KR101651662B1 - Method of culturing peanut callus for increasing resveratrol content - Google Patents

Abstract

The present invention relates to a method for culturing peanut callus for increasing resveratrol content, and more particularly, to a method for culturing callus derived from peanut leaf under specific conditions to increase the content of resveratrol. (A) inducing a peanut callus from a peanut leaf; (B) directing the peanut callus to an MS medium supplemented with at least one growth regulator selected from the group consisting of auxin and cytokinin; (C) irradiating ultraviolet rays (UVC) for 5 to 40 minutes at an intensity of 0.5 to 1.0 mW / cm ² after culturing for 20 to 30 days; And (D) subculturing at intervals of 21 to 28 days. A method for culturing peanut callus for increasing resveratrol content is provided.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for culturing peanut callus for increasing resveratrol content,

The present invention relates to a method for culturing peanut callus for increasing resveratrol content, and more particularly, to a method for culturing callus derived from peanut leaf under specific conditions to increase the content of resveratrol.

Resveratrol is a type of polyphenols found in many plants, including vertebrates such as audi, peanuts, grapes, raspberries, and cranberries. It has anticancer and strong antioxidant action and is known to lower serum cholesterol. Resveratrol is a kind of stilbene that is produced by stilbene synthase (STS), an enzyme in plants, and exists as two structural isomers of cis and trans. Resveratrol is known to have anticancer and potent antioxidant action, and is known to lower serum cholesterol. It is also known to have antiviral, neuroprotective, anti-inflammatory, anti-aging and life-prolonging effects.

In particular, trans-resveratrol has recently been attracting attention as phytoestrogen as the female hormone estrogen-like action is revealed (Gehm, BD et al., Proc. Natl. Acad Sci., 94: 14138-14143, 1997). On the other hand, resveratrol derivatives have been reported to be potent inhibitors of tyrosinase enzymes that promote the synthesis of skin melanin pigment (Iida, K. et al., Planta Med., 61: 425-428 , 1995; Shimizu K. et al., Planta Med., 64: 408-412, 1998).

Accordingly, research for using resveratrol contained in natural products has been continued. However, the content of resveratrol contained in grape or peanut was not large enough to utilize it, and studies for increasing the content thereof have been made actively.

Korean Patent Laid-Open Publication No. 2009-0097729 discloses a method for increasing the content of resveratrol in peanuts by treating fungi in germinated peanuts with fungi. Korean Patent Publication No. 2011-0080901 entitled " Method for cultivating peanut sprout herb containing a large amount of resveratrol "includes the step of cultivating peanut sprout herb seeds at 26 ° C to 30 ° C under a dark condition maintaining a relative humidity of 80 to 90% ≪ RTI ID = 0.0 > a < / RTI > method of cultivating peanut sprouts in large amounts containing resveratrol.

In the past, researches have been carried out to increase the content of resveratrol mainly by treating peanut seeds or cultivating peanut buds obtained by germinating them. The present inventors have found that the callus derived from the leaves of peanuts can be greatly increased in the content of resveratrol by culturing and treating the calli under specific conditions, thus completing the present invention.

It is an object of the present invention to provide a method for culturing a peanut callus derived from a peanut leaf to increase the content of resveratrol having an excellent physiologically active effect.

It is another object of the present invention to provide a culture medium of peanut callus derived from peanut leaves used for increasing the resveratrol content.

In order to achieve the above object, according to the present invention,

(A) inducing a peanut callus from a peanut leaf;

(B) directing the peanut callus to an MS medium supplemented with at least one growth regulator selected from the group consisting of auxin and cytokinin;

(C) irradiating ultraviolet rays (UVC) for 5 to 40 minutes at an intensity of 0.5 to 1.0 mW / cm ² after culturing for 20 to 30 days; And

(D) a step of subculturing at intervals of 21 to 28 days, wherein a culture of peanut callus for increasing resveratrol content is provided.

The step (A) may be performed as follows.

(a1) immersing peanut seeds in 70% ethanol for 30 to 60 seconds, taking out them, and immersing them in an aqueous solution of 2% sodium hypochlorite for 15 to 20 minutes;

(a2) washing with sterilized distilled water and inducing germination in a dark room by sterilization with MS medium (3% sucrose, 0.4% gelrite, pH 5.8); And

(a3) inducing a callus by directing a piece of rice cake of a herbivorous plant 20 to 30 days after the tooth to the MS medium supplemented with at least one growth regulator selected from the group consisting of auxin and cytokinin.

The step (A) may be performed as follows.

(a1) immersing the leaves of the adult under growth in a natural state for 30 to 60 seconds in 70% ethanol, taking out the resulting leaves, and immersing them in an aqueous solution of 0.5% sodium hypochlorite for 15 to 20 minutes;

(a2) washing with sterile distilled water; And

(a3) inducing callus by directing the adult leaf slice to an MS medium supplemented with at least one growth regulator selected from the group consisting of auxin and cytokinin.

 In step (B), IBA (indolebutyric acid) is used as the auxin as the growth regulator, and kinetin is used as the cytokinin. In this case, the concentration of IBA (indolebutyric acid) may be 0.5-5 mg / l, and the concentration of kinetin may be 0-5 mg / l. Preferably, IBA (Indolebutyric acid) is 0.5-3 mg / May be included at a concentration of 0 to 1 mg / l.

In the step (C), ultraviolet rays having a wavelength of 280 to 100 nm are used as ultraviolet rays (UV-C).

According to the present invention, there is provided a method for cultivating peanut callus derived from peanut leaf to which a growth regulator having a concentration of 0.5 to 3 mg / l of IBA (indolebutyric acid) and 0 to 1 mg / l of kinetin is added to a basic MS medium A culture medium for use is provided.

Since the callus culturing method derived from the peanut leaf of the present invention can increase the intracellular resveratrol content, it is possible to mass produce and industrialize useful components of peanuts containing a high content of resveratrol from the cultured calli, There is an effect of promoting the production of functional foods and cosmetics and pharmaceuticals containing resveratrol as an active ingredient.

Figure 1 is a photograph of a peanut callus derived from a peanut leaf according to one embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention relates to a culture method in which a callus derived from a peanut leaf is cultured to obtain resveratrol in a high content. In the case of using only leaves that are above the ground, it is not necessary to collect the underground part, so that the plant can grow continuously and the utilization of the remaining ground surface tissue even after the ground harvesting of the peanut can be enhanced.

According to one embodiment of the present invention, there is provided a method of producing peanut callus comprising: (A) inducing a peanut callus from a peanut leaf; (B) directing the peanut callus to an MS medium supplemented with at least one growth regulator selected from the group consisting of auxin and cytokinin; (C) irradiating ultraviolet rays (UVC) for 5 to 40 minutes at an intensity of 0.5 to 1.0 mW / cm ² after culturing for 20 to 30 days; And (D) subculturing at intervals of 21 to 28 days. A method for culturing peanut callus for increasing resveratrol content is provided.

The step (A) of inducing peanut callus from the peanut leaf may be carried out using a nutritional plant obtained by germinating the peanut seed as described below, or using an adult under growing in a natural state.

According to one embodiment of the present invention, peanut seeds are first immersed in 70% ethanol for 20 to 40 seconds, taken out, and then added to a 2% by weight aqueous solution of sodium hypochlorite for 15 to 20 minutes, more preferably 15 minutes It is immersed and sterilized.

Disinfection of seeds for aseptic germination results in destruction of tissue when prolonged time is taken according to disinfection treatment time and disinfection with appropriate treatment time because various fungi and bacteria of surface tissue are not completely disinfected when the time is short, It is important. According to one embodiment of the present invention, the endothelial tissue of the peanut seed is preserved and is a method for completely sterilizing the epidermal tissue. First, it is immersed in 70% ethanol for 30 seconds, taken out and dissolved in an aqueous solution of 2% by weight sodium hypochlorite Immerse for 15 minutes.

After the disinfection, the sterilized medicinal solution remaining on the epidermis is washed three times with sterilized distilled water to prevent the penetration into the endothelial cells. Sterilized seeds were germinated in a dark room by sterilization with MS medium (3% sucrose, 0.4% gelrite, pH 5.8) The callus induction plant is used for callus induction for about 20 days after tooth decay.

The cotyledon of Cotyledon, which is about 20 days after tooth decay, is cut into a size of 5mm to 5mm, and the cuticle is guided by placing it on the medium.

According to one embodiment of the present invention, as the callus induction medium, MS medium having the composition shown in Table 1 below is used as a basic medium.

Ingredients Content (mg / L) CoCl ₂ .6H ₂ O 0.025 CuSO ₄ .5H ₂ O 0.025 FeNaEDTA 36.35 H ₃ BO ₃ and H ₂ O 6.20 KI 0.83 MnSO ₄ 22.30 Na ₂ MoO ₄ .2H ₂ O 0.25 ZnSO ₄ .7H ₂ O 8.60 CaCl ₂ .2H ₂ O 440.00 KH ₂ PO ₄ 170.00 KNO ₃ 1900.00 MgSO ₄ .7H ₂ O 370 NH ₄ NO ₃ 1650.00 Glycine 2.00 Myo-Inositol 100.00 Nicotinic acid 0.50 Pyridoxine HCl 0.50 Thiamine HCl 0.10

At least one growth regulator selected from the group consisting of auxin and cytokinin is added to the medium as a growth regulator. Growth regulators are organic compounds, not nutrients, which means substances that inhibit, promote, or regulate plant physiological processes in small amounts. Growth regulators can be broadly divided into five categories: auxin, cytokinin, gibberellin, ethylene, and abscisic acid.

Oxine is known to be involved mainly in root formation, and 2,4-D (2-4-dichlorophenoxy acetic acid), NAA (Naphthaleneacetic acid), IAA (Indolacetic acid) and IBA (Indolebutyric acid) are examples. Cytokinin is an essential growth regulator for plant cell cultures with auxin. Typically kinetin, 6-furfurylaminopurine, 6-benzylaminopurine, zeatin or 2- ip).

In the present invention, auxin and / or kinetin is used as a growth regulator. According to one embodiment of the present invention, IBA is used as the auxin as the growth regulator, and kinetin is used as the cytokinin. In this case, the concentration of IBA (indolebutyric acid) may be 0.5 to 5 mg / l, and the concentration of kinetin may be 0 to 5 mg / l, more preferably 0.5 to 3 mg / l of IBA (indolebutyric acid) Was found to be effective in the callus induction or culture when contained at a concentration of 0 to 1 mg / l.

According to another embodiment of the present invention, the step (A) of inducing the peanut callus from the peanut leaf may be carried out using an adult growing in a natural state as follows.

According to one embodiment of the present invention, an adult undergoing growth in a natural state is arbitrarily cut to an appropriate size, immersed in 70% ethanol for 30 seconds, taken out, and immersed in an aqueous 0.5% by weight sodium hypochlorite solution for 15 minutes. After disinfection, it is washed three times with sterilized distilled water to prevent penetration of the remaining disinfectant into inner cells. The seedlings grown in the natural state were also sterilized and cut into 5 x 5 mm sections, and the cuticle inducing medium was used to induce the peanut callus.

After culturing for about 2 to 3 weeks under the conditions of the callus induction medium, when the cells of the tissue undergo cell division and callus, which is a lump of cells, is formed, the callus alone is separated and cultured on a new medium under the same conditions as the callus induction medium .

The callus culture medium may further include protein hydrolysates derived from plants and microorganisms, and may further include casein enzymatic hydrolysates. According to one embodiment of the present invention, the casein enzymatic hydrolyzate is preferably contained at a concentration of 0.1 to 30 g / L.

In addition, the culture medium of the peanut callus of the present invention may further comprise a surfactant, and the surfactant is preferably Pluronic F-68, PEG, PVA, PPG, more preferably Pluronic F-68, PEG 1,000 Or PEG 8,000, and may be included at a concentration of 0.1 to 10 g / L.

When cultured in the culture medium for 20 to 30 days and then grown to a predetermined size, ultraviolet light is irradiated to amplify intracellular resveratrol content. At this time, the wavelength, intensity, and time of ultraviolet light to be irradiated are important factors. Care should be taken when irradiating ultraviolet light for too long, since the amount of resveratrol may be reduced. According to one embodiment of the present invention, the callus is irradiated with ultraviolet C (UV-C) having a wavelength of 280 to 100 nm at an intensity of 0.5 to 1.0 mW / cm ² for 5 to 40 minutes. More preferably 10 to 20 minutes. It was confirmed that the content of resveratrol was greatly increased due to ultraviolet irradiation under the above conditions.

By subculturing at 21 to 28 days after irradiation with ultraviolet light, resveratrol increased peanut callus can be obtained.

[Example]

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 1: Callus induction using peanut leaf

The seeds (Arachis hypogaea L.) used in the present invention were purchased from the Jecheon traditional medicine center in Chungcheongbuk-do and the adults were grown under natural conditions at Jecheon farm.

Germination rate of peanut seeds according to surface disinfection time

Seed disinfection and adult disinfection for aseptic germination are disrupted by prolonged disinfection treatment time, and when the time is short, various fungi and bacteria of the surface tissue are not completely disinfected, Disinfection.

In the present invention, the endothelial tissue of the peanut seeds is preserved and is completely sterilized by dissolving the skin tissue in 70% ethanol for 30 seconds, and then taken out to prepare a solution of sodium hypochlorite in an amount of 5, 10, 15, And immersed for 30 minutes. After the disinfection, it was washed three times with sterilized distilled water to prevent the penetration of the disinfectant remaining in the epidermis into the endothelial cells.

The sterilized seeds were germinated in the dark room aseptically by denting each 10 pieces in the MS medium (3% sucrose, 0.4% gelrite, pH 5.8) shown in Table 1, and the results are shown in Table 2 below.

time 5 minutes 10 minutes 15 minutes 30 minutes Germination rate 0% 10% 60% 20%

As shown in Table 2, the germination rate of peanuts was significantly lowered due to damage of the endothelial tissue during 30 minutes of sterilization compared to that of the conventional silk seeds having a seed sterilization time of about 30 minutes. In addition, the disinfection of 5 minutes and 10 minutes did not completely sterilize the epidermal tissue, and the degree of contamination was severe. In other words, it was confirmed that adequate disinfection time for in - flight cultivation of peanut seeds was about 15 minutes.

Disinfection of adults under natural conditions

In the natural state, the adult undergoing growth was arbitrarily cut to a suitable size, immersed in 70% ethanol for 30 seconds, taken out and immersed in a 0.5% by weight sodium hypochlorite aqueous solution for 15 minutes. After disinfection, Lt; RTI ID = 0.0 > sterile < / RTI > distilled water.

Sectioning

Twenty - five days after sowing, the cotyledon of the plant was cut into 5 × 5 mm pieces. The step of disinfection is omitted because the seedlings are already germinated aseptically.

Among the sterilized tissues grown in the natural state, the leaves were cut into 5 × 5 mm pieces.

Selection of medium for callus induction

30 g / l sucrose and 0.4% gelrite were added to the basal MS medium of Table 1, and IBA and kinetin were treated at the ratios shown in Tables 3 and 4 below. The degree of callus induction by each treatments was observed and subcultured every 4 weeks.

The results are shown in Table 3 and Table 4 as a result of observing the callus induction after cotyledon of Cotyledon and adult leaves of the seedling plants by treating the growth regulators with concentration.

In adult leaf cuttings, callus was induced from cuts at the central vein of the cuticle (see Fig. 1).

(%) IBA (mg / l) 0.5 One 3 5 Kinetin
(mg / l) 0 16 15 8 5 0.5 4 5 5 2 One 8 3 2 0 3 0 5 3 4 5 4 0 2 4

(%) IBA (mg / l) 0.5 One 3 5 Kinetin
(mg / l) 0 32 37 35 12 0.5 23 29 24 16 One 38 11 9 0 3 2 7 5 2 5 0 0 3 2

As shown in Tables 3 and 4, particularly when the concentration of IBA (Indolebutyric acid) is in the range of 0.5 to 3 mg / l and the concentration of kinetin is in the range of 0 to 1 mg / l, .

Example 2: Culture for Increasing Resveratrol Content in Peanut Leaf Callus

After incubation for about 3 weeks in medium supplemented with 0.5 mg / l of IBA (indolebutyric acid) and 1 mg / l of kinetin in the basic MS medium of Example 1, the cells of the tissue were cell- When the callus was grown, the callus alone was separated and plated on a fresh medium of the same composition. After 30 days of culture, the cells were incubated for a certain period of time (0 min, 1 min, 5 min, 10 min, 20 min, 30 min, 60 minutes) Using the ultraviolet C light energy, the intensity was 0.5 ~ 1.0 mW / cm ² .

Test Example 1: Analysis of resveratrol content

The peanut leaf-derived calli collected in Example 2 was lyophilized and pulverized. For the resveratrol quantitative analysis, samples were extracted with 80% methanol aqueous solution at room temperature for 10 minutes using an ultrasonic extractor. The extracted sample was used as a sample for analysis by filtration with a membrane filter (pore size 0.45 or 0.2 micrometer).

The HPLC instrument for resveratrol analysis was a Waters HPLC system model with a device equipped with a binary pump, autosampler and photodiode array detector.

For quantitative analysis of resveratrol content in peanut callus, standard calibration curve and HPLC analysis conditions were established using standard materials. Various solvent combinations and solvent gradient conditions were established to establish optimal separation conditions. The solvent conditions for separation were a combination of 0.01 M phosphoric acid H ₂ O and acetonitrile solvent as a 65:35 isocratic solvent combination with time slope Solvent conditions were set. Absorbance was measured at 325 nm by a photodiode array detector for detection of resveratrol. The column for separation was a Mightysil RP-18 GP 250-4.6. The HPLC standard calibration curve for resveratrol of peanut callus was prepared by dissolving 1-2 mg resveratrol in methanol and sequentially diluting it with concentration (40, 20, 10, 5, 2.5, 1.25 μg / mL) A calibration curve was prepared.

Table 5 shows the results of comparing the resveratrol content with the irradiation time of the ultraviolet C series light energy in Example 2.

Time (minutes) 0 One 5 10 20 30 40 60 Resveratrol
Content (ug / g) 0 1.38 15.61 35.21 21.01 15.35 6.79 0.38

As shown in Table 5, when the peanut callus was cultured under the same conditions as above, the intracellular resveratrol content was amplified when irradiated with ultraviolet light. When the irradiation time was 5 to 40 minutes, the effect was excellent. Especially, the resveratrol content was increased in 10 to 20 minutes.

Claims (6)

(A) (a1) immersing leaves of an adult under growing in a natural state for 20 to 40 seconds in 70% ethanol, taking out the resulting leaves and immersing them in an aqueous solution of 0.5% sodium hypochlorite for 15 to 20 minutes;
(a2) washing with sterile distilled water; And
(a3) inducing peanut callus from peanut leaves by directing adult leaf slices onto MS medium supplemented with 0.5 mg / l of indolebutyric acid (IBA) and 1 mg / l of kinetin;
(B) digesting the peanut callus into MS medium supplemented with 0.5 mg / l of indolebutyric acid (IBA) and 1 mg / l of kinetin;
(C) irradiating ultraviolet rays (UVC) having a wavelength of 280 to 100 nm for 10 minutes at an intensity of 0.5 to 1.0 mW / cm ² after culturing for 20 to 30 days; And
(D) subculturing at intervals of 21 to 28 days. delete delete delete delete delete

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