KR101857005B1 - Production method of Oplopanax elatus containing germanium - Google Patents

Abstract

The present invention relates to a production method of a germanium-containing gibbous adventitious roots, an antioxidative and skin whitening cosmetic composition containing germanium-containing germanium adducts produced by the above-mentioned method, and germanium-containing gibbous adventitious roots as active ingredients will be. In the present invention, when cultured through the method of treating an organic acid and a chelate and a two-step culture method in the cultivation of the Aspergillus oryzae, the accumulation and production of organic germanium in the adventitious muscle are effectively performed. , Antioxidant activity and total phenol content were increased. By using the adventitious roots produced by the method of the present invention, it is possible to develop high-functional raw materials by the production of organic germanium as well as the beneficial components of the peach tree, and to confirm the skin whitening and antioxidant-promoting effects, .

Description

Production method of Oplopanax elatus containing germanium containing germanium [

The present invention relates to a method for producing germanium containing germanium, and more particularly to a method for producing germanium ores containing germanium, a germanium-containing germanium ores produced by the method, and a germanium- The present invention relates to a cosmetic composition for antioxidant and skin whitening, comprising as an active ingredient an epidermal root gangrene root.

Oplopanax elatus Nakai, belonging to the Araliaceae plant, is a deciduous shrub with a length of about 1m. It has been used mainly as a treatment and emollient for fever, cough and inflammation in Korean herbarium. It is also called ginseng (ginseng).

Chestnut tree In (Oplopanax sp.) Plant Panax leg o 'Douce as native in the United States and Canada (Oplopanax horridus ), O. japonicus ( O. japonicus ), which is native to Japan, And O. elatus , native to Korea, China and Russia. Studies on physiological activity and active ingredients have been focused on Oplopanax horridus , and studies on Japanese and Korean pteridophytes have been insufficient.

In addition, domestic peat extracts are known to be toxic and safe natural products. Therefore, the possibility of development as cosmetics, medicines and functional foods is very high. At present, however, the native forests are endangered due to artificial indiscriminate over-exploitation, and they are designated and managed as a second-level protection in Korea, China and other countries. Therefore, it is required to study methods to improve existing efficacy for various industrial applications such as medicine, food, and cosmetics industry of peach tree, as well as a method for mass production of peach tree by tissue culture method in laboratory have.

On the other hand, Germanium (germanium) in 1930 in the French and Spanish border Lourdes (Lourdes) spring water is very effective in treating various diseases, germanium content is known to be very high as a result of the analysis, its medical efficacy is known . The characteristics of germanium can be divided into organic germanium and inorganic germanium. It is reported that organic germanium is safe, but inorganic germanium causes dysfunction of the kidney and liver. After that, it is reported that the inorganic germanium Studies on organic germanium, which does not remain in the body but exhibits pharmacological action, have been actively studied.

In the case of organic germanium, there are various pharmacological actions such as promotion of oxygen supply, purification of blood, promotion of in vitro excretion of heavy metals in the body, antitumor, antimutagenesis, immunity enhancement, therapeutic effect of viral infection, arthritis and antiinflammation, heavy metal detoxification, There is a report. Various studies using microorganisms and yeasts have been carried out for the organic germaniumization of inorganic germanium. In recent years, studies using rice, lettuce, bean sprouts and ginseng have been reported to develop plants containing organic germanium , Is insufficient. In particular, medicinal plants such as ginseng, Ganoderma lucidum, and Myeongilbyeo have effective ingredients in the human body. In addition to efforts to promote the production thereof, researches on in vitro culture by plant tissue culture have been actively conducted for production and synthesis of useful substances.

Korean Patent Registration No. 0413613 discloses a method for mass production of germanium-containing plants by plant tissue culture, but no production method of germanium-containing gabbrosomes containing germanium of the present invention has been disclosed.

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems, and it is an object of the present invention to provide a method for cultivating germanium ores in a culture medium containing inorganic germanium, . The present invention was carried out in a two-step culture method in which the organic acid and the chelate were treated and the adventitious root growth was carried out for 3 weeks and then the additional culture was carried out in a liquid medium supplemented with inorganic germanium for 3 weeks The present inventors have accomplished the present invention by confirming that the organic germanium accumulation and production in the adventitious root is effectively performed and that the germanium irregular root which has accumulated the germanium has the effect of increasing skin whitening, antioxidant activity and total phenol content.

In order to solve the above-described problems, the present invention provides a method for producing germanium rhizome root containing germanium, which comprises culturing a rhizome of the genus Rhizoma in a liquid medium supplemented with inorganic germanium.

In addition, the present invention provides germanium-containing pea germs produced by the above method.

In addition, the present invention provides a cosmetic composition for antioxidation and skin whitening, wherein the germanium-containing gilt-eutexia root gum is an effective ingredient.

By using the adventitious roots produced by the method of the present invention, it is possible to develop high-functional raw materials by the production of organic germanium as well as the beneficial components of the peach tree, and to confirm the skin whitening and antioxidant-promoting effects, .

Fig. 1 is a photograph showing the appearance of a seedling plant after 8 weeks of cultivation in a medium containing germanium at different concentrations (0, 2.5, 5, 10, 25, 50, 100 and 250 mg / to be. IG represents inorganic germanium, and OG represents organic germanium.
FIG. 2 is a graph showing inhibitory activity of tyrosinase according to the presence or absence of germanium treatment (0, 50 mg / liter) of plantlet root and adventitious root. Positive control: kojic acid 0.75 mM.
FIG. 3 is a graph showing antioxidative activities of plantlet root and adventitious root according to germanium treatment (0, 50 mg / liter). (A): DPPH free radical inhibitory activity, (B): ABTS free radical inhibitory activity. Positive control: ascorbic acid.
FIG. 4 is a graph showing total phenol and total flavonoid contents according to germanium treatment (0, 50 mg / liter) of plantlet root and adventitious root.

In order to achieve the object of the present invention,

(a) inducing adventitious roots by cultivating leaves, stems, roots or calli of a seedling plant;

(b) inoculating the induced adventitious roots in a liquid medium to proliferate; And

(c) further culturing the proliferated adventitious roots in a liquid medium supplemented with inorganic germanium. The present invention also provides a method for producing germanium-containing adventitious roots.

In the method for producing germanium adventitious roots containing germanium according to an embodiment of the present invention, the induction of the rootstock root of step (a) may be carried out in a medium containing 0.5 to 1 mg / l of indole butyric acid (IBA) , Preferably 0.5 mg / l IBA-containing medium, but is not limited thereto. The medium may be a solid medium or a liquid medium.

In step (a), the induction of the Aspergillus oryzae can be performed in a 1/2 MS medium containing 10 to 30 g / L sucrose, but is not limited thereto.

The step (a) may cultivate the leaves, stalks, roots or calluses of the proliferated mammary plants, preferably the roots of the mammary plants, but the present invention is not limited thereto.

The step (a) may preferably be carried out under dark conditions. In light conditions, it is characterized by almost no induction of adventitious roots in all parts of leaves, stems and roots.

In the method for producing germanium adventitious roots containing germanium according to an embodiment of the present invention, the growth of the glutinous adventitious roots of step (b) is carried out by adding 10 to 30 g / l of sucrose and 0.5 to 1 mg / ≪ / RTI > to about 6.3 in 1/2 MS liquid medium.

The step (b) may be carried out in a bioreactor at 23 to 27 ° C under a dark condition, but the present invention is not limited thereto.

In the method for producing germanium adventitious roots containing germanium according to an embodiment of the present invention, the inorganic germanium in the step (c) may be added at a concentration of 10 to 60 mg / l, 50 mg / L, but the present invention is not limited thereto.

In the method for producing germanium adventitious roots containing germanium according to an embodiment of the present invention, the oropharyngeal proliferation period of the step (b) is 2.5-3.5 weeks, and the additional cultivation period of the step (c) But is not limited thereto.

In the method for producing germanium adventitious roots containing germanium according to an embodiment of the present invention, the adventitious propagation of step (b) is performed for 3 weeks, and then 50 mg / l of inorganic germanium in step (c) It may be preferable to carry out further cultivation in the liquid medium for 3 weeks. In this way, it is possible to increase the growth of gnomere adventitia and the absorption efficiency of germanium.

In the method for producing germanium ores according to an embodiment of the present invention, the liquid medium of step (c) may include a germanium absorption promoter, and the germanium absorption promoter is preferably phosphoric acid ), Ethylenediamine tetraacetic acid (EDTA), and nitrilotriacetic acid (NTA), and more preferably phosphoric acid, but is not limited thereto.

The concentration of the germanium absorption promoter may be 0.05-0.5 mM, preferably 0.1 mM, but is not limited thereto.

Therefore, the method for producing germanium ores according to one embodiment of the present invention is preferably

(a) Leaf, stem, root, or callus of a seedling plant is cultured for 5 to 7 weeks in a 1/2 MS medium containing 10 to 30 g / L sucrose and 0.5 to 1 mg / Inducing;

(b) inoculating the induced adventitious germ to a 1/2 MS liquid medium at a temperature of 23 to 27 ° C, a pH of 5.3 to 6.3 containing 10 to 30 g / l of sucrose and 0.5 to 1 mg / Weekly proliferation; And

(c) further culturing the proliferated adventitious germ in a liquid medium supplemented with 0.05-0.5 mM phosphoric acid and 10-60 mg / l inorganic germanium for 2.5-3.5 weeks, but the present invention is not limited thereto.

In addition, the present invention provides germanium-containing pea germs produced by the above method.

The present invention relates to an Oplopanax sp. Plant, which comprises Oplopanax elatus , Oplopanax horridus , And O. japonicus , preferably, but not limited to, Oplopanax elatus .

In addition, the present invention provides a cosmetic composition for antioxidation and skin whitening, wherein the germanium-containing gilt-eutexia root gum is an effective ingredient.

Since the glutinous rhizome containing germanium, which is an active ingredient of the cosmetic of the present invention, has a tyrosinase-inhibiting activity, it can be used for skin whitening purposes and has antioxidative activity, so that it can be used for antioxidant use.

The cosmetics of the present invention can be used in cosmetics such as soft longevity, astringent lotion, nutritional lotion, nutritional cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, ≪ / RTI > body essence, and the like.

The cosmetic composition may further contain, in addition to the active ingredient of the present invention, a lipid, an organic solvent, a solubilizing agent, a thickening agent and a gelling agent, a softening agent, an antioxidant, a suspending agent, a stabilizer, a foaming agent, It may be advantageous to add one or more pharmaceutically acceptable excipients such as water, ionic or nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, barrier agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, And may contain adjuvants conventionally used in the cosmetics field, such as any other ingredients used.

In the cosmetic composition of the present invention, the composition of the present invention may be added in an amount of 1 to 15% by weight, preferably 2 to 10% by weight, based on the cosmetic composition usually contained.

The cosmetic composition of the present invention may further comprise functional ingredients other than the above-mentioned effective ingredients. The addition of the raw material which helps the skin health further enhances the effect of the composition of the present invention. In addition, when the raw material that helps the skin health is added, the skin safety due to the combined use, ease of formulation, The stability of the users can be considered. The additional functional ingredient is a functional ingredient disclosed in the Korean Food and Drug Administration. For example, oil, sugar, polyol and the like are not limited to the known raw materials that contribute to skin health.

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.

Materials and methods

1. Cultivation of Pinus densiflora and adventitious roots

The materials used in this study were cabbage seedlings grown on a laboratory farm at Kangwon National University and cultivated in 1/3 MS medium containing 1% sucrose and 1% agar for 6 months or longer. Stem and root parts were completely differentiated and seedling plants of 3cm or more in length were used as irregular root induction materials.

For the induction of adventitia adventitia on the liquid medium, the root of the seedling seedlings was inoculated with 0.5 to 0.8 cm long sections in a 1/2 MS liquid medium supplemented with IBA at a concentration of 0.5 mg / l and cultured for 6 weeks . 1% sucrose as a carbon source was added to a 1/2 MS liquid medium supplemented with IBA at a concentration of 0.5 mg / l and the pH was adjusted to 5.8.

For the mass proliferation of adductor limbalis, the adventitious roots induced by liquid culture were cut to length of 0.5 ~ 0.8cm. Each of the treatments was divided into 100 ml of 250 ml Erlenmeyer flasks, and each of the flakes was inoculated at a rate of 100 to 135 rpm. Each of the flasks was inoculated in an amount of about 30 mg each, and cultured for 6 weeks under dark conditions. Adrenal gland formation patterns were observed. All experiments were performed in 3 replicates.

IBA 0.5 mg / L was added as a plant growth regulator to the 1/2 MS basal medium, and 3% sucrose was added as a carbon source. The pH was adjusted to 5.8 to prepare 15 L of The medium was cultured for 6 weeks under the dark condition by inoculating 2g of.

2. In-flight cultivation Germanium treatment of seedling plant

(1) Optical pigment content by germanium treatment

(G [O (Ge (═O) CH ₂ CH ₂ CO ₂ H] ₂ , SIGMA-ALDRICH) was added to a 1/2 MS solid medium containing 1% sucrose for the production of organic germanium using a seedling plant. , And inorganic germanium (GeO ₂ , SIGMA-ALDRICH) at 0, 2.5, 5, 10, 25, 50, 100 and 250 mg / And cultured for 8 weeks.

In order to investigate the effect of germanium on the growth of the Pinus densiflora plant, weights of leaves, stems and roots were measured. In order to investigate the photosynthetic efficiency, the optical pigment chlorophyll a, chlorophyll b and carotenoid contents were measured. 0.1 g of the leaves of the seedlings of each of the treatments was extracted with 10 ml of acetone in cold dark condition for 2 days, and the absorbance was measured at 663.2, 646.8 and 470.0 nm after filtration. And the content thereof was measured.

Carotenoid content: [(1000A ₄₇₀ -1.82a-85.02b) / 198]

Chlorophyll a (μg / ml) = [12.25 A _663.2 -2.79 A _646.8 ]

Chlorophyll b (μg / ml) = [21.50 A _646.8 -5.10 A _663.2 ]

(2) Growth and organic germanium production ability by germanium treatment

In order to confirm the accumulation of germanium and germanium production ability by germanium type and concentration treatments, it was separated for each part of the seedling plant and shade for 5 days or more. Powder samples were prepared by using a mortar bowl and 0.5 g of 35% nitric acid, 10 ml of nitric acid was added, and the mixture was allowed to stand at room temperature for 6 hours, and 1 ml of hydrogen peroxide was added thereto, followed by preliminary decomposition using microwave. The wet decomposition product was again diluted to 50 ml with tertiary distilled water and used for analysis using ICP-OES (Inductively Coupled Plasma Optical Emission Spectrometer).

3. Germanium treatment of apical adventitia

(1) Growth and organic germanium production ability by germanium treatment

2.5, 5, 10, 25, 50, 100 and 250 mg / l in a 1/2 MS (3% sucrose) liquid medium supplemented with 0.5 mg / The slices produced in this study were 0.5 ~ 0.8 cm long and 40 mg inoculated into Erlenmeyer flasks and cultured for 6 weeks. The fresh weight and dry weight of Pinus rigida were measured and the germanium production ability was evaluated by ICP analysis.

(2) chelate treatment and two-step cultivation for improving organic germanium production ability

Chelate treatment and two - step culture method were devised to improve organic germanium production ability. First, citrate, phosphoric acid and chelating agent, EDTA, were added to 1/2 MS (3% sucrose) liquid medium supplemented with 0.5 ㎎ / ℓ IBA to examine the effect of chelate on the growth of adventitious roots. (ethylenediaminetetraacetic acid), ethylene glycol tetraacetic acid (EGTA), nitrilotriacetic acid (NTA) and mio-inositol at concentrations of 0.1, 1 and 10 mM, respectively. And cultured for 6 weeks.

To investigate the effect of chelating agents on the germanium production ability, citrate, phosphoric acid, chelating agent EDTA (1: 1), and phosphate buffered saline (EDTA) were added to a liquid medium of 1/2 MS (3% sucrose) supplemented with 0.5 mg / l IBA supplemented with 25 mg / l of inorganic germanium , EGTA, NTA, and mio-inositol were added at 0.1 and 1 mM, respectively. Then, inoculated with Euglena equilibria and incubated for 6 weeks, growth and germanium production were investigated.

A two - step culture method was devised and treated as a method to facilitate germanium absorption ability and growth of adventitious roots. After incubation for 6 weeks, the cells were cultured for 1, 2, 3, 4, and 5 weeks in germanium-free growth medium and 0.5 mg / l IBA supplemented with 25 mg / And cultured in a 1/2 MS (3% sucrose) liquid medium added thereto was used for analysis.

4. Physiological Activity and Substance Analysis

(1) Measurement of whitening and antioxidant activity

In order to compare the possibility of the functional material of the genus Orthoptera, root portions of adventitious roots and seedlings were analyzed for germanium - free (0 ㎎ / ㎖) and inorganic germanium - treated (50 ㎎ / ㎖).

The whitening activity was evaluated by inhibition of tyrosinase. Tyrosinase inhibition was measured by the Tyrosinase Inhibitory Screening Kit (BioVision, USA) at 25 ° C. and absorbance at 510 nm.

Antioxidant activity was measured by DPPH free radical scavenging activity and ABTS free radical scavenging activity by diluting the concentrated sample with 100%, 500, and 1,000 ppm of 80% EtOH.

The DPPH free radical scavenging method was measured by modifying the method of Xiong et al. (1996, biological and pharmaceutical bulletin. 19: 1580-85). 100 μl of 0.15 mM DPPH was added to each well of a 96-well plate and 100 μl of each sample was added to each well. After incubation at room temperature for 30 minutes in a dark place, the absorbance at 519 nm was measured. RC ₅₀ (占 퐂 / ml), which is a 50% reduction concentration.

ABTS free radical scavenging method was modified by the method of Roterta et al. (1999, Free radical biology & medicine, 26 (9/10): 1231-1237). 7.5mM ABTS (2,2-azinobis (3 -ethyl benzothiazoline-6-sulfonic acid)) solution and the 2.6mM KPS (potassium persulphate) solution 1: allowed to stand for 12 hours and mixed with cow 1, the radical ABTS ⁺ for turquoise And the solution was refrigerated. The radial stock solution was diluted to 1: 1 with the extinction coefficient of 0.70 ± 0.03 using 1XPBS, and 10 μl of each sample solution was added to 190 μl of the reaction solution. The reaction solution was reacted in the dark at room temperature for 10 minutes and then measured at 740 nm Respectively.

(2) Analysis of antioxidants

Antioxidant analysis was carried out by total phenol content, total flavonoid content and phenol quantitative analysis using UPLC.

Total phenol content was measured by modifying the Folin-ciocalteu assay. 100 μl of the extract prepared at a concentration of 1,000 ppm was mixed with 50 μl of Folin-ciocalteu reagent and stabilized at room temperature. Then, 300 μl of 20% sodium carbonate was added and reacted at room temperature for 15 minutes. Then, 1 ml of distilled water was added And the absorbance was measured at 740 nm. Standard curves were prepared using gallic acid as a reference material and expressed as mg / g GAE (gallic acid equivalent).

Total flavonoid content was determined by modifying the method of Moreno et al. (2000, J. Ethnopharmacology 71: 109-114). 100 μl of 10% aluminum nitrate and 100 μl of 1 M potassium acetate were mixed, and 500 μl of the sample was added thereto. After reacting at room temperature for 40 minutes, the absorbance was measured at 415 nm. Standard curves were generated using quercetin as a reference material followed by quercetin equivalents (QE).

Example  1. Optical pigment content of germanium treated plants in cabbage cultivation

However, there is no report that the absorption capacity and the pattern of the germanium are actually analyzed. Then, the plant was introduced into the cabin, and the seedling plant completely differentiated into leaves, stems and roots at a plant height of 3 cm or more was placed in a medium treated with germanium and incubated for 2 months. The optical pigment Content analysis and absorption of absorbed germanium were analyzed to analyze absorption capacity and migration pattern.

To confirm the efficiency of smooth growth and photosynthesis by germanium treatment, optical pigment analysis was performed. Chlorophyll a and chlorophyll b showed similar tendency and increased with the addition of germanium, the highest at the concentration of 10 ㎎ / ℓ of inorganic germanium, 25 ㎎ / ℓ of organic germanium and the decreasing tendency at the higher concentration (Table 1).

The chlorophyll absorbs the light energy and transfers it to the photosynthesis system. The carotenoid contributes energy to the photosynthesis system and avoids the damage of the photosynthesis system when the light energy is exceeded. The carotenoid content is the same as the chlorophyll content and the inorganic germanium is 10 ㎎ / ℓ At the concentration of 25 ㎎ / ℓ, organic germanium showed the highest value at the concentration higher than the concentration of 25 ㎎ / ℓ (Table 1).

The ratio of chlorophyll a and b content to germanium treatment of the Pinus densiflora plant was higher than 2.6: 1 and decreased rapidly at high concentration of inorganic germanium (Table 1).

The photosynthetic efficiency of chlorophyll decreased rapidly at higher concentrations when treated with germanium. The chlorosulphate showed a tendency to decrease more rapidly than that of carotenoid during the stress or aging process. (Table 1).

Example  2. Growth and organic germanium production by in vitro culture of germanium-treated seedling plants

In order to investigate the effect of germanium on the seedling plants, treatment with inorganic germanium and organic germanium, respectively, was found to be more effective in the inorganic germanium treatment than in the organic germanium treatments (see FIGS. 1, 2 and 3) Table 3).

On the other hand, the organic germanium production ability of P. germanium was confirmed by the ICP analysis by the germanium content in the body. The accumulation of organic germanium was more actively carried out when the inorganic germanium was treated. When the germanium accumulation amount was multiplied by the germanium accumulation amount during the drying of the germanium plant, / ℓ in the roots of the treated germanium at 40.87 ± 3.04 ㎍ / plantlet (Table 3).

The accumulation of inorganic germanium did not accumulate at the roots, but the accumulation of organic germanium at the roots was not accumulated. The concentration of 5 ㎎ / ℓ was transferred to the roots and stems at the concentration of 5 ㎎ / , And organic germanium was produced by the transfer and accumulation of germanium to the stem and leaf in the inorganic and organic germanium treatment of 10 mg / L or more. As the concentration of germanium treatment increased, the accumulation of organic germanium also increased. As a result, both organic and inorganic germanium treatments were accumulated at a concentration of 100 ㎎ / ℓ and decreased at 250 ㎎ / ℓ (Table 3).

Organic germanium was more effective in the transition and accumulation of leaves, especially at high concentration of 250 ㎎ / ℓ (Table 3).

As for the growth of the seedling seedlings by germanium treatment, growth of inorganic germanium at 10 ㎎ / ℓ and organic germanium at 25 ㎎ / ℓ showed more growth than germanium non - treatment, and growth at high concentration (Table 2 and Table 3).

Example  3. Growth and organic germanium by germanium treatment of apical adventitia Production capacity

Organic germanium mass production and evaluation of the efficacy of functional germanium were carried out by treating inorganic germanium, which had good absorption and production efficiency, in the seedling plant by concentration in adventitious culture.

The germanium treatment in the Pinus densiflora plant increased the growth and photosynthetic efficiency at low concentrations and the accumulation and production of germanium at high concentration treatments. Growth was poor in the Pinus densiflora cultivation, , And organic germanium accumulation and production in the adductor muscle increased (Table 4). In the high concentration of 50 ㎎ / ℓ inorganic germanium treatment, it showed the highest accumulation and production of organic germanium, but the growth rate was one third of the untreated.

In the production of organic germanium using this seedling, the efficiency was more than 9 times higher than that of root in the root of the root. Cultivation of seedlings is obtained through subculture of more than 5 months and treatment period of 2 months. However, it is possible to produce organic germanium in 6 weeks of cultivation of adventitious root, and it can be cultured for 6 weeks. Germanium mass production system became possible.

Example  4. Organic germanium Production capacity  For promotion Chelate  Treatment and 2-step culture

In order to enhance the organic germanium production ability of the adductor gibbous root, chelate which plays a role of enhancing the absorption ability by forming a ligand of a heavy metal and an organic acid which functions to absorb and stabilize the ion were treated.

Phosphoric acid, citric acid, nitrilotriacetic acid, ethylenediamine tetraacetic acid (EDTA), myo-inositol and ethylene glycol-bis (2-aminoethylether) -N, N , N ', N'-Tetraacetic Acid) were added to the media to 0.1, 1, and 10 mM concentrations.

The growth of adventitious roots was investigated in fresh weight, growth rate, and dry weight for 6 weeks by adding only organic acid and chelate to the growth medium without treatment with germanium.

As a result, in all treatments except phosphoric acid, 10 mM of high concentration treatment reached only 1/3 level in the living body, and there were no treatments showing enhanced effect on the growth of the control. In particular, all the treatments in the dry condition were less than 1/2 of the untreated level, and organic acid and chelate treatment decreased the growth of the aspergillus oryzae (Table 5).

To investigate the effect of organic acid and chelate treatment on the absorption and production of germanium, 25 ㎎ / ℓ of inorganic germanium was added to the seedling growth medium and treated with 0.1 and 1 mM of organic acid and chelate.

The growth of Pinus densiflora in the medium treated with organic acid or chelate and germanium was slightly enhanced in the citrate or myo - inositol treatment at 0.1 mM compared to the control treated with germanium alone. (Table 6).

In the case of 0.1 mM citric acid, 0.1 mM of myo-inositol and 0.1 mM of phosphoric acid showed a similar increase in fresh weight (Table 6).

The germanium accumulation was enhanced in NTA 0.1, 1 mM and 0.1 mM EDTA compared to the control, but the final germanium production per flask was significantly lower than that of the final organ germanium, (See Table 6).

In order to reduce the effect of organic germanium uptake on the adductor germanium uptake and to reduce the adverse effects on the growth of adventitious roots, a two - stage culture method, which induces the differentiation and growth of adventitious roots and treats germanium - added medium Respectively.

For this purpose, 1, 2, 3, 4, and 5 weeks were cultured for 1, 2, 3, 4, and 5 weeks in the seedling growth medium and the growth medium was treated with inorganic germanium at a concentration of 50 ㎎ / ℓ for the rest of 6 weeks.

The germanium uptake was the highest in the experimental group treated with 3 weeks of germanium untreated and the highest in the experimental group after 3 weeks of germanium treatment, (Table 7).

In this study, it was confirmed that the two - step cultivation method was more effective than the organic acid and chelate treatment when cultured in the 3 week amniotic growth medium for 3 days and germanium accumulation for the growth of adventitious roots and germanium accumulation.

Example  5. Measurement of whitening activity of germanium-containing adventitious roots

In order to investigate the effects of the production of organic germanium on physiological activity and the content of organic germanium, inorganic germanium treatment (0 ㎎ / ℓ) and inorganic germanium treatment (50 ㎎ / ℓ) Respectively.

The whitening activity of untreated control plants of rootstocks and adventitious roots was slightly higher than that of control roots, which was 1.4 times higher in roots of plant root and 1.9 times higher in irregular root through organic germanium accumulation and production. The highest whitening activity was observed at a concentration of 50 ppm in the germanium-treated amygdalus adrenal gland, indicating a tyrosinase inhibitory activity of 31.4% (FIG. 2).

Example  6. Measurement of antioxidative activity of germanium-containing adriamycin

Antioxidant activity was measured by RC ₅₀ (mg / ml), which is required for 50% scavenging of DPPH and ABTS free radicals.

Comparison of antioxidative power between root and adventitious roots of Pinus densiflora plant showed that the adventitious roots had superior antioxidant power over the root of the plant root in both DPPH and ABTS free radical inhibitory activity (Fig. 3).

The antioxidant activity of germanium was increased by germanium treatment in both roots and adventitious roots of Pinus densiflora plant. The antioxidant capacity of the plant was increased at the roots of the seedling, but the antioxidant activity of the adventitious root was superior to that of the germanium-free treatment (Fig. 3).

Example  7. Analysis of antioxidants in germanium-containing root ganglion

In the analysis of antioxidants, the contents of phenolic compounds and flavonoids having strong antioxidant activity of plants were measured and quantitatively analyzed by UPLC analysis.

Total phenol contents and total flavonoid contents were analyzed, and flavonoid contents were similar in all treatments. The total phenolic content of the adventitious roots was higher than that of the seedling root, and the total phenol content was increased by the germanium treatment (Fig. 4).

Claims (10)

(a) Leaf, stem, root, or callus of a seedling plant is cultured for 5 to 7 weeks in a 1/2 MS medium containing 10 to 30 g / L sucrose and 0.5 to 1 mg / Inducing;
(b) inoculating the induced adventitious germ to a 1/2 MS liquid medium at a temperature of 23 to 27 ° C, a pH of 5.3 to 6.3 containing 10 to 30 g / l of sucrose and 0.5 to 1 mg / Weekly proliferation; And
(c) further culturing the proliferated adventitious germ in a liquid medium supplemented with 0.05-0.5 mM phosphoric acid and 10-60 mg / l inorganic germanium for 2.5-3.5 weeks. The germanium- . delete delete delete delete delete delete delete A germanium-containing gabbro adventitious root produced by the method of claim 1. delete

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