KR101496790B1 - Method for increasing growth and bioactive compound content of Eurycoma sp. adventitious root - Google Patents

Abstract

The present invention relates to a method for the treatment of Eurycoma sp.) inoculating a plant leaf, stem or root with MS medium containing auxin to induce adventitious roots and then cultivating the induced adventitious roots. The present invention relates to a method for cultivating a plant of the genus Leucillus of the genus Leucidae, which is produced by the method of the present invention and the method of the present invention, wherein the growth of the plant is promoted and the content of the physiologically active substance is increased. ( Eurycoma longifolia ) adventitious root can be effectively mass-produced, and the glassy coral rhizobia obtained by the culturing method of the present invention ( Eurycoma longifolia ) The biomass and physiologically active substance of the adventitious root can be used industrially because they can be used for the production of cosmetics, foods, spices, beverages and nourishing tonics.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for increasing the content of physiologically active substances in a plant, adventitious root}

The present invention relates to a method of increasing the growth and the physiologically active substance content of the glass coma adventitious roots in plants, and more particularly coma in glass (Eurycoma sp.) inoculating a plant leaf, stem or root with MS medium containing auxin to induce adventitious roots and then cultivating the induced adventitious roots. And an increased amount of growth and physiologically active substance produced by the above method.

Eurycoma longifolia is a flowering plant of Simaroubaceae, also known as Tongkat Ali, and is a major producer of Indonesia, Malaysia and Vietnam. It is a kind of tall, slender shrub found mainly in sloping and deep jungles of Southeast Asia such as Malaysia, and is a wild plant in the rainforest of a million-year history. The roots of the vitamins are used as traditional remedies for fever and malaria. The pharmacological tests of various ingredients extracted from the vitamins have shown that beneficial ingredients for malaria prevention and treatment, growth hormone formation and anti-ulcer Respectively. It is also known to be effective in the treatment of diabetes, treatment of tuberculosis, treatment of hypertension and low blood pressure, and is also effective in increasing testosterone levels, restoring fatigue, increasing muscle mass and strengthening muscles (Rajeev et al., 2010, Fitoterapia 81, 669-679 ).

Plant tissue culture technology is the most common way to produce large amounts of plant cells and organs throughout the year (Lee et al., 2008, Kor J Hort Sci Technol 26, 306-312). Therefore, in order to supply glass comas as a raw material for industrial market that requires uniform quality control, mass production using plant tissue culture technology can be said to be an effective production method satisfying the demand of the consumer. In recent years, cells of medicinal plants (Lee et al, 2006, J Plant Biol 49, 427-431), somatic embryos (Shohael et al., 2008, Biochem Eng J 38, 270-273) Acta Physiol Plant 31, 219-222) have been actively studied to produce physiologically active substances using tissue culture techniques. The cultivation of adventitious roots allows the growth of the cultures to be rapid, continuous growth, stable supply, and easy commercialization (Kim et al, 2004, J Plant Biol 47, 336-56). In addition, since the environment is easily controlled in the case of in-vitro culturing using physiological active materials using tissue culture technology, it is possible to produce physiologically active substances of uniform quality throughout the year, and also to increase the content artificially (Zhao et al., Biotechnol Adv 23, 283-333). The method using this is called elicitation, and a biological elicitor or an abiotic elicitor is used to make a lupine pool ( Scopolia parviflora ) (Kang et al., 2004, Plant Sci 166, 745-751), ginseng ( Panax ginseng ) (Ali et al., 2005, Plant Sci 169, 83-92) and Echinacea purpurea ) (Wu et al., 2007, J Plant Biol 50, 636-643) and the like have been actively studied to increase the content of physiologically active substances in the adventitious roots of various medicinal plants.

In the present invention, by using a plant tissue culture technique, the growth and physiologically active substance content of Eurycoma longifolia ).

Korean Patent No. 0842420 discloses a method for mass production of adventitious roots of a plant belonging to the genus Echinacea using a bioreactor and Korean Patent Registration No. 1018843 discloses a method for propagating a plant of the genus Pelagonium However, there is no description about a method for increasing the content of the physiologically active substance and the growth of the root canal of the present invention.

The present invention is derived by the request as described above, in the present invention, indole butyric acid (indole butyric acid, IBA) or naphthalene acetic acid (α-Naphtalene acetic acid, NAA) determining the auxin concentration in the glass coma ronji polyamic (Eurycoma longifolia ) adventitious root culture medium conditions were optimized, and the culture medium was used for the determination of Eurycoma longifolia ) plants, the growth of adventitious roots and the content of physiologically active substances were increased, thereby completing the present invention.

In order to solve the above problems, the present invention is a glass in a coma (Eurycoma sp.) inoculating a plant leaf, stem or root with MS medium containing auxin to induce adventitious roots and then cultivating the induced adventitious roots. ≪ / RTI >

In addition, the present invention relates to a method for producing an Eurycoma sp.) Provides plant apex.

When the adventitious root culture method of the present invention is used, the growth is promoted and the content of the physiologically active substance is increased. Eurycoma longifolia ) can be mass produced effectively. Further, the glass coma ronji polyamic (Eurycoma obtained by the culture method of the present invention longifolia ) The biomass and physiologically active substance of the adventitious root can be used industrially because they can be used for the production of cosmetics, foods, spices, beverages and nourishing tonics.

1 is a glass coma ronji polyamic (Eurycoma longifolia ) indicating the process of induction and proliferation of adventitious roots. The adventitious roots derived from the leaves, stems and roots of the inflorescence were sequentially grown in petri dishes, flasks and bioreactors.
FIG. 2 shows the results of measuring the effect of the type and concentration of auxin in the initial culture medium on the growth of free corn borer muscles at 7 weeks after the incubation of the glass beads with the flask. (A) live body weight, and (B) irregular muscle shape. IBA; Indole butyric acid, NAA; Naphthalene acetic acid.
FIG. 3 shows the results of measuring the effect of the medium type on the growth of the glass coma amygdaloidal muscle at 7 weeks after the incubation of the amniotic cavity with the bioreactor. (A) a bioreactor culture appearance, and (B) fresh weight.
FIG. 4 shows the influence of the medium type on the pH of the medium when the adventitious root was cultured 7 weeks after the incubation of the glass beads with the bioreactor. b ^z represents the mean value within 5% of Duncan's multiple range test.
FIG. 5 shows the effect of the type of medium on the electrical conductivity (EC) of the adventitious root culture after 7 weeks of incubation with the biochemical reactor. b ^z represents the mean value within 5% of Duncan's multiple range test.
FIG. 6 shows the influence of the kind of medium upon the adventitious root culture on the water potential of the medium after 7 weeks from the incubation of the glass coma with the bioreactor. b ^z represents the mean value within 5% of Duncan's multiple range test.
Figure 7 shows the process of production of the glass coma oblique root. 20 ℓ bioreactor is proliferated and produced by mass production, drying process and production process of final sales stage using factory bioreactor.

According to an aspect of the invention there is provided a glass coma in (Eurycoma sp.) inoculating a plant leaf, stem or root with MS medium containing auxin to induce adventitious roots and then cultivating the induced adventitious roots. ≪ / RTI >

The induction of the adventitious roots can be carried out by a method commonly used in the art. Indorphous butyric acid (IBA), indole acetic acid (IAA), naphthalene acetic acid (NAA), kinin (kinin), or benzyl amino purine (BAP) may be added to the adventitious root induction medium. It does not. Preferably, adventitious roots can be derived from the plant in 3/4 MS medium supplemented with 1 to 5 mg / l of IBA and 10 to 50 g / l of sugar, more preferably 2 to 4 mg / l of IBA and 20 to 40 g / The adventitious roots can be induced from the plant in 3/4 MS medium supplemented with 3 mg / l of the medium, and most preferably 3/4 MS medium supplemented with 3 mg / l IBA and 30 g / But is not limited thereto.

In the method according to one embodiment of the present invention, the cultivation of the adventitious roots can be performed preferably for 40 to 60 days, and most preferably for 49 days, but is not limited thereto.

In the method according to one embodiment of the present invention, the auxin may be indole butyric acid (IBA) or naphthalene acetic acid (NAA), and the IBA treatment concentration is preferably 1-5 mg / l, more preferably 2 to 4 mg / l, most preferably 3 mg / l, and the NAA treatment concentration is preferably 3 to 9 mg / l, more preferably 4 to 6 mg / l, 5 mg / l, but is not limited thereto.

In a method according to an embodiment of the present invention said Eurycoma sp. Plants are selected from the group consisting of Eurycoma longifolia), glass coma Bahia Kula other (Eurycoma apiculata), glass coma hareuman Diana (Eurycoma harmandiana) or glass can be a polyamic coma Lahti (Eurycoma latifolia), and most preferably from glass coma ronji polyamic (Eurycoma longifolia ). < / RTI >

In the method according to an embodiment of the present invention, the physiologically active substance refers to a substance that enhances or inhibits the function of a living body. The phenolic compound, isoflavone, beta glucan, rutin, genistein, glycitein, zeaxanthin, polyphenol, syringin, propolis, resveratrol, ), Triterpen, lycopene, quercetin, saponin, octacosanol, phytosterol, ginsenoid, anthocyanin, catechin, or gamma Gamma linolenic acid, and the like, and most preferably, the physiologically active substance may be a phenolic compound or a flavonoid, but is not limited thereto.

The present invention also provides an Eurycoma sp. Plant adventitious root produced by the method and having increased growth and bioactive material content. The advantage in combed (Eurycoma sp.) Plants adventitious root can contain adventitious roots of the plant in a coma glass, preferably glass coma ronji polyamic (Eurycoma longifolia ) plant, but is not limited thereto.

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

Materials and methods

One) Adrenaline  Induction and proliferation

The leaves, stems or roots of the in-vitro cultured plants were cut to induce the glass coma irregularities, and then 1 / 4-1MS medium supplemented with 1.0-5.0 mg / l of indole butyric acid (IBA) and 10-50 g / (Murashige and Skoog, 1962, Physiol Plant 15, 473-497).

The induced adventitious roots were cut at intervals of 0.5 to 1.5 cm and maintained and proliferated using Petri dishes, flasks or bioreactors containing the same medium and used as experimental materials. The medium was adjusted to pH 5.8 with 1N NaOH or 1N HCl, sterilized at 121 ° C and 1.2 atm. All adventitious roots were continuously cultured at 6 ± 8 week intervals in dark conditions maintained at 22 ± 1 ° C (Fig. 1).

2) Auxin (Plant Growth Regulator)  Type and concentration of glass beads Adrenaline  Effects on growth and production of biologically active substances

In order to investigate the types and concentrations of auxin that are suitable for the growth of free radicals and accumulation of physiologically active substances, indole butyric acid (IBA) and α-naphthalene acetic acid were added to MS medium supplemented with 30 g / NAA) were treated with 1, 3, 5, 7 and 9 mg / L, respectively, for 7 weeks.

The medium was adjusted to pH 5.8 with 1N NaOH, and 100 ml each was dispensed into an Erlenmeyer flask having an air volume of 250 ml. The medium was sterilized at 121 ° C and 1.2 atm. Cultures were inoculated with adventitious roots at an inoculum density of 5.0 g / ℓ based on the live weight, followed by shake culture at a rate of 100 rpm in a dark condition maintained at 22 ± 1 ° C, and repeated five times for each treatment group. After 7 weeks of culture, the ratio of live weight to dry weight was measured for growth, and total phenolic (phenolic) and flavonoid contents were measured as physiologically active substances.

3) The type of medium is glass beads Adrenaline  Effects on growth and production of biologically active substances

In order to investigate the types and concentrations of auxin that are suitable for the growth and physiologically active substance accumulation in the free comas, 3mg / ℓ of IBA and 30g / ℓ of IBA, which showed the most effective growth of adventitious roots and accumulation of physiological active materials One MS, 3/4 MS, B5, DKW (Driver-Kuniyuk-Walnut) and WPM (Woody plant medium) medium were used. The medium was treated with different concentrations of inorganic substances to cultivate the glass coma root for 7 weeks.

The medium was adjusted to pH 5.8 with 1N NaOH, and then 2 L each was dispensed into a bioreactor having an air volume of 3 L and sterilized at 121 캜 and 1.2 atm. The cultures were inoculated with adventitious roots at an inoculum density of 5.0 g / ℓ based on the live weight, and cultured in air volume of 0.05 vvm (Air volume culture volume ^-1 min ^-1 ) under the dark condition maintained at 22 ± 1 ° C., Repeated three times per treatment group. After 7 weeks of culture, the ratio of live weight to dry weight was measured for growth, and total phenolic (phenolic) and flavonoid contents were measured as physiologically active substances. The pH, electrical conductivity (EC) and water potential of the media were measured to determine the physiological characteristics of other adventitious roots.

4) Growth measurement

After 7 weeks of cultivation, the adventitious root, from which the medium was removed, was subjected to moisture removal using a desiccant paper, and then live weight was measured. The ratio of dry weight to fresh weight was determined by Shohael et al. (Shohael et al., 2006 Process Biochem 41, 1179-1185) Was calculated using the following equation according to the method.

Percentage of building to live weight = (in final harvested building) / (final harvested living weight) × 100

5) Measurement of physiologically active substance content

0.1 g of the dried irregular root was immersed in 60 to 80% (w / v) methanol and heated at 80 ° C for 2 hours using a reflux condenser (LS-2050-S10, LS-TECH, And extracted. The extract was filtered using a filter paper (Advantec 110 mm, Toyo Roshi Kaisha Ltd., Japan) and then 50 ml was used to prepare a final extract.

Foline-Ciocalteu Reagent Based on the Foline-Ciocalteu Method (Foline and Ciocalteu, 1927 J Biol Chem 27, 627-650), which is converted to molybdenum blue by reducing the total phenolic compound of the extract, Ali et al. Enz Microbial Technol 39, 982-990) was used to measure the total phenolic compound content of the adventitious roots. After adding 2.55 ml of distilled water to 0.05 ml of the extract and reference material solution, 0.1 ml of a 2N Foline-Ciocalteu reagent solution (10-fold dilution; Sigma chemical Co., St. Louis, Mo., USA) was added. After 6 minutes, 20% (w / v) Na ₂ CO ₃ Solution was added, and after standing for 30 minutes in a dark state, the absorbance was measured at a wavelength of 760 nm using a spectrophotometer. Garlic acid (Sigma Chemical Co., St. Louis, Mo., USA) was used as the reference material, and the total phenolic compound content in the adventitious roots was expressed in mg / g · DW.

The total flavonoid content of adventitious roots was measured according to the method of Sakanaka et al. (Sakanaka et al., 2005 Food Chem 89, 569-575) based on the colorimetric method. 1.25 ml of distilled water was added to 0.25 ml of the extract and reference material solution, and 5% (w / v) NaNO ₂ 0.075 ml of the solution was added. After 6 minutes, 10% (w / v) AlCl ₃ 0.15 ml of the solution was added, and the mixture was allowed to stand for 5 minutes, and then 0.5 ml of 1N NaOH solution was added. The distilled water was added so that the final volume of the mixed solution became 2.5 ml, and the absorbance was measured at a wavelength of 510 nm using a spectrophotometer. (+/-) catechin standard solution (Sigma Chemical Co., St. Louis, Mo., USA) was used as a reference material, and the total flavonoid content of the adventitious root was expressed in mg / g · DW.

6) pH Wow EC  Measure

The adventitious root-free medium was filtered using a filter paper (Advantec 110 mm, Toyo Roshi Kaisha Ltd., Japan). The pH and electrical conductivity (EC) of the adventitious root culture medium were then measured using a pH meter (pH-Electrode SenTix 41, WTW GmbH, Weilhein, Germany) and an EC meter (TetraCon 325, WTW GmbH, Weilhein, Germany) Respectively.

7) moisture Potential  Measure

The medium collected from the culture container was used as an analytical sample after removing impurities using a filter paper (Advantec 110 mm, Toyo Rosihi Kaisha Ltd., Japan). The analytical sample was diluted 5 ml each time, placed in a sample chamber, sealed, and measured using a water potentiometer (WP4 Potentia Meter, Decagon, Washington, USA). The water potential meter was calibrated at room temperature for 25 minutes at room temperature for 30 minutes and then repeated 5 times for each treatment.

Example  One. Auxin  The type and concentration of the glass Composition Eurycoma  longifolia ) Adrenaline  Effects on growth and production of biologically active substances

Eurycoma longifolia ). In order to increase the growth of the adventitious roots and physiologically active substance contents, the growth of the adventitious roots and the physiologically active substance contents were measured after treatment with auxin, a plant growth regulator. Indole butyric acid (IBA) and naphthalene acetic acid (NAA), which are representative oxine species, were treated at different concentrations of 1, 3, 5, 7 and 9 mg / ℓ for 7 weeks. A medium not treated with auxin was used as a control. After 7 weeks of culture, the ratio of live weight to dry weight was measured for growth, and total phenolic (phenolic) and flavonoid contents were measured as physiologically active substances.

As a result of measuring the amount of physiologically active substance in the adventitious roots cultured with different concentrations of IBA and NAA, it was found that the adventitious roots cultured for 7 weeks in medium supplemented with IBA and NAA, (Table 1). Especially, total phenolic compounds and total flavonoid contents increased by 26% and 34%, respectively, in IBA 3mg / ℓ treated group, and total physiologically active substance contents in 1g of the building were significantly increased compared to the control. Total phenolic compounds and total flavonoid contents were increased by 24% and 41%, respectively, compared to the control, even at the treatment of 5 mg / l NAA, and the total amount of physiologically active substance contained in 1 g of the building was significantly increased compared with the control. As the concentration of IBA and NAA increased, the phenolic compounds and flavonoid contents of the adventitious roots increased significantly compared to the control group.

In addition, as shown in Fig. 2, when adventitious roots were cultured by treating IBA and NAA at different concentrations, it was found that both the live weight and the dry weight of the burnt root cultured for 7 weeks in the medium supplemented with IBA and NAA were increased Respectively. As a result, it was found that the optimum growth rate of IBA was 3 ~ 20%, and that of IBA was 3 ~ 7 mg / L was determined as the appropriate concentration.

As a result, it was confirmed that the amount of growth of free corpus callosum and the amount of physiologically active substance were increased by treatment with IBA and NAA, and the type and concentration of optimal auxin suitable for continuous culturing of free cormorant rhizome were determined to be 3 mg / l of IBA.

z represents the mean value test within the 5% level by Duncan's multiple range test.

NS; No significant.

* Significance; P? 0.1

** significance; P? 0.01

*** Significance; P? 0.001

Example  2. The type of medium is glass beads Ronzio Piola ( Eurycoma longifolia ) Adrenaline  Effects on growth and production of biologically active substances

MS, 3/4 MS, B5, DKW (Driver-Kuniyuk-Walnut) and WPM (Woody plant medium) media were used to determine the type and concentration of medium suitable for in-flight mass production of the glass coma. The medium was treated with 3 mg / l of IBA selected as optimal auxin in the preceding experiment, treated with 3% of sucrose, adjusted to pH 5.7, cultured in a bioreactor for 7 weeks, and the growth rate and physiologically active substance content Respectively. After 7 weeks of culture, the ratio of live weight to dry weight was measured for growth, and total phenolic (phenolic) and flavonoid contents were measured as physiologically active substances.

As shown in Table 2 and FIG. 3, the growth of adventitious roots according to the type of the medium was measured. The fresh weight and the dry weight of the glass-coma irregular root cultured using the bioreactor for 7 weeks under the condition of 3/4 MS medium and B5 medium Respectively. The fresh weight and the dry weight of the adventitious root cultured on 3/4 MS medium were the highest, 31.07 g / ℓ and 3.22 g / ℓ, respectively, and it was confirmed that it was higher than that of MS medium.

As a result of measuring the physiologically active substance content of adventitious roots by the type of medium during the incubation with the bioreactor at 7 weeks after culturing, the total phenolic compounds (phenolic) and the total phenolic compounds The flavonoid contents were 7.04 mg / g · DW and 1.95 mg / g · DW, respectively, and the contents of physiologically active substances were increased compared to the adventitious roots cultured in MS medium (Table 3). In particular, the content of physiologically active substance in the free radical of the coma cultured on 3/4 MS medium was higher than the content of physiologically active substance in the adventitious root cultured in B5 medium and was determined as the optimum medium composition.

z represents the mean value test within the 5% level by Duncan's multiple range test.

z represents the mean value test within the 5% level by Duncan's multiple range test.

Example  3. Depending on the type of medium Adrenaline  Physiological characteristics measurement

The pH, electrical conductivity (EC) and water potential of the adventitious roots were measured to determine the physiological characteristics of the adventitious roots according to the type of culture medium. The adventitious roots were removed from the medium using a filter paper, and then used as an analytical sample.

As shown in FIG. 4, the pH of the culture medium was about 5 ~ 6, which was about 5 ~ 6, after 7 weeks of culture.

After 7 weeks of culture, the effect of the medium type on the electrical conductivity and water potential of the glass-coma amygdala cultured on a bioreactor was measured, and the electrical conductivity of the 3/4 MS medium was found to be about 4.0 mS / cm 5), and the water potential was about -0.3 MPa (FIG. 6).

In the case of glass comas that generally use roots, mass production is difficult in the natural state due to the temperature condition in Korea. Therefore, the present invention intends to increase the availability of the glass coma by producing the physiologically active substance in large quantities in the form of a plant-like plant in the form of adventitious roots (Fig. 7). The adventitious roots derived from leaves, stems and roots of the plant of the present invention are sequentially grown using a Petri dish, a flask and a bioreactor, and the kind and concentration of auxin suitable for in-flight mass production of the glass coma root are determined Respectively. It is believed that the use of the method of the present invention for growing a glass coma adventitious root can effectively mass-produce the glass coma irregular root of which the growth and physiologically active substances are promoted.

Claims (8)

Leaves, stems or roots of Eurycoma longifolia plants are inoculated into 3/4 MS medium supplemented with 2-4 mg / l indole butyric acid (IBA) and 20-40 g / l sugar, A method for increasing the growth and phenolic or flavonoid content of Eurycoma longifolia plantar roots, which comprises culturing the induced adventitious roots for 40 to 60 days. delete delete delete delete delete delete A plant oropharyngeal of Eurycoma longifolia with increased growth and phenolic or flavonoid content produced by the method of claim 1.

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