homogenization and fixation of the panting callus was unexpectedly simple, but in the process of taking a newly differentiated callus from the dead callus, it also had the effect of paralleling the homogenization process naturally.

Example 3. On HPLC by Tiarelic acid analysis

In the callus proliferation process, the final callus line for each tissue was selected for HPLC analysis in consideration of whether the callus was proliferated, condition, growth rate, and shape. HPLC analysis is as follows. Extraction method; A solvent (methanol) was added at a concentration of 1.25 mg/ml in the dry sample weight of each sample, and extracted for 20 minutes with an ultrasonic extractor at room temperature, and the extracts were combined by repeating a total of 3 times. The concentration of the extraction sample for analysis was concentrated by combining the methanol extract, and then alcohol was added to adjust the concentration to 20 mg/ml, and 2 to 10 ul each were injected during the analysis. UHPLC, Waters Acquity UPLC; Column, Acquity UPLC BEH C18 1.7um 2.1*100mm; Solvent, acetonitrile (CAN) (0.1% formic acid) + H ₂ O (0.1% FA); Flow rate, 0.4 ml/min; Detection, 210 nm; Injection volume, 2 µl of standard material, 10 µl of sample.

As a result of the analysis, the content of tiarelic acid obtained from the roots of pantweed grown in the growth chamber was 67.9 ㎍/100 mg, whereas the content of tiarelic acid obtained from callus obtained through treatment with different plant growth regulators in this experiment was about 4 It is a double increase, and specifically, TP1: 272 µg/100mg, TP6: 255 µg/100mg, and TP7: 169 µg/100mg were shown (Table 1).

Through this experiment, three types of callus were obtained, including callus with a content that was 4 times higher than that of the original plant. Interestingly, the treatment with the highest content was TDZ 0.1 + pickle, not the generally predictable root-derived callus. It is a leaf-derived callus derived under the condition of 0.2 mg/l Loram. Therefore, the leaf-derived callus TP1 was deposited with the Korea Research Institute of Bioscience and Biotechnology on January 08, 2018, as the deposit number is KCTC 18650P.

Korea Research Institute of Bioscience and Biotechnology KCTC18650P 20180108

Claims (8)

comprising the steps of: (a) by gasping a germinated in germination medium was sterilized and then the seeds of the plant pool (Tiarella polyphylla), induction of stem, leaf and root;
(b) Stem, leaves and roots were cultured in MS medium supplemented with 0.05 to 0.15 mg/l TDZ (Tidiazuron) and 0.15 to 0.25 mg/l picloram. Inducing and proliferating the derived callus, respectively;
(c) subculturing the proliferated stem, leaf, and root-derived callus in a callus growth medium to proliferate each; And
(d) Tiarelic derived from panting plants comprising the step of selecting callus having excellent tiarelic acid productivity by measuring the content of tiarelic acid for calli derived from stems, leaves and roots grown by subculture. A method for producing callus for mass production of Tiarellic acid. delete The method of claim 1, wherein the callus growth medium in step (c) is an MS medium to which 0.05 to 0.15 mg/l TDZ (Tidiazuron) and 0.15 to 0.25 mg/l picloram are added. . The method of claim 1, wherein the callus having excellent tiarelic acid productivity in step (d) is derived from leaves. A callus for mass production of tiaric acid derived from a plant of Tiarella polyphylla prepared by the method of any one of claims 1, 3 and 4. Callus for mass production of tiaric acid derived from Tiarella polyphylla plant whose deposit number is KCTC 18650P. delete Mass production of Tiarella polyphylla plant-derived tiarelic acid containing MS basic medium supplemented with 0.05~0.15mg/l TDZ (Tidiazuron) and 0.15~0.25mg/l picloram as an active ingredient For callus induction and growth medium composition.

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