KR102140050B1 - Production method of Lycoris radiata adventitious roots for producing galantamine - Google Patents

Abstract

The present invention relates to a method for producing Lycoris radiata adventitious roots for the production of galantamine. By using the production method of Lycoris radiata adventitious roots according to the present invention, Lycoris radiata adventitious roots can be mass-produced in vitro year round, and thus galantamine, a useful ingredient used as a raw material for dementia treatment, can be continuously produced. Therefore, the present invention may be advantageously utilized in related industrial fields.

Description

Production method of Lycoris radiata adventitious roots for producing galantamine}

The present invention relates to a method of producing oxalic acid roots for the production of galantamine.

Galantamine (galantamine) is an alkaloid-derived herbal ingredient extracted from the plant Amaryllidaceae, and has been used for a long time as a folk medicine to treat various symptoms of muscle disease or central nervous system, mainly in Eastern Europe. Since then, it has been proven effective in symptoms of cognitive impairment associated with dementia and is mass-produced and sold as a drug for treating dementia.

Narcissus plants that also produce galantamine in Korea, Seoksan (flowers, Lycoris radiata ), Lycoris squamigera MAX., Lycoris chinensis Traub., Lycoris koreana NAKAI., Narcissus tazetta var. chinensi ), but there are no studies on the production of galantamine using these plants. Accordingly, it was judged that various studies for the production of raw materials would be needed to develop a treatment for dementia using native plants that produce galantamine.

The adventitious root is a root originating from a stem or leaf, and the root originating from a seed is generally called a root, and in a relative sense, it is called a root root. It has been reported that the research method using the in-flight induction method is a suitable research method for natural product research due to the high rate of proliferation of the adventitious root and the production rate of secondary metabolites. In addition, it has been reported that in recent years, as interest in natural medicines increases, mass production of useful secondary metabolites is required industrially, and in order to produce products of a certain quantity and quality, induction of arrhythmias is reported to be the most suitable method with cell culture.

In the present invention, the production of galantamine through in-flight culture was attempted by inducing the arrhythmia from the callus of Seoksan, a native Korean daffodil plant.

On the other hand, Korean Patent Publication No. 2016-0021371 discloses'a composition for external application for skin having an anti-inflammatory and anti-aging effect containing a narcissus plant cell culture extract and a manufacturing method thereof', and Korean Patent No. 0786371 discloses'galantamine Manufacturing method of', but has not been described with respect to the production method of oxalic acid root for the production of galantamine of the present invention.

The present invention was derived by the above-mentioned needs, and the present inventor synthesizes callus derived from the bulbs of oxalic acid (flowers), 2,4-D, dicamba, picloram or queen of synthetic auxins. As a result of investigating the induction of adventitious roots by culturing in a medium containing various concentrations of chlorak (Quinclorac), the induction rate of oxalic adventitious roots and the average number and elongation of adventitious adventitious muscles in the Dicamba 0.1 mg/L treatment group were different from the untreated control It was confirmed that it was higher than the synthetic auxin treatment. In addition, as a result of analyzing the growth and galantamine production of oxalic acid roots in various types of media containing Dicamba, IAA, IBA, and NAA, the growth and galantamine production of oxalic acid roots were superior to other types of media in MS media. By confirming, the present invention was completed.

In order to solve the above problems, the present invention comprises the step of culturing Calcium ( Lycoris radiata ) callus in a medium containing Dicamba (Dicamba: 3,6-dichloro-2-methoxybenzoic acid) Gives

In addition, the present invention provides seoksan irregular roots produced by the above production method.

In addition, the present invention is the step of inoculating and growing the oxalic acid roots in a liquid medium containing Dicamba, IAA (Indole-3-acetic acid), IBA (indole-3-butyric acid) and NAA (1-naphthaleneacetic acid). It provides a method for producing galantamine (galantamine) from seoksan malteun, including.

The present invention has established the basis for the mass production system of oxalic acid roots by identifying in-flight culture conditions from induction of oxalic acid roots. In addition, by using the method for producing oxalic malnutrition according to the present invention, since it is possible to mass-produce oxalic malignant in-flight throughout the year, it is possible to continuously produce galantamine, a useful ingredient used as a raw material for the treatment of dementia, which is useful in related industries. It could be.

In order to achieve the object of the present invention, the present invention comprises the step of culturing lactic acid ( Lycoris radiata ) callus in a medium containing Dicamba (Dicamba: 3,6-dichloro-2-methoxybenzoic acid), Provide a production method.

In the method for producing oxalic acid root according to an embodiment of the present invention, the dicamba may be preferably contained in the medium at a concentration of 0.01 to 0.4 mg/L, and more preferably 0.05 to 0.15 mg/L. It may be contained in the medium at a concentration, and more preferably, may be contained in the medium at a concentration of 0.1 mg/L, but is not limited thereto.

In addition, the method of producing the oxalic acid roots of the present invention specifically,

(a) 1.5 to 2.5 mg/L of 2,4-D (2,4-Dichlorophenoxyacetic acid) and 0.3 to 0.7 mg/L of TDZ (Thidiazuron: 1-phenyl-) of the bulbs of Lycoris radiata Inducing callus by culturing in a medium containing 3-(1,2,3-thiadiazol-5-yl) urea); And (b) inducing the negative root by incubating the callus of the induced oxalate in a medium containing 0.01-0.4 mg/L of Dicamba; and more specifically,

(a) inducing callus by incubating the bulbs of oxalic acid in a solid medium containing 1.5 to 2.5 mg/L of 2,4-D and 0.3 to 0.7 mg/L of TDZ for 4 to 8 weeks; And (b) inducing malignant muscle by incubating the callus of the induced oxalate in a solid medium containing 0.01 to 0.4 mg/L of Dicamba for 4 to 8 weeks by cancer culture, and most specifically,

(a) inducing callus by culturing the bulbs of sterile oxalic acid at a temperature of 25° C. for 6 weeks in a solid medium containing 2.0 mg/L 2,4-D and 0.5 mg/L TDZ; And (b) inducing malignant muscle by incubating the callus of the induced oxalic acid at a temperature of 25° C. for 6 weeks in a solid medium containing 0.1 mg/L of Dicamba; but it is not limited thereto.

In addition, but not limited to, the method for producing the oxalate arrhythmia is selected from the group consisting of Indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA) in addition to Dicamba. It may further include one or more auxins, specifically, the medium of step (b) in addition to 0.01 to 0.4 mg/L of Dicamba in addition to 4.5 to 5.5 mg/L of IAA or 2.5 to 3.5 mg/L of IBA It may include, preferably, the medium of step (b) may include 5 mg/L of IAA or 3 mg/L of IBA in addition to 0.1 mg/L of Dicamba, but is not limited thereto. .

The present invention also provides oxalic acid roots produced by the above production method.

The present invention also contains dicamba, indole-3-acetic acid (IAA), indole-3-butyric acid (IAB) and 1-naphthaleneacetic acid (NAA) in the oxalic acid roots produced by the production method of the present invention. It provides a method of producing galantamine (galantamine) from seoksan malignant root, comprising the step of inoculating and growing in a liquid medium.

In the production method of galatamin according to an embodiment of the present invention, the Dicamba, IAA, IBA and NAA are respectively 0.01 to 0.4 mg/L, 4.0 to 6.0 mg/L, 2 to 4 mg/L and It may be contained in the medium at a concentration of 0.01 to 1 mg/L, more preferably, Dicamba, IAA, IBA and NAA in order of 0.05 to 0.15 mg/L, 4.5 to 5.5 mg/L, and 2.5 to 3.5, respectively. It may be contained in a concentration of mg/L and 0.05 to 0.15 mg/L, and more preferably, Dicamba, IAA, IBA, and NAA are each 0.1 mg/L, 5 mg/L, 3 mg/L in order. And 0.1 mg/L, but is not limited thereto.

In addition, in the method for producing galatamin of the present invention, the liquid medium inoculating the oxalic acid root may be preferably MS medium or SH medium, but is not limited thereto. The lactic acid roots proliferated in the MS medium and SH medium are superior to the lactic acid roots grown in other types of media (eg, 1/2 MS, 1/2 B5, B5, 1/2 SH, etc.), and the production of galantamine is superior. It is characteristic.

The method for producing galatamin from oxalate root according to the present invention may further include a process of separating and purifying the useful substance galatamin from the proliferated oxalate root, the separation/purification method being known in the art. Technology is available.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate the present invention, the content of the present invention is not limited to the following examples.

Example 1. Seoksan callus induction

After immersing the bulbs of Lycoris radiata in 70% (v/v) ethanol solution for 1 minute, 0.1 ml of Tween 20 was added to the 4.5% (v/v) sodium hypochlorite solution, followed by surface sterilization for 10 minutes. . After washing three times in sterile water, 2,4-D (2,4-Dichlorophenoxyacetic acid) 2 mg/L and TDZ (Thidiazuron: 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea) Seven bulbous fission tissues were placed on MS (Murashige & Skoog, 1962) solid medium treated with 0.5 mg/L. Thereafter, the cells were cultured in a dark state at 25°C for 6 weeks to induce callus.

Example 2. Induction of Seoksan irregular root

In order to induce arrhythmia in the oxalic acid callus induced by the method of Example 1, 2,4-D, Dicamba, 3,6-dichloro-2-methoxybenzoic acid, Picloram, 4-Amino- MS treated with 0, 0.1, 0.5, 1.0, 2.0 and 4.0 mg/L of 3,5,6-trichloro-2-pyridinecarboxylic acid and Quinclorac (3,7-Dichloro-8-quinolinecarboxylic acid), respectively. After incubation for 6 weeks in a cancerous state at 25°C in a solid medium, induction of adventitious roots was investigated (Table 1).

As a result, the decamba 0.1 mg/L treatment group showed the highest induction rate of 77% of adventitious roots, an average number of induced adventitious roots of 3.9, and an average of 1.33 cm of induced adventitious roots compared to the untreated control group and other synthetic auxin treatments. .

Dicamba 0.1 mg/L treated with three types of auxin IAA (Indole-3-acetic acid), IBA (indole-3-butyric acid) and NAA (1-naphthaleneacetic acid) 0.1, 0.5, 1.0, 3.0, respectively. As a result of investigating the effect on induction of adventitious muscle after incubating callus in a cancer state at 25°C for 6 weeks in MS solid medium treated with 5.0 mg/L, the adventitious induction rate was Dicamba 0.1 mg/L and NAA 3.0 or 5.0 mg Except in the case of mixed /L treatment, the control group Dicamba 0.1 mg/L was higher overall than the treatment alone (Table 2), and the adventitious root induction was 0.1 mg/L Dicamba and 3 mg/L of IBA. In the case of mixed treatment, it was the highest with an average of 5.4, and the root length extension was highest at 1.8 cm in the case of dicamba 0.1 mg/L and IAA 5 mg/L.

Example 3. Selection of medium for culture of arrhythmia

The oxalic acid roots induced by treatment with Dicamba 0.1 mg/L from the oxalate callus were 1/2 MS mixed with Dicamba 0.1 mg/L, IAA 5 mg/L, IBA 3 mg/L and NAA 0.1 mg/L, MS, 1/2 B5 (Gamborg et al., 1968), B5, 1/2 SH (Schenk and Hildebrandt, 1972) or SH Ergonomic flask (100 mL) containing 30 ml of body weight in 4 g of body weight in the dark state Incubated for 4 weeks.

Effect of Medium on Growth of Seoksan Unregulated Roots Badge type In the building of irregular root (g/ 30 ml) MS 0.83 ± 0.04 a 1/2 ms 0.76 ± 0.02 b B5 0.72 ± 0.03 b 1/2 B5 0.68 ± 0.01 c SH 0.73 ± 0.01 b 1/2 SH 0.75 ± 0.02 b

As a result, as shown in Table 4, the growth rate of oxalate roots was highest in the MS medium, 0.83 g/30 mL in the building. And the production of galantamine from the roots cultured in each medium was investigated by HPLC (high-performance liquid chromatography) analysis. For HPLC analysis, add 3 ml of 1% H ₂ SO ₄ (water/H ₂ SO ₄ , 99:1 v/v) to a 15 ml conical tube containing 0.1 g of oxalic acid root sample and vortex for 2 minutes. After standing at room temperature (28°C) for 24 hours, it was centrifuged for 12.000 rpm, 4°C, and 10 minutes. After filtering the centrifuged crude extract with a 0.45 μm, PTFE hydrophilic syringe filter (13 mm in diameter), it was put into a brown vial for HPLC and HPLC was performed under the conditions shown in Table 5 below. The galantamine standard sample (Sigma Aldrich Korea (U)) was dissolved in tertiary distilled water, diluted to 4 concentrations (0.125, 0.0625, 0.0312, and 0.01562 mg/ml), and area values were obtained through HPLC analysis. To obtain a calibration curve. The equation for the calibration curve was y = 505.77x-2.3471 (R ² = 0.9945).

HPLC analysis conditions HPLC equipment Futecs Co. Column OptimaPak C18-51002546
(250 mm x 4.6 mm Id, particle size 5 μm) Detector 285 nm Column temperture 30 ℃ Mobile phase Solvent A (50 mM ammonium formate)
Solvent B (acetonitrile) Fow rate 1.3 ml/min Injection volume 20 μl Time 32 min
Gradient conditions 0 ~ 17 min solvent B 1.5% 17 ~ 32 min solvent B 65%

As a result of HPLC analysis, lactic acid roots cultured in MS medium showed an average production of galantamine of 0.0204 mg/g Dry Weight, and lactic acid roots cultured in SH medium averaged 0.0221 mg/g Dry Weight, and lactic acid cultured in other types of media It was confirmed that galantamine production was higher than the root muscle (Table 6). The medium suitable for the production of galantamine using oxalic acid roots was considered as the MS medium in consideration of the amount of galantamine production in the building.

Claims (7)

(a) 1.5 to 2.5 mg/L of 2,4-D (2,4-Dichlorophenoxyacetic acid) and 0.3 to 0.7 mg/L of TDZ (Thidiazuron: 1-phenyl-) of the bulbs of Lycoris radiata Inducing callus by culturing in a medium containing 3-(1,2,3-thiadiazol-5-yl) urea); And
(b) inducing the negative root by culturing the callus of the induced oxalic acid in a medium containing 0.01 to 0.4 mg/L of Dicamba (Dicamba: 3,6-dichloro-2-methoxybenzoic acid); Characterized by a method of producing Seoksan irregular roots. delete The method of claim 1, wherein the medium of step (b) is characterized in that it comprises an IAA of 4.5 to 5.5 mg/L or an IBA of 2.5 to 3.5 mg/L in addition to 0.01 to 0.4 mg/L of Dicamba. Method of producing Seoksan irregular root. delete Liquid medium containing dicamba, indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA) for oxalic acid roots produced by the production method of claim 1 or 3. As a method of producing galantamine (galantamine) from seoksan malignant root, comprising the step of inoculating and multiplying,
The Dicamba, IAA, IBA and NAA are contained in the liquid medium at a concentration of 0.01 to 0.4 mg/L, 4 to 6 mg/L, 2 to 4 mg/L and 0.01 to 1 mg/L in order, respectively. How to do. delete [6] The method of claim 5, wherein the liquid medium is MS medium or SH medium.