KR20180137883A - Method for increasing effective component content of Echinacea sp. adventitious root by elicitor treatment - Google Patents

Abstract

The present invention relates to a method for culturing an adventitious root of a plant of the genus Echinacea with an increased content of useful components, comprising the steps of: sterilizing the plant of the genus Echinacea; inducing and propagating the adventitious root from a petiole explant of the sterilized plant of the genus Echinacea; and culturing the adventitious root by treating the adventitious root with an attractant in a bioreactor. The method of the present invention can produce an adventitious root of Echinacea with enhanced content of useful components by attractant treatment, and thus can increase the utility of Echinacea.

Description

The present invention relates to a method for increasing the content of useful components of the adventitious roots of plants belonging to the genus Echinacea by treating the attractants with an effective component content of Echinacea sp. adventitious root by elicitor treatment}

The present invention relates to a method of treating an attractant to increase the content of useful components of the adventitious roots of a plant of the genus Echinacea.

Echinacea is a perennial herbaceous plant belonging to the Asteraceae family. Echinacea has eight species and several species. Echinacea purpurea , E. angustifolia , and E. pallida , which are widely grown in North America, are now industrialized in Europe and China. Scale cultivation.

Echinacea is an important medicinal plant for Native Americans and early migrants and is known to have effects on wounds, burns, snake or insect bites or colds, toothache, tonsillitis, rabies, paralysis, sepsis. Echinacea is also used in the form of extracts, tea, tonic, tablets and ointments, and is also used as a cosmetic raw material for skin protection. Echinacea is currently used as a non-specific immune stimulator for respiratory infections, urinary tract infections, skin diseases and many other inflammatory processes. It is also known to increase macrophages and kill cancer cells.

It has been reported that the immune stimulating function of echinacea is due to the lipophilicity and polarity of extracts including alkamides, caffeic acid derivatives, glycoproteins, polysaccharides, and lipidic fractions such as alkamides, , And that the alkamid fraction promotes phagocytosis in vivo and in vitro. It has been reported that alkamides of echinacea are mainly isobutylamides of highly unsaturated carboxylic acids with olefinic or acetylenic bonds and inhibit cyclooxygenase and 5-lipoxygenase as anti-inflammatory agents, It has been reported that echinacoside and polysaccharide echinacin have antimicrobial activity and antifungal activity, and many studies have control of the immune system. Echinacea, however, is not able to meet demand with only harvesting of wild species, so more efficient production methods are required.

The present invention is an improvement of the conventional technique for the adventitious mass production of plants of the genus Echinacea. In the process of inducing and propagating adventitious roots using Echinacea purpurea and producing adventitious roots using a bioreactor, In order to produce the adventitious roots of Echinacea plant containing the useful components higher than the basic culture process by adding the attractant into the reactor.

Korean Patent No. 0842420 discloses a method for mass production of adenomyas of a plant belonging to the genus Echinacea using a bioreactor, and Korean Patent No. 1078888 discloses a cosmetic composition comprising a tissue-cultured Echinacea roots extract, A method for producing the same has been disclosed. However, there is no description about a method for treating the attractant of the present invention to increase the useful ingredient content of the adventitious roots of the plant of Echinacea genus.

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a process for producing adventitious roots of Echinacea purpurea, which comprises culturing adventitious roots in a bioreactor, Methyl jasmonate, or a combination thereof), the present inventors have completed the present invention by culturing the adventitious roots of Echinacea purpurea with an increase in the total content of polyphenol compounds as compared to the untreated control.

In order to solve the above-mentioned problems, the present invention provides a method of treating a plant of the genus Echinacea, Inducing and propagating adventitious roots from the petiole explant of the sterilized Echinacea plant; And culturing the adventitious germ in a bioreactor by treating with an attractant, wherein the content of the useful ingredient is increased.

In addition, the present invention provides the adventitious roots of plants of the genus Echinacea cultured by the above method, wherein the content of the useful ingredient is increased.

The method of the present invention can produce echinacea adventitious roots that can increase the content of the useful ingredient by the treatment with an attractant, and thus it is considered that the utility of echinacea can be enhanced. In addition, the method of the present invention may be applied to an in-vitro culture method of various wild medicinal plants containing useful components to further enhance the value of wild medicinal plants.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a flow chart of a method for culturing adventitious roots of a plant belonging to the genus Echinacea in which the content of a useful ingredient is increased by an attractant treatment.
Fig. 2 is a photograph showing the adventitious roots derived from the Echinacea leaf blade.
Fig. 3 is a photograph showing the growth of the Echinacea adductor muscle in the solid medium (left) and the bioreactor (right), respectively.

In order to achieve the object of the present invention,

Sterilizing the plant of the genus Echinacea;

Inducing and propagating adventitious roots from the petiole explant of the sterilized Echinacea plant; And

And culturing the adventitious germs in a bioreactor by treating with an attractant, wherein the content of the useful ingredient is increased.

In the culturing method of the present invention, the plant of the Echinacea genus is selected from the group consisting of Echinacea purpurea , E. angustifolia , E. pallida , It may be E. atrorubens , E. paradoxa , E. sanguinea , E. serotina or E. simulata , and preferably May be, but is not limited to, Echinacea purpurea.

In the culturing method of the present invention, the useful component may be a polyphenol compound, but is not limited thereto.

The adriamycin culture of the present invention is carried out by using (1) 1x MS (Murashige & Skoog) medium containing 50 g / L sucrose and 1 mg / L Indole-3-butyric acid, (5) incubation temperature is 20 ± 1 ° C, (6) incubation period is 5 weeks, and (3) But is not limited thereto.

In the culturing method of the present invention, the attractant may be at least one selected from the group consisting of carbon dioxide, yeast extract, chitosan and methyl jasmonate, but is not limited thereto. The attractant may preferably be a mixture of yeast extract, chitosan and methyl jasmonate, but is not limited thereto.

In the method of culturing the adventitious roots according to an embodiment of the present invention, when carbon dioxide is used as an attractant to increase the content of the polyphenol compound as a useful ingredient, carbon dioxide is added at a concentration of 0.05 to 2.5% And may be supplied at a concentration of preferably 0.5 to 1.0%. However, the present invention is not limited thereto.

When the yeast extract or chitosan is used as an attractant to increase the content of the polyphenol compound as a useful ingredient in the method of culturing the adventitious roots according to an embodiment of the present invention, To 150 mg / L, preferably at a concentration of 80 to 120 mg / L, more preferably at a concentration of 100 mg / L, but the present invention is not limited thereto .

In the method of culturing the adventitious roots according to an embodiment of the present invention, when methyl jasmonate is used as an attractant to increase the content of the polyphenol compound as a useful ingredient, To 250 μM, preferably at a concentration of 180 to 220 μM, and more preferably at a concentration of 200 μM. However, the present invention is not limited thereto.

In the method of cultivating the adventitious roots of the present invention, when two or more kinds of yeast extract, chitosan and methyl jasmonate are mixed and used as attractants, the content of polyphenols is significantly increased compared with the case of the single treatment of each attractant, Synergetic effect on the usage was confirmed.

The present invention also provides the adventitious roots of plants of the genus Echinacea cultured by the above method, wherein the content of the useful ingredient is increased. The adventitious roots of the Echinacea plant having an increased content of the useful component according to the present invention may be the adventitious roots of Echinacea purpurea with an increased total content of polyphenols including caffeic acid derivatives, It is not limited.

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

Example  One. Adrenaline  Induction and proliferation

Echinacea purpurea used in the present invention was an actual seedlings germinated with seed purchased in Canada, which is a native plant. For the purpose of surface sterilization, the petiole portion of the plant was cut to an appropriate size, and then immersed in 70% ethanol for 30 seconds, sterilized by shaking in 4% sodium hypochlorite for 5 minutes, and then washed with sterilized water (0.5 ~ 1 cm) and used as a culture material.

1 mg / L of indole butylic acid (IBA), sucrose of 50 g / L, and 2.5 g / L of gelrite were added to induce the adventitious roots from the leaf segment of the plant. (MS) (Murashinge and Skoog) medium (pH 5.8) and cultured for 5 weeks in an incubator at 20 ± 1 ° C. The adventitious roots induced in the solid medium were cut at about 1 cm from the apex and inoculated on the same medium to continuously grow adventitious roots (FIG. 2). The adventitious roots selected in the solid medium were inoculated into an aerobic submerged bioreactor having a total volume of 5 L, 4 L of a gel-free liquid medium was added, and cultured in a culture room at 20 ± 1 ° C for 5 weeks, (Fig. 3). As the liquid medium, a medium consisting of 1 mg / L of IBA and 50 g / L of sucrose was used as the MS basic medium.

Example 2: Investigation of the effect of inducing agent on the increase of the components of the adventitious roots in the bioreactor culture

4 L each of 5 L bioreactor was inoculated into a 1 × MS basal medium containing 1 mg / L of IBA and 50 g / L of sucrose, and then inoculated with 7.0 g of adventitious root. The amount of air supplied into the bioreactor was 0.1 vvm, and the incubation temperature was 20 ± 1 ° C. In order to maximize both the growth of adventitious roots and the accumulation of useful components, a two stage culture was performed in a bioreactor. The adventitious root was cultivated for 4 weeks in the bioreactor, and after 4 weeks, induction agent (yeast extract, chitosan, methyl jasmonate) was treated at various concentrations for 1 week. However, in the case of carbon dioxide, the period of feeding into the bioreactor was preliminarily experimented (1 to 4 weeks after inoculation). As a result, it was confirmed that the treatment was carried out one week after incubation, Respectively. The effects of total polyphenolic compounds and caffeic acid derivatives on the growth of adventitious roots were investigated.

At each step, the growth of adventitious roots and the content of polyphenol compounds are measured as follows. The body weight of the adriamycin was measured by weighing the harvested adventitious roots with a stainless steel sieve, separating the medium, removing the water with a clean tissue and measuring the weight. The weight of the building was measured after drying for 2 days at 60 ° C. The growth rate of adventitious roots was calculated using the following equation.

Growth rate = [(among the buildings of the last grown cormorant) - (among the buildings of the initial cormorant)] /

The contents of caffeic acid derivatives were analyzed by extracting the dried plant material with 80% methanol. The content was measured by adding Folin-Ciocalteu reagent and absorbance at 760 nm based on gallic acid. HPLC analysis of caffeic acid derivative content was carried out using XTerraRP 18 column and water and acetonitrile as mobile phase at 330 nm.

2-1. Effect of Carbon Dioxide Concentration on the Increase of Ingredients in the Root Root

To investigate the effect of the carbon dioxide concentration in the bioreactor on the increase of the components of the adventitious root, the carbon dioxide feed was treated at 0.03, 0.5, 1.0, 2.0 and 5.0%. Bioreactor culture conditions were the same as in the above example.

Growth of adventitious roots and content of polyphenol compounds Carbon dioxide concentration (%) Fresh weight (g / L) Building (g / L) Growth rate (%) Total polyphenol compound content (mg / g DW) 0.03 (control) 74.8 ± 1.8 11.1 ± 0.14 10.1 55.04 0.09 0.5 76.8 ± 2.4 12.4 ± 0.22 11.4 62.81 + 0.07 1.0 75.7 ± 2.1 11.2 ± 0.18 10.2 61.63 + - 0.10 2.0 62.9 ± 1.9 10.5 ± 0.21 9.5 56.42 + 0.11 5.0 51.2 ± 1.6 7.6 ± 0.17 6.6 52.54 ± 0.05

Caffeic acid derivative content carbon dioxide
Concentration (%) Caffeic acid derivative content (mg / g DW) Captalic acid
caftaric acid Chlorogenic acid
(chlorogenic acid) Chicory acid
(cichoric acid) Sum 0.03 3.54 + 0.17 4.68 ± 0.08 26.68 ± 0.26 34.90 ± 0.12 0.5 4.34 ± 0.21 5.26 ± 0.04 33.34 + - 0.41 42.94 + 0.22 1.0 4.12 ± 0.15 5.09 ± 0.06 32.90 ± 0.16 42.11 ± 0.08 2.0 3.88 ± 0.10 4.91 ± 0.06 30.48 ± 0.20 39.27 + - 0.15 5.0 3.38 ± 0.05 4.55 + 0.07 25.92 + 0.08 33.85 + 0.17

As a result of examining the amount of carbon dioxide which is suitable for increasing the active ingredient of Echinacea adductor muscle, total polyphenol compound content and total caffeic acid derivative content of echinacea adventitious roots were increased in the other treatments except the 5% carbon dioxide treatment treatments 1 and Table 2). The total polyphenol compound content was 62.81 mg / g DW, and the total caffeic acid derivative content was 42.94 mg / g DW. As a result, Respectively, by 1.14 times and 1.23 times, respectively.

2-2. Effect of Yeast Extract on the Increase of Dietary Ingredients

In order to investigate the effect of the yeast extract on the increase of the components of the adventitious roots, the cells were treated with 100, 200 and 400 mg / L, respectively, four weeks after the culture, and the bioreactor was cultivated in the same manner as in the above example.

Growth of adventitious roots and content of polyphenol compounds Yeast extract concentration (mg / L) Fresh weight (g / L) Building (g / L) Growth rate (%) Total polyphenol compound content (mg / g DW) 0 72.1 ± 2.2 11.0 + 0.11 10.58 56.23 + 0.015 100 71.3 ± 1.8 10.8 ± 0.10 10.37 63.78 + 0.07 200 68.4 ± 3.2 10.7 ± 0.15 10.26 57.50 + - 0.10 400 59.1 ± 3.4 8.5 ± 0.13 7.95 54.12 + - 0.11

Caffeic acid derivative content Yeast extract concentration (mg / L) Caffeic acid derivative content (mg / g DW) Captalic acid
caftaric acid Chlorogenic acid
(chlorogenic acid) Chicory acid
(cichoric acid) Sum 0 3.34 ± 0.06 5.06 ± 0.08 27.12 ± 0.26 35.52 ± 0.09 100 4.12 + 0.03 5.35 + 0.04 34.31 + - 0.41 43.78 ± 0.12 200 3.82 + 0.11 5.23 + 0.11 32.60 ± 0.26 41.65 + - 0.25 400 3.68 ± 0.09 5.11 + 0.07 32.29 ± 0.26 41.08 ± 0.19

High concentration of yeast extract (400 mg / L) inhibited the growth of Echinacea adriamycin but the low concentration of yeast extract showed similar growth to the untreated control. The total content of polyphenol compounds was 63.78 mg / g DW and the total amount of caffeic acid derivatives was 43.78 mg / g DW, which was higher than that of the control (1.13 Fold and 1.23 fold, respectively (Table 3 and Table 4).

2-3. Effect of chitosan on the increase of the components of the adventitious roots

In order to investigate the effect of chitosan on the increase of the components of the adventitious root, the cells were treated with 100, 200 and 400 mg / L, respectively, four weeks after the adventitious root culture, and the bioreactor culture conditions were the same as in the above example.

Growth of adventitious roots and content of polyphenol compounds Chitosan concentration (mg / L) Fresh weight (g / L) Building (g / L) Growth rate (%) Total polyphenol compound content (mg / g DW) 0 73.4 ± 2.2 11.2 ± 0.24 10.43 55.93 + - 0.23 100 66.3 ± 1.8 10.4 ± 0.11 9.61 63.22 + -0.31 200 51.4 ± 1.3 7.6 ± 0.17 6.76 54.30 ± 0.15 400 40.1 ± 2.0 6.5 ± 0.13 5.63 51.53 + - 0.26

Caffeic acid derivative content Chitosan concentration (mg / L) Caffeic acid derivative content (mg / g DW) Captalic acid
caftaric acid Chlorogenic acid
(chlorogenic acid) Chicory acid
(cichoric acid) Sum 0 3.55 + - 0.12 5.11 + 0.04 26.41 + - 0.15 35.07 ± 0.14 100 4.08 ± 0.09 5.27 ± 0.07 33.57 ± 0.22 42.92 + 0.17 200 3.22 ± 0.05 4.93 + 0.11 25.60 ± 0.12 33.75 ± 0.09 400 3.03 ± 0.16 4.66 ± 0.21 22.42 + 0.09 30.11 ± 0.28

In all chitosan - treated groups, the fresh weight and the dry weight of Echinacea nois were decreased compared to the control. However, the content of total polyphenol compounds as a useful ingredient was found to be higher in the 100 mg / L and 200 mg / L chitosan treated groups than in the control group, except for the 400 mg / L treated group. Especially, in the 100 mg / L chitosan treated group, And total caffeic acid derivatives were found to be higher than those of the control (Table 5 and Table 6).

2-4. Effect of methyl jasmonate on the increase of the components of the adventitious roots

In order to examine the effect of methyl jasmonate on the increase of the components of the adventitious root, the cells were treated with 100, 200, and 400 μM, respectively, after 4 weeks of culturing. Bioreactor culture conditions were the same as those in the above example.

Growth of adventitious roots and content of polyphenol compounds Methyl jasmonate concentration ([mu] M) Fresh weight (g / L) Building (g / L) Growth rate (%) Total polyphenol compound content (mg / g DW) 0 73.8 ± 1.4 11.7 ± 0.21 11.06 54.71 ± 0.16 100 67.2 ± 1.8 10.4 ± 0.09 9.72 64.27 + - 0.14 200 65.6 ± 1.7 10.1 ± 0.16 9.41 65.12 + - 0.21 400 53.1 ± 0.8 7.9 ± 0.11 7.14 62.15 + 0.17

Caffeic acid derivative content Methyl jasmonate concentration
(μM) Caffeic acid derivative content (mg / g DW) Captalic acid
caftaric acid Chlorogenic acid
(chlorogenic acid) Chicory acid
(cichoric acid) Sum 0 3.35 + 0.08 5.10 + - 0.11 26.32 + - 0.12 34.77 + 0.11 100 4.78 ± 0.23 5.42 ± 0.16 35.62 ± 0.18 45.82 ± 0.20 200 5.22 ± 0.19 5.97 ± 0.20 36.16 ± 0.12 47.35 + 0.14 400 4.33 ± 0.06 5.36 ± 0.09 31.44 0.09 41.13 ± 0.08

In all treatments of methyl jasmonate, both live weight and dry weight of Echinacea nois were decreased compared to the control. The total polyphenol compound content was 65.12 mg / g DW and the total caffeic acid derivative content was 47.35 mg / g DW in 200 μM of methyl jasmonate. g DW and increased by 1.2 and 1.4 times, respectively, compared to the control (Table 7 and Table 8).

2-5. The Effect of Injection Mixture Treatment on the Increase of the Ingredients of Orthotropic Root

The inventors of the present invention have found that when the attractants (yeast extract, chitosan, methyl jasmonate) are treated singly, when these attractants are mixed in combination, and when all of the attractants are mixed, The effects of the adventitious roots on the growth of useful components were investigated. After 4 weeks from the incubation of the bioreactor, (1) yeast extract 100 mg / L, (2) chitosan 100 mg / L, and (3) methyl jasmonate 200 μM were individually and mixed and the bioreactor culture conditions were the same Respectively.

Growth of adventitious roots and content of polyphenol compounds Treatment Fresh weight (g / L) Building (g / L) Growth rate (%) Total polyphenol compound content (mg / g D.W) 0 74.5 ± 1.1 11.2 ± 0.16 10.42 53.14 + 0.22 1 (yeast extract) 72.1 ± 0.9 10.8 ± 0.08 10.02 63.92 + 0.17 2 (chitosan) 69.5 ± 0.06 10.7 ± 0.09 9.92 63.27 ± 0.13 3 (methyl jasmonate) 64.6 ± 1.7 10.2 ± 0.21 9.41 65.52 + - 0.11 1 + 2 71.8 ± 0.05 11.1 ± 0.14 10.33 72.74 ± 0.19 1 + 3 68.5 ± 1.3 10.6 ± 0.07 9.82 80.12 ± 0.26 2 + 3 66.7 ± 1.2 10.4 ± 0.15 9.62 74.03 + - 0.15 1 + 2 + 3 59.4 ± 0.07 8.6 ± 0.08 7.78 82.52 ± 0.21

Caffeic acid derivative content Treatment Caffeic acid derivative content (mg / g D.W) Captalic acid
caftaric acid Chlorogenic acid
(chlorogenic acid) Chicory acid
(cichoric acid) Sum 0 3.32 ± 0.15 5.08 ± 0.09 26.75 ± 0.15 35.15 + 0.14 One 4.05 + 0.11 5.32 ± 0.08 34.54 0.11 43.91 + 0.09 2 4.01 + 0.07 5.11 + - 0.14 33.60 ± 0.13 42.72 + 0.13 3 5.43 + 0.16 6.04 ± 0.11 36.42 ± 0.09 47.89 + - 0.12 1 + 2 6.20 0.16 6.84 ± 0.19 37.20 ± 0.17 50.24 + 0.04 1 + 3 6.43 + 0.16 7.98 ± 0.13 41.42 ± 0.09 55.83 ± 0.08 2 + 3 6.23 ± 0.09 7.71 ± 0.05 40.44 + - 0.12 54.38 ± 0.06 1 + 2 + 3 7.45 ± 0.16 8.24 + 0.11 41.65 ± 0.09 57.34 ± 0.15

(Table 9 and Table 10) showed that the efficacy of Echinacea adventitious roots was significantly increased in both the single treatment and the mixed treatment of each attractant compared to the control. The total content of polyphenol compounds was 82.52 mg / g DW and the total amount of caffeic acid derivatives was 57.34 mg / g DW Respectively, which was 1.55 times and 1.63 times higher than that of the untreated control group, respectively.

Claims (9)

Sterilizing the plant of the genus Echinacea;
Inducing and propagating adventitious roots from the petiole explant of the sterilized Echinacea plant; And
And culturing the adventitious germ in a bioreactor by treating with an attractant, wherein the content of the useful ingredient is increased. The method according to claim 1, wherein the plant belonging to the genus Echinacea is Echinacea purpurea , wherein the content of the useful ingredient is increased. The method according to claim 1, wherein the useful ingredient is a polyphenol compound, wherein the content of the useful ingredient is increased. [3] The method according to claim 1, wherein the attractant is at least one selected from the group consisting of carbon dioxide, yeast extract, chitosan and methyl jasmonate. [2] The method according to claim 1, wherein the attractant is a mixture of a yeast extract, chitosan and methyl jasmonate, wherein the content of the useful ingredient is increased. [5] The method according to claim 4, wherein the carbon dioxide is supplied at a concentration of 0.05 to 2.5% after 1 week from the beginning of the adventitious root culture of a plant belonging to the genus Echinacea, wherein the content of the useful ingredient is increased. [Claim 5] The yeast extract or chitosan according to claim 4, wherein the yeast extract or chitosan is fed at a concentration of 50 to 150 mg / L four weeks after the start of the adventitious root culture of the plant belonging to the genus Echinacea. Lt; / RTI > The method according to claim 4, wherein the methyl jasmonate is supplied at a concentration of 150 to 250 μM after 4 weeks from the beginning of the adventitious root culture of a plant belonging to the genus Echinacea. . 8. The adventitious root of the plant of the genus Echinacea cultivated by the method of any one of claims 1 to 8, wherein the content of the useful ingredient is increased.

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