KR100413394B1 - Extracting method of polyprenol and cultivating method of plant with it - Google Patents

Abstract

The present invention relates to a plant growth regulator for steaming and an extraction method thereof, and provides a plant growth regulator for steaming comprising polyprenol represented by the following formula (1).

By using the material of the present invention, it is possible to obtain a high productivity increase effect at a lower price than the existing growth regulators, and to increase the yield effect not only in the fruit and vegetable field but also in the larger grain field, which will contribute to the future agricultural development. Can be.

Description

Extracting method of polyprenol and cultivating method of plant with it}

The present invention relates to a plant growth regulator for steaming and an extraction method thereof, and more particularly, to a plant growth regulator capable of increasing the effect of plant growth, and a method for extracting such plant growth regulators from plants.

Currently, the world's population is increasing by 100 million people every year, but the world's farmland is limited, and therefore high density intensive production for maximum production is required. In particular, in Korea, since farmland is small due to the size of farmland, it is important to produce high quality products by increasing the output per unit area. In recent years, various agricultural chemicals and fertilizers have been produced by the development of the chemical industry, resulting in an increase in agricultural productivity. However, the abuse of such chemicals has caused problems such as ecosystem destruction and environmental pollution, and therefore, research and development on pesticides that can satisfy all safety aspects regarding toxicity and environmental pollution problems are required.

Pesticides are largely divided into insecticides, fungicides, herbicides and plant growth regulators. Insecticides, fungicides, herbicides, etc. are used to prevent the reduction of crops, while plant growth regulators play a role in increasing productivity and marketability by utilizing various physiological activities such as maturation, prevention of fallout, and reduction of shedding along with the increase of the plant itself. Therefore, the importance is increasing. Plant growth regulators are a type of plant hormone that is synthesized in plants and then migrated to affect each tissue and differentiation at extremely low concentrations. Plant hormones are synthesized in many parts and their effects are relatively wide.

On the other hand, unlike other insecticides, herbicides and fertilizers, such plant growth regulators have the characteristics of promoting the growth and development of plants in small amounts, and auxin (gixin), gibbereelic acid (cybbereelic acid), cytokinin ( cytokinin), abscisic acid, ethylene and bracinolide are known. Gibberellins and oxins are generally used as growth regulators for fruiting, but they are not limited to fruit growing and are not known. In addition, Rice, etc. (Rice et al, Science 195: . 1339-1341, 1977) , and the bar is confirming that the growth promoting effect of separating a substance called tree cones tanol (triacontanol), etc Joe (Joe et al, Science. The Ministry of Technology's research report "Study on Plant Growth Regulators" (1983) has applied this to corn, barley, rice and tomatoes. In addition, the yield increased by 14.8% -41% for rice, 83% for cabbage, and 108.4% for radish, but was also very expensive, and attempted to synthesize them to overcome these economic problems. A method for synthesizing tricontanol was developed by Rao et al. , Organic preparations and procedures International 24: 67-70, 1992, but has not yet been commercialized.

On the other hand, the recent research on the development of microorganisms for promoting plant growth, but does not show the effect as much as chemicals. In addition, attempts have been made to develop a transgenic plant using genetic engineering techniques to show the increase effect, but the increase in effect is not large and safety problems have not been proved and thus have not been put to practical use.

In response to these demands, the present inventors have completed the present invention by showing that the polyprenole substance stably enhances the effect of crop growth while conducting research on plant growth regulators.

An object of the present invention is to provide a plant growth regulator that can enhance the effect of increasing the plant.

Another object of the present invention is to provide a method for extracting the plant growth regulator from a plant.

1 is a chromatogram showing the result of separating the extract extracted from the cotton leaf using high pressure liquid chromatography (HPLC).

Figure 2 shows the mass spectrum (MASS spectrum) results of undecaprenol determined by the liquid chromatography mass spectrometer (HPLC-MASS) extracted from the cotton leaf.

Figure 3 shows the mass spectrum results of dodecaprenol obtained from the liquid extracted from the cotton leaf was confirmed using a liquid chromatography mass spectrometer (HPLC-MASS).

Figure 4 shows the NMR spectrum (NMR spectrum) results of the extract extracted from the cotton leaves obtained using proton-NMR.

Figure 5 shows the NMR spectrum results of the extract extracted from the cotton leaves obtained using ¹³ C-NMR.

In order to achieve the above object of the present invention, the present invention provides a method for growing a plant by applying a polyprenol represented by the formula (1) to the plant or its seeds.

In the above formula, n is preferably an integer of 9 to 12.

In addition, the present invention, in order to achieve the other object of the present invention,

1) extracting an organic solvent soluble substance from the plant leaf containing the polyprenol represented by the formula (1) using an organic solvent soluble in the polyprenol;

2) adding the organic solvent soluble material to the potassium hydroxide solution and further adding pyrogallol to the potassium hydroxide solution;

Removing the organic solvent from the solution by using anhydrous neutral salt and drying; And

And 4) purifying polyprenol from the dried organic solvent soluble material.

Hereinafter, the present invention will be described in detail.

Polyprenol having the structural formula of Formula 1 is a linkage of isoprenoids, isoprenoids are included in more than 30,000 compounds in a large group in nature, and is responsible for various biochemical functions. That is, it acts as a component of quinones in the electron transport system, components of microbial cell membranes, carotenoids, photosynthetic pigments such as chlorophyll, and hormones such as gibberellin and brassinosteroids (Taniguchi et al. , Proc. Natl. Acad). Sci. USA 97: 13172-13177, 2000). In addition, polyprenol is well soluble in organic solvents such as ethanol, chloroform, hexane and acetone, and is non-toxic and has a long term storage for 6-12 months (Stone et al ., Biochem. J. 102: 325-330, 1967).

The polyprenol having a structural formula of Formula 1 has the characteristic that four double bond carbons in the molecular structure may have cis-type and trans-type structures. The present invention is characterized by the use of such polyprenol as a plant growth regulator for steaming. In the polyprenol having the structural formula (1), n is an integer of 9 to 12, specifically, decaprenol, undecaprenol, dodecaprenol and tridecaprenol.

In using the polyprenol as a plant growth regulator for increasing water, fruit, such as tobacco, grapes, strawberries, tomatoes, cucumbers, corn, potatoes, sprouts, etc .; It can be applied to cereals, such as rice, barley, millet, wheat, chrysanthemum, roses, lilies, and specifically, in the present invention, it is applied to wheat, corn, and cucumbers.

In addition, the present invention is characterized by providing a method for extracting the polyprenol from the plant.

As a method of extracting the polyprenol from the plant, a method known in the art may be used, and specifically, in the present invention, an extraction method using an organic solvent is used. Looking specifically at the polyprenol extraction method of the present invention, first, cotton (cotton plant), horse chestnut (horse chestnut), tobacco (tobacco plant), lords and ladies (silver birch), ginkgo (ginko) Organic solvent solubles are separated from plants, such as soybean leaves, preferably cotton leaves, using organic solvents. Methanol, ethanol, propanol and benzene may be used as the organic solvent, but it is preferable to use ethanol and benzene.

Thereafter, an aqueous potassium hydroxide solution is added to the organic solvent soluble material. The reason why the aqueous potassium hydroxide solution is added is that polyprenol, which is generally found in plants, exists in the form of an ester combined with acetic acid, so it is necessary to make it in the form of alcohol through acid or base hydrolysis. It is also preferred to add pyrogallol together with potassium hydroxide solution. The organic solvent formed during the extraction process is removed and dried. The drying method is a method known in the art, that is, dried using anhydrous neutral salts such as anhydrous sodium sulfate, anhydrous magnesium sulfate and the like. The organic layer remaining after drying is concentrated, only the polyprenol is purified, and a general chromatography method is used as the purification method. As the chromatographic method, methods known in the art, such as LC, silica gel, Sephadex, LH-20, and the like, may be used. In the present invention, silica gel chromatography was used. In addition, in the chromatographic purification, a general solvent combination may be used as the developing solvent, but a hexane and hexane ethyl acetate combination is used in the present invention.

As a method for determining whether the purified polyprenole is a polyprenol according to the present invention having the structural formula represented by Formula 1, a known method known in the art may be used, and specifically, in the present invention, HPLC, NMR , MASS and IR spectral analysis. The polyprenol that can be isolated from the plant, specifically cotton, is undecaprenol (n = 10) or dodecaprenol (n = 11).

Thereafter, the yield of the plants treated with the plant growth regulator comprising polyprenol was compared with the yield of the plant control group not treated with the plant growth regulator. As a result of this comparison, when the plant growth regulator comprising polyprenol according to the present invention is treated to plants, it was confirmed that there is a 10% to 70% increase in water effect.

In one embodiment of the present invention, polyprenol was formulated to confirm the effect of water extraction of the extracted polyprenol. The polyprenol may be used as a formulation by mixing with a commonly used emulsifier, preferably Triton X 1000 may be used as an emulsifier.

In addition, as a method of treating the polyprenol solution to the plant seed, a general treatment method such as spraying may be used, but in the present invention, a seed immersion method is used. By using the seed immersion method as described above it is possible to considerably reduce labor costs, and also minimize the problems that may occur due to the wrong treatment time.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Isolation of Extract from Plant Leaves

10 g of dried cotton leaves were treated with 100 ml of 96% ethanol and 100 ml of benzene three times to separate soluble organic solvents. The extract was added to 90 ml of a solution containing 200 mg of pyrogallol and 50% sodium hydroxide and stirred at room temperature for one hour. The solution was diluted with distilled water and the separated benzene layer was separated using a separatory funnel. After washing with distilled water, anhydrous sodium sulfate (anhydrous Na ₂ SO ₄ ) was added and dried, and the remaining solution was concentrated using a rotary evaporator (Buchi R-205).

Example 2 Purification of Polyprenol from Extract

The extract extracted in Example 1 was purified to 90% or more purity. Using a combination of hexane and hexane ethyl acetate as the developing solvent, the material was separated by silica gel chromatography. Hexane was initially used as the developing solvent, and then the solvent was developed while increasing the polarity by increasing the amount of ethyl acetate. Each fraction was separated by UV absorbance measurement to obtain a UV profile, and then stored at low temperature by combining the same absorbance.

Afterwards, the chemical structure was analyzed by HPLC, MASS, NMR, and IR spectral analysis to confirm that the material was polyprenol.

First, the material was analyzed by high pressure liquid chromatography (instrument: Waters alliance system, column: μ-Bondapak 3.8X300 mm, mobile phase: 100% acetonitrile, moving rate: 1.0 ml / min), and the results were analyzed. 1 is shown.

Based on the above results, mass chromatography was performed using a liquid chromatography mass spectrometer (VG BIOTECH platform, ion source: ESI, resolution: 1000, mass range: 2-3000 (m / z)). 2 and 3 are shown.

In addition, the material was analyzed by using proton NMR and carbon ( ¹³ C) NMR in an NMR (FT-NMR (600 MHz), AVANCE 600, Bruker) instrument, and the results are shown in FIGS. 4 and 5, and IR The results of spectral analysis (MKh 1310 instrument, 150 ° C., 50 Vt) were as follows.

IR (KBr, cm ^-1 ): 3333, 2962, 2926, 1666

As a result of the analysis, it was confirmed that the purified material was polyprenol, specifically, n = 10 undecaprenol was obtained in 44.9% yield and n = 11 dodecaprenol in 42.4% yield. .

Example 3 Formulation of Polyprenol

The polyprenol extracted in Example 2 was used to formulate the material to verify the effect of plant growth. 10 mg of the material and 10 drops of Triton (Triton X 1000) as an emulsifier were mixed and then carefully mixed. The mixture was added to 1 L of distilled water.

Example 4 Verification Effect

4-1) Verification effect of increase in wheat

The wheat seeds were placed in a pail to 1 g or 10 g per tonne of polyprenol solution prepared respectively, and mixed for 3-5 minutes with a rotational speed of 68 revolutions per minute. The mixed seeds were then dried for 2-3 hours and seeded to 180-200 kg per hectare of land. The crop yield was calculated after 30 days, and the average value was repeated four times. As shown in Table 1, when compared with the control group not treated with polyprenol, the yield increased more than 40% compared to the control group for winter wheat, and the yield increased 12-23% compared to the control group for summer wheat. .

Yield Comparison of Wheat Rating Polyprenol (g) / wheat seed (ton) Wheat yield and growth rate Yield (ton / ha) % Increase Winter wheat One 2.82 43 10 2.87 46 Control 1.97 - Summer wheat One 6.28 23 10 5.71 12 Control 5.11 -

4-2) Verification of the effect of increase in maize

Except for using corn seeds instead of wheat seeds, the effect of polyprenol was verified according to the same method as Example 4-1), and the results are shown in Table 2 below.

Corn Yield Comparison Rating Polyprenol (g) / corn seed (ton) Corn yield and growth rate Yield (ton / ha) % Increase Corn hybrids1 One 4.82 -5.7 10 6.30 23.3 100 6.83 33.9 Control 5.11 - Corn hybrids2 One 8.60 0.5 10 8.62 1.2 100 9.42 10.5 Control 8.56 -

4-3) Verification effect of increase in cucumber

Except for using cucumber seeds instead of wheat seeds, the effect of polyprenol was verified according to the same method as Example 4-1), and the results are shown in Table 3 below.

Yield comparison of cucumbers Polyprenol (g) / cucumber seed (ton) Cucumber yield and growth rate Yield (ton / ha) % Increase 10 3.88 12.1 100 5.79 67.3 Control 3.46 -

As shown in Table 1, the polyprenol-treated wheat showed a 10% or more increase effect compared to the control mills not treated with polyprenol. In addition, as shown in Table 2, when corn treated with polyprenol showed a 10% or more increase effect compared to the untreated control. In the case of cucumber, when the seed immersed in the polyprenol solution was 100g, as shown in Table 3, the yield increase effect was more than 60%, and the cucumber was found to be an excellent yielding effect compared to other crops.

As described in detail above, the inventors have shown that plant growth regulators comprising polyprenol are effective in plant growth, in particular in water vapor. In addition, by using the polyprenol separated from the extract of the plant, it is possible to obtain a high productivity increase effect at a lower price than the existing growth regulators, and to increase the effect in the grain field, which is larger in the market than the fruit vegetable sector. Can contribute to agricultural development.

Claims (9)

A method of growing a plant by applying polyprenol represented by the following formula (1) to a plant or its seed. <Formula 1> (N is an integer of 9 to 12 in Formula 1) A method of growing a plant by applying polyprenol undecaprenol, dodecaprenol or a mixture thereof to the plant or seeds thereof. According to claim 1 or 2, wherein the plant is tobacco, grapes, strawberries, tomatoes, cucumbers, corn, potatoes and bean sprouts selected from the group consisting of sprouts; Cereals selected from the group consisting of rice, barley, corn, sorghum and wheat; Or, applied to a flower selected from the group consisting of chrysanthemums, roses and lilies. The method of claim 3, wherein the polyprenol is extracted from any one selected from the group consisting of cotton, horse chestnut, tobacco, Tiannan, birch, ginkgo and soybean. 1) extracting an organic solvent soluble substance from a plant leaf containing polyprenol represented by the following formula (1) using an organic solvent in which the polyprenol is soluble; 2) adding the organic solvent soluble material to the potassium hydroxide solution and further adding pyrogallol to the potassium hydroxide solution; 3) removing the organic solvent from the solution using anhydrous neutral salt and drying; And And 4) purifying the polyprenol from the dried organic solvent soluble material. <Formula 1> (N in Formula 1 is an integer of 9 to 12.) 6. The method of claim 5, wherein the plant is any one selected from cotton, horse chestnut, tobacco, tiannan, birch, ginkgo or soybean. 6. The method of claim 5, wherein the organic solvent of step 1) is ethanol or benzene. 6. The method of claim 5 wherein the drying of step 3) is by anhydrous sodium sulfate. 6. The method of claim 5, wherein the developing solvent used in the purification process of step 4) is hexane and hexane ethyl acetate.