KR102544450B1 - Method for increasing phenylpropanoids content in Pimpinella brachycarpa - Google Patents

Abstract

The present invention relates to a method for increasing the content of phenylpropanoids in C. japonica. When 0.05 to 0.15 mM methyljasmonate is sprayed 2 to 4 times every 3 days to the leaves or stems of C. catechin hydrate, chlorogenic acid, caffeic acid, When the content of p-coumaric acid and benzoic acid is significantly increased, and 0.05 to 0.15 mM salicylic acid is sprayed 2 to 4 times every 3 days, catechin hydrate, chlorogenic acid, caffeic acid, The contents of p-coumaric acid and ferulic acid are significantly increased, and when 0.2-0.4% (w/v) chitosan is sprayed 2-4 times every 3 days, catechin hydrate, chlorogenic acid, caffeic acid, p-coumaric acid , Ferulic acid, benzoic acid, and quercetin contents are remarkably increased, so through the present invention, a large amount of chamnamul with increased phenylpropanoid content having excellent antioxidant, anticancer, anti-inflammatory and cardioprotective activities is easily and economically You can get it.

Description

Method for increasing phenylpropanoids content in Pimpinella brachycarpa}

The present invention relates to a method for increasing the content of phenylpropanoids in japonica by treating an elicitor, specifically methyljasmonate, salicylic acid or chitosan.

Pimpinella brachycarpa Nakai is an aromatic perennial plant of the dicotyledonous plant umbel family Ubelaceae. It grows mainly in the shade of trees in Korea and northeast China and is an edible plant with a high preference for its unique fragrance. Chamnamul, also called san parsley because of its strong water parsley scent, is mainly used as vegetables for wraps, vegetables, and tempura.

Antioxidant activity, antimutagenic and alcohol-induced liver damage protective effects, high cholesterol inhibitory activity, antithrombotic activity, antibacterial activity, and anticolorectal cancer activity have been reported as studies on the useful physiological activities of C.

On the other hand, phenylpropanoids (phenylpropanoids) is a kind of chemical substances found in plants and is a phenolic substance with three carbon chains attached to an aromatic ring. Changes in physiological activity appear differently depending on the substitution of hydroxyl and methoxyl groups in the basic skeleton.

Phenylpropanoids are synthesized from the amino acid phenylalanine, and numerous phenolic acids and flavonoids are synthesized through various pathways. There are over a thousand types of phenylpropanoids, such as catechins in green tea, resveratrol in wine, and quercetin in apples or onions. Flavonoids found in fruits and isoflavones found in soybeans are also a type of this substance. In addition, since it is a pigment of plants produced by photosynthesis and a component of bitter taste, it is often found in foods that are vivid in color and have an astringent or bitter taste, such as grapes.

Phenylpropanoids perform various physiological functions in plants and play a key role in defense mechanisms against biotic or abiotic stresses such as pathogens, insects, low temperature, high temperature, salt damage, and ultraviolet rays. It has been reported to have an effect on anticancer and cardioprotective activities.

On the other hand, elicitors refer to various organisms or chemicals capable of inducing physiological reactions of plants. Plants respond to stress factors by influencing plant metabolism, increasing the synthesis of related substances, and activating mechanisms similar to defense responses to pathogen infections such as viruses and bacteria or environmental changes such as ultraviolet rays, low temperatures, and high temperatures. The first elicitors were described in the early 1970s, and since then many studies on inducing defense responses in plants and plant cells have been conducted.

However, since there is no study on the phenylpropanoid component and elicitor treatment of C. japonica, the present inventors tried to confirm the change in the phenylpropanoid component of C. chinensis according to the elicitor treatment.

As a prior art related to the enhancement of phenylpropanoid content, Korea Patent No. 0858002 discloses a biosynthetic inducer and biosynthetic method of phenylpropanoid amide compounds in plants using methanol as an attractant, but the elicitor of the present invention , More specifically, a method for increasing the phenylpropanoid content in Chamnamul by treating with methyljasmonate, salicylic acid or chitosan has not been disclosed.

The present invention, which was derived from the above request, provides a method for increasing the content of phenylpropanoids in Chamnamul, including the step of treating the elicitor in Chamnamul, and the elicitor 0.05 ~ 0.15mM Methyljasmonate is treated 2-4 times every 3 days, 0.05-0.15 mM salicylic acid is treated 2-4 times every 3 days, or 0.2-0.4% (w/v) chitosan is treated 2-4 times every 3 days. When Chamnamul was grown using a method for increasing the phenylpropanoid content in Chamnamul, which included the step of treating it four times, the phenylpropanoid component contained in Chamnamul was significantly increased compared to that grown by the conventional method. By confirming, the present invention was completed.

In order to solve the above problems, the present invention provides a method for increasing the content of phenylpropanoids in C.

In addition, in the present invention, 0.05 to 0.15 mM methyljasmonate or 0.05 to 0.15 mM salicylic acid is treated 2 to 4 times at 3-day intervals or 0.2 to 0.4% (w / v) chitosan is treated with 3 It provides a method for cultivating Cham sprouts with enhanced phenylpropanoids content, including the step of treating 2 to 4 times at daily intervals.

In addition, the present invention provides Chamnamul with increased phenylpropanoid content cultivated by the above method.

In addition, the present invention provides a composition for increasing the content of phenylpropanoids in C. japonica containing, as an active ingredient, any one elicitor selected from methyljasmonate, salicylic acid and chitosan.

The present invention relates to a method for increasing the content of phenylpropanoids in C. japonica. When 0.05 to 0.15 mM methyljasmonate is sprayed 2 to 4 times every 3 days to the leaves or stems of C. catechin hydrate, chlorogenic acid, caffeic acid, When the content of p-coumaric acid and benzoic acid is significantly increased, and 0.05 to 0.15 mM salicylic acid is sprayed 2 to 4 times every 3 days, catechin hydrate, chlorogenic acid, caffeic acid, The contents of p-coumaric acid and ferulic acid are significantly increased, and when 0.2-0.4% (w/v) chitosan is sprayed 2-4 times every 3 days, catechin hydrate, chlorogenic acid, caffeic acid, p-coumaric acid , the content of ferulic acid, benzoic acid and quercetin is remarkably enhanced.

1 is a result of confirming the change in the content of phenylpropanoid in C. japonica by treating the elicitors of methyljasmonate (A), salicylic acid (B), or chitosan (C) once every 3 days for a total of 9 days at each concentration. . Control is a group that has not been treated with an elicitor.

The present invention relates to a method for increasing the content of phenylpropanoids in Chamnamul, comprising the step of treating the elicitor in Chamnamul.

The elicitor is methyl jasmonate, salicylic acid or chitosan, but is not limited thereto.

The elicitor may be treated 1 to 4 times at 3-day intervals, preferably 2-4 times at 3-day intervals, and more preferably 3 times at 3-day intervals, but limited thereto It is not.

The elicitor can be applied to any part of the plant, and is preferably sprayed on the leaves or stems of the plant, but is not limited thereto.

The elicitors methyl jasmonate and salicylic acid can be treated at a concentration of 0.05 to 0.6 mM, and chitosan can be treated at a concentration of 0.05 to 0.6% (w / v), preferably methyl jasmonate (methyl jasmonate), salicylic acid (salicylic acid) can be treated at a concentration of 0.05 ~ 0.15mM, chitosan can be treated at a concentration of 0.2 ~ 0.4% (w / v), more preferably methyl Jasmonate (methyl jasmonate), salicylic acid (salicylic acid) can be treated at a concentration of 0.1mM, chitosan can be treated at a concentration of 0.3% (w / v), but is not limited thereto.

Specifically, the method for increasing the phenylpropanoid content in Chamnamul of the present invention is to treat 0.05-0.15mM methyljasmonate 2-4 times at 3-day intervals, preferably 0.1mM methyljasmonate 3 times. It is processed three times at intervals of one day, but is not limited thereto.

In the above method, when methyljasmonate is treated with an elicitor, the content of at least one phenylpropanoid selected from among catechin hydrate, chlorogenic acid, caffeic acid, p-coumaric acid, and benzoic acid in Chamnamul is increased.

In addition, the method of increasing the phenylpropanoid content in Chamnamul of the present invention is specifically, 0.05 to 0.15 mM salicylic acid is treated 2 to 4 times at 3-day intervals, preferably 0.1 mM salicylic acid at 3-day intervals. It is to process sashimi, but is not limited thereto.

In the above method, when salicylic acid is treated as an elicitor, the content of one or more phenylpropanoids selected from among catechin hydrate, chlorogenic acid, caffeic acid, p-coumaric acid and ferulic acid in Chamnamul is increased.

In addition, the method of increasing the phenylpropanoid content in Chamnamul of the present invention is specifically, 0.2 to 0.4% (w / v) chitosan is treated 2 to 4 times at 3-day intervals, preferably 0.3% (w / v) /v) Chitosan is treated three times at 3-day intervals, but is not limited thereto.

In the above method, when chitosan is treated with an elicitor, the content of one or more phenylpropanoids selected from catechin hydrate, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, benzoic acid, and quercetin in Chamnamul is increased.

In addition, in the present invention, 0.05 to 0.15 mM methyljasmonate or 0.05 to 0.15 mM salicylic acid is treated 2 to 4 times at 3-day intervals or 0.2 to 0.4% (w / v) chitosan is treated with 3 It relates to a cultivation method of Chamnamata with enhanced phenylpropanoids content, including the step of treating 2 to 4 times at daily intervals.

In addition, the present invention relates to Chamnamul with increased phenylpropanoid content cultivated by the above method.

When methyl jasmonate is treated as an elicitor, the content of one or more phenylpropanoids selected from catechin hydrate, chlorogenic acid, caffeic acid, p-coumaric acid, and benzoic acid in Chamnamul is increased, and salicylic acid as an elicitor When treated, the content of one or more phenylpropanoids selected from among catechin hydrate, chlorogenic acid, caffeic acid, p-coumaric acid and ferulic acid in Chamnamul is increased, and when chitosan is treated with an elicitor, catechin hydrate, chlorogenic acid in Chamnamul , caffeic acid, p-coumaric acid, ferulic acid, benzoic acid and quercetin are characterized in that the content of one or more phenylpropanoids selected from is increased.

In addition, the present invention relates to a composition for increasing the content of phenylpropanoids in C. japonica, which contains, as an active ingredient, any one elicitor selected from methyljasmonate, salicylic acid and chitosan.

When the composition is treated with Chamnamul, the content of one or more phenylpropanoids selected from catechin hydrate, chlorogenic acid, caffeic acid, and p-coumaric acid is increased.

Hereinafter, the present invention will be described in more detail using preparation examples and examples. These Preparation Examples and Examples are only for explaining the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited thereto.

1. Materials and Methods

1) Treatment of plant materials and elicitors

Seedlings of Pimpinella brachycarpa Nakai were planted in a shaded house on a 120-150 cm wide ridge at 15-20 cm intervals in all directions, and then cultivated for 60 days. After that, an aqueous solution of methyl jasmonate to which 0.1% (v/v) Tween 20 was added was sprayed on the leaves and stems of C. japonica at concentrations of 0.1 mM, 0.3 mM, and 0.5 mM using a sprayer. It was sprayed once every 3 days, and harvested for comparison of useful components on the 3rd, 6th, and 9th days of spraying. The salicylic acid aqueous solution, which is another elicitor, has a concentration of 0.1 mM, 0.3 mM, and 0.5 mM, and the chitosan aqueous solution has a concentration of 0.1% (w / v), 0.3% (w / v), 0.5% (w / v). It was sprayed at the concentration of v) and proceeded in the same way as above.

2) Phenylpropanoid extraction and HPLC analysis

Each of the harvested Chamnamul was dried for 3 days at -70 ° C using a freeze dryer. For extraction and content analysis, the dried plants were finely ground with a grinder and made into a powder form, and then 100 mg was measured and transferred to a 15 mL conical tube. 80% (v / v) methanol (Samchun chemical, Seoul, Korea) was used as the extraction solvent, and 2mL was dispensed into the tube, mixed with vibration for 5 minutes, and then ultrasonically extracted at 37 ° C. for 1 hour (WUC-06H, DAIHAN scientific, Wonju, Korea). After ultrasonic extraction, centrifugation (12,000 rpm, 10 minutes, 4 ℃), and then the supernatant was filtered using a PVDF 0.45㎛ syringe filter (Advantec, Tokyo, Japan), and the filtrate was used as a sample for HPLC. used

The analysis conditions for phenylpropanoids are shown in Table 1 below, and for each component, a standard calibration curve was prepared as shown in Table 2 below using the average value of the area values occupied by peaks in the chromatogram. Standards used were all purchased from Sigma-Aldrich (Sigma-Aldrich Co., St. Louis, MO, USA). In the chromatogram of the extracts of Parsnip persimmon analyzed under the same conditions, the concentrations of the components in the extracts of Parsnip persimmon were determined by substituting the peak area value at the same retention time (Rt) as the standard product into the prepared standard calibration curve.

HPLC conditions for analysis of phenylpropanoid content HPLC Futecs HPLC system NS-4000
(Futecs Co. Ltd., Daejeon, Korea) column OptimaPak C18-51002546
(250mm x 4.6mm ID, particle size 5 μm)
(RStech Co. Ltd., Daejeon, Korea) mobile phase A solvent (0.15% (v/v) acetic acid water)
B Solvent (methanol) flow rate 1.0mL/min column temperature 30℃ injection volume 10 μl UV condition 280 nm Gradient elution time (minutes) A solvent (%, v/v) Solvent B (%, v/v) 0 - 1 95 5 1 - 9 85 15 9 - 24 80 20 24 - 54 70 30 54 - 65 55 45 65 - 75 44 56 75 - 80 40 60 80 - 90 20 80 90 - 105 95 5

standard calibration curve phenylpropanoids ceremony ^R2 Catechin Hydrate
(catechin hydrate) y=28025.39x-2223.87 0.9997 chlorogenic acid
(chlorogenic acid) y=52135.70x-57351.97 0.9991 caffeic acid
(caffeic acid) y=101427.93x-19999.04 0.9990 p-coumaric acid
(p-coumaric acid) y=172376.91x-2288.50 0.9991 ferulic acid
(ferulic acid) y=115029.24x-16713.48 0.9995 benzoic acid
(Benzoic acid) y=16424.67x+1917.49 0.9996 Routine
(rutin) y=25662.41x-3352.89 0.9990 Quercetin
(quercetin) y=40520.94x+12547.82 0.9994

3) Statistical analysis

Phenylpropanoid content values were expressed as mean ± standard deviation of triplicate. Phenylpropanoid values were analyzed using IBM SPSS statistics 26 software (IBM Corp., Armonk, NY, USA), and statistical significance was determined by Duncan's multiple range test (DMRT) at a significance level of p≤0.05. was evaluated by one-way analysis of variance (ANOVA) using .

2. Experimental results

1) Changes in the content of phenylpropanoids in C. japonica by treatment with methyl jasmonate (MeJA)

As a result of analyzing the phenylpropanoid content of the methyljasmonate-treated Chamnamoreum by HPLC, as shown in Table 3, catechin hydrate, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, benzoic acid, rutin and quercetin were found. A total of eight phenylpropanoids were detected.

As a result of comparing the sum of all detected contents of catechin hydrate, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, benzoic acid, rutin, and quercetin according to the concentration and duration of methyljasmonate treatment, 3 days at a concentration of 0.1 mM The phenylpropanoid content of the group treated with methyljasmonate for a total of 9 days at intervals was the highest at 13.78 mg per 1 g of dry weight (FIG. 1).

Different letters a-g in a column in Table 3 indicate a statistically significant difference, p < 0.05.

2) Changes in the content of phenylpropanoids in chamomile plants according to salicylic acid (SA) treatment

As a result of analyzing the phenylpropanoid content of the salicylic acid-treated C. japonica by HPLC, as shown in Table 4, catechin hydrate, chlorogenic acid, caffeic acid, p-coumaric acid, and ferulic acid increased, but benzoic acid, rutin, and quercetin decreased.

As a result of comparing the sum of all detected contents of catechin hydrate, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, benzoic acid, rutin and quercetin according to salicylic acid treatment concentration and duration, total The phenylpropanoid content of the group treated with salicylic acid for 9 days was the highest at 14.14 mg per 1 g of dry weight (FIG. 1).

Different letters a-f in the columns in Table 4 indicate statistically significant differences, p < 0.05.

3) Changes in phenylpropanoid content of Chamnamu according to chitosan treatment

As a result of analyzing the phenylpropanoid content of the chitosan-treated japonica by HPLC, all substances except for rutin tended to increase, as shown in Table 5.

As a result of comparing the sum of all detected catechin hydrate, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, benzoic acid, rutin and quercetin contents according to chitosan treatment concentration and period, at a concentration of 0.3% (w / v) The phenylpropanoid content of the group treated with chitosan for a total of 9 days at 3-day intervals was the highest at 14.63 mg per 1 g of dry weight (FIG. 1).

Different letters a-f in the columns in Table 5 indicate statistically significant differences, p < 0.05.

Claims (15)

A method for increasing the content of phenylpropanoids in Chamnamu, comprising treating Chamnamul with any one of the elicitors composed of methyl jasmonate, salicylic acid and chitosan. . delete The method according to claim 1, wherein the elicitor is treated 1 to 4 times at 3-day intervals. The method according to claim 1, wherein the elicitor is sprayed on leaves or stems. The method according to claim 1, wherein the methyljasmonate and salicylic acid have a concentration of 0.05 to 0.6 mM. The method of claim 1, wherein the chitosan is at a concentration of 0.05 to 0.6% (w/v). The method according to claim 1, wherein the elicitor is treated with 0.05 to 0.15 mM methyljasmonate 2 to 4 times at 3-day intervals. The method of claim 7, wherein the phenylpropanoid is selected from catechin hydrate, chlorogenic acid, caffeic acid, p-coumaric acid and benzoic acid. A method for enhancing the content of phenylpropanoids in Chamnamul, characterized in that at least one. The method according to claim 1, wherein the elicitor is treated with 0.05 to 0.15 mM salicylic acid 2 to 4 times at 3-day intervals. 10. The method of claim 9, wherein the phenylpropanoid is selected from among catechin hydrate, chlorogenic acid, caffeic acid, p-coumaric acid and ferulic acid. A method for enhancing the content of phenylpropanoids in Chamnamul, characterized in that at least one selected. The method according to claim 1, wherein the elicitor is treated with 0.2-0.4% (w/v) chitosan 2-4 times at 3-day intervals. The method of claim 11, wherein the phenylpropanoid is catechin hydrate, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, A method for enhancing the content of phenylpropanoids in Chamnamul, characterized in that at least one selected from benzoic acid and quercetin. 0.05~0.15mM methyljasmonate or 0.05~0.15mM salicylic acid is treated 2~4 times every 3 days, or 0.2~0.4% (w/v) chitosan is treated 2~4 times every 3 days on the leaves or stems of C. 4 times of treatment step; phenylpropanoids (phenylpropanoids) content is enhanced Cultivation method of Chamnamu containing. Chamnamul with increased phenylpropanoid content cultivated by the method of claim 13. A composition for enhancing the content of phenylpropanoids in C. japonica, containing as an active ingredient any one elicitor selected from methyl jasmonate, salicylic acid and chitosan.

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