KR20190136631A - Marker compound for diagnosis of plants withering to death and method of diagnosing growth sate using the same - Google Patents

Abstract

The present invention relates to a marker material for diagnosing withering of plants and a method for diagnosing growth of plants using the same. Among secondary metabolites, separation purified from an extract of abies Koreana which is a variety of needleleaf trees growing in Korea, a trans-piceid component or a resveratrol component shows expression ratio difference caused by non-biological environmental factors such as average temperature, precipitation, wind speed, and the like by habitat and time. Thus, the component is used as a marker material for diagnosing withering of plants, and a method for diagnosing growth of plants using the same is established. Accordingly, the method can be usefully used for protecting and managing evergreen needleleaf forest in national parks which are weak to weather change.

Description

Marker material for solid photo of trees and method for diagnosing growth of trees using same {{MARKER COMPOUND FOR DIAGNOSIS OF PLANTS WITHERING TO DEATH AND METHOD OF DIAGNOSING GROWTH SATE USING THE SAME}

The present invention relates to an indicator material for photographing trees and a method for diagnosing the growth of trees using the same. More specifically, among seedwood secondary metabolites separated and purified from Abie Koreana extract, which is a domestic conifer species, ( Trans -Piceid) or Resveratrol component shows difference in expression level according to environmental factors such as average temperature, precipitation, and wind speed depending on the habitat and time period, and the growth of trees and indicators for photorealism of trees using the same It is about a diagnosis method.

As part of the conservation project for the protection of evergreen coniferous forests in national parks that are vulnerable to climate change, tree rings analysis studies on dead trees have been conducted to identify the causes of collective deaths of evergreen conifers in the national park.

A tree ring analysis study secures growth information before the tree dies, and the tree ring tree tree analysis will help to identify the causes of dead trees in living trees and to establish a scientific management system for evergreen conifers.

In general, plants produce more than 4,000 triterpenoids secondary metabolites, which, unlike carbohydrates and proteins, do not directly participate in growth, proliferation, or life support, but they do not contribute to their own survival. Organic compounds produced to create favorable conditions are commonly referred to as plant secondary metabolites.

Since the main purpose of the plant to produce secondary metabolites is to have exclusivity as it is self-defense and the exclusion of competitors, there are some strong toxic or hallucinogenic components, but if used well, it is very beneficial to the human body. There are many ingredients to say.

Thus, secondary metabolites of plants are small molecule compounds that have specific physiological activities but are not involved in basic metabolic processes.

In the early days when plant secondary metabolites began to attract attention, polyphenols, which are widely known for their antioxidant effects, were mentioned, but recently, polyphenols are divided into catechin, anthocyanin, resveratrol, quercetin, and tannin, depending on their characteristics. As many diverse plant secondary metabolites are used for each purpose.

In addition, secondary metabolites such as alkaloids, steroids, terpenoids, and phenolics produced by plants include pharmaceuticals, dyes, pigments, flavorings, pesticides, hormones, and food additives. It is used. Thus, although many compounds have been synthesized by the development of organic synthesis technology, plants are still in the spotlight as a source of new substances.

However, plant secondary metabolites are directly extracted from plants because of their complex biosynthetic pathways, but they are low in content and show a great difference in productivity depending on season, place, climate, cultivation conditions, plant parts, etc. I can't.

Abies Species, a conifer species, tends to prefer cold and humid environments, and has long been considered a natural material for materials such as resins, and commercially available pulp wood, Christmas trees, wildlife habitats, timber, Increasingly important as a resource, including fragrances and natural medicine. Accordingly, there is a growing interest in habitat differentiation and biodiversity as a result of human activity and climate change.

Also, among Abies Species, Abies Koreana, Korean fir) is a perennial evergreen conifer tree of pineaceae, which grows at 500 ~ 2,000m above sea level in Halla, Jirisan, Deogyusan, and Mudeungsan, and is a special plant that grows only in Korea. Until recently, the physiological activity of secondary metabolites isolated from spherical trees has been reported to be applied to various fields using antibacterial, antibacterial and antioxidant activities, but using spherical tree extracts (Patent Document 1), There is only a report of the degree to which a method for separating a single component of a secondary target material from an extract (Patent Document 2) is proposed.

Therefore, the present inventors have tried to utilize the secondary metabolites of plants that are produced to make the conditions favorable for the survival of the plants themselves, as a result, the spherical tree leaves according to the habitat area and dead zones and periods with good growth of the spherical trees in the national park By extracting the spherical tree extract, and analyzing the correlation by environmental factors such as average temperature, precipitation, wind speed, and quantify the expression amount of the spheroid secondary metabolites separated from the spherical tree extract, The present invention was completed by confirming that it can be utilized as an indicator material for diagnosing the growth or dead environment of trees.

Republic of Korea Patent Publication No. 2015-0057333 (2015.05.28 published) Republic of Korea Patent Publication No. 2014-0141593 (released Dec. 10, 2014)

An object of the present invention is to provide an indicator material for photographic algae using spherical tree secondary metabolites purified from Abie Koreana extract.

It is another object of the present invention to provide a method for diagnosing growth of trees using indicator material for photodiagnosis of trees.

In order to achieve the above object, the present invention utilizes a spherical tree secondary metabolite separated and purified from Aies Koreana extract, which is represented by a trans- Piceid component represented by Formula 1 or Formula 2 It provides a marker for planting of dead trees using resveratrol.

Formula 1

Formula 2

The present invention 1) sample preparation step of washing and drying the spherical tree collected by habitat;

2) filling the prepared sample in a test tube divided by habitat and extracting by sequential solvent circulation to obtain a spherical tree extract for each habitat; and

3) HPLC analysis of the spherical tree extract for each habitat, using the indicator of the trans- Piceid component represented by the formula (1) or the resveratrol component represented by the formula (2) as a reference material in the analysis process, It provides a method for diagnosing the growth of trees consisting of determining whether trees die or not according to the presence of the indicator material.

In the growth diagnostic method of the present invention, the spherical tree collected by the habitat in step 1) is preferably collected in July to October.

In addition, in step 2), the spherical tree extract is a spherical tree leaf extract, and in step 2), the sequential solvent circulating method is a single organic solvent is sequentially added from the non-polar solvent to the polar solvent, preferably hexane, ethyl acetate and The organic solvent selected from the group consisting of methanol is sequentially added to obtain a spherical tree extract.

According to the present invention, there is provided a spherical tree extract containing secondary metabolites from the extract of Abie Koreana , and by environmental factors such as average temperature, precipitation, and wind speed by habitat and time in the spheroid secondary metabolites Trans- Piceid component or Resveratrol component, which showed a difference in expression level, may be used as an indicator for photophyllosis of globular tree.

Therefore, by establishing a method for diagnosing the growth of trees, which are used as indicator materials for photorealism of trees, they can be usefully used for the protection and management of evergreen coniferous forests in national parks that are vulnerable to climate change.

1 is a calibration graph for quantifying the expression level of secondary metabolites contained in the spherical tree extract of the present invention,
2 It is the result of quantifying the expression amount according to time and region for the seed component selected as the indicator for the photogram of the present invention,
Figure 3 is a result of quantifying the expression amount according to time and region for the resveratrol component selected as an indicator material for a photogram of the present invention,
4 is a result of analyzing the correlation between the seasonal average, monthly average, monthly average, previous month's average precipitation data and the amount of change in secondary metabolites for the seed component and resveratrol component selected as the index photo material for photomontage of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention utilizes purified spherical tree secondary metabolites separated from Aies Koreana extract, but utilizes a trans- Piceid component represented by the following Chemical Formula 1 or a resveratrol component represented by the Chemical Formula 2 Provide an indicator material for a photomontage.

Formula 1

Formula 2

The present invention 1) sample preparation step of washing and drying the spherical tree collected by habitat;

2) filling the prepared sample in a test tube divided by habitat and extracting by sequential solvent circulation to obtain a spherical tree extract for each habitat; and

3) HPLC analysis of the spherical tree extract for each habitat, using the indicator of the trans- Piceid component represented by the formula (1) or the resveratrol component represented by the formula (2) as a reference material in the analysis process, It provides a method for diagnosing the growth of trees consisting of determining whether trees die or not according to the presence of the indicator material.

In step 1), the habitat is for subalpine ecosystem analysis, in the embodiment of the present invention, Yeongsil (Horse Spring, altitude 1500-1650m), Wiseoum (Eorimok, altitude 1650-1700m) in Hallasan National Park, azalea Three fields of the field (Seongpanak, altitude 1500-1650m), and three places in the Jirisan National Park (Jurim, altitude 1500-1600m) and Banyabong (Rimyeong-ryeong, altitude 1400-1500m) are designated and collected. For example, spherical trees may be harvested if they are native to Korea.

The harvesting time is divided into spring (April), summer (July) and autumn (October), and the trees are produced by trans- Piceid or Resveratrol, a secondary metabolite. When conducting a photographic group, it is easy to diagnose if the harvest time is collected in July or October.

In addition, the sample site is carried out by preventing the damage of the entire plant due to the collection and collecting the leaves of the spherical tree for ease of collection. Therefore, the globular tree extract in step 2) is a globular leaf extract.

In addition, while the sample prepared in step 2) is filled in a test tube, a cylindrical filter for soxhlet (Soxhlet) extraction is filled in a (thimble filter).

After sequential extraction by solvent circulation, the entire sample filled in the cylindrical filter is in contact with the evaporated solvent and the solvent is cooled again for a long time, so as to separate by the polarity and solubility difference of the solvent. do.

At this time, in the sequential solvent circulation method, a single organic solvent is sequentially added from the nonpolar solvent to the polar solvent to obtain a spherical tree extract. Preferably, an organic solvent selected from the group consisting of hexane, ethyl acetate and methanol is sequentially added.

In particular, hexane is useful for separating substances such as lipids / fatty acids, and thus, by extracting the fat using hexane from the leaves of conifers containing a lot of essential oils, the substance is added in the order of ethyl acetate and methanol. It can be easily extracted and separated.

More preferably, after extraction of lipids / fatty acids through hexane, ethyl acetate and methanol are added and extracted.

In addition, in the ethyl acetate or methanol extract of the spherical tree extract, the seed component represented by Formula 1 or the resveratrol component represented by Formula 2 is separated and purified.

The spheroidal tree secondary metabolites are expressed in the spherical tree extract obtained in step 2). At this time, the component analysis through HPLC using known secondary metabolites contained in the spherical tree as a standard, Taxifolin, Kempferol-3-glucoside, p-coumaric acid, 4-Hydroxybenzoic acid, and gallic acid. Protocatechuic acid and dehydroabietic acid have been identified, and maltol, trans- Piceid and Resveratrol components, in addition to the above standards, are spectroscopically analyzed. Confirmed.

In addition, as a result of analysis of spherical tree leaf extracts collected from Halla and Jirisan to examine the correlation between the secondary metabolites contained in spheroidal tree extracts, Pseed ( piceid, resveratrol, maltol, protocatechuic acid, 4-hydroxybenzoic acid, taxifolin, dehydroabietic acid ), The expression of para-coumaric acid (p-coumaric acid), camphorol-3-glucoside (keampferol-3-glucoside) was confirmed.

In addition, the correlation analysis showed that the expression level of thaxipoline was highly correlated with the temperature condition of two months before the sample rats, and the expression level of para-coumaric acid was two months ago, and that of the seed and resveratrol components The amount of expression was analyzed to be affected by the conditions of precipitation at the time of sampling.

1 is a calibration graph for quantifying the amount of expression of the secondary metabolites contained in the extract of the spherical tree, the expression amount for the seed component and resveratrol component showing a significant difference in the expression amount in the climate change of the secondary metabolites Digitize

In FIGS. 2 and 3 As confirmed, the seed component and the resveratrol component selected as the indicator group for the photographic group of the present invention, each component detection and increase tendency is confirmed in Yeongsil and Wittseom of Hallasan National Park, and Seseok of Jirisan National Park. In the Rhododendron fields of the National Park and the Banyabong Peak of Jirisan National Park, the expression level of the seed component and the resveratrol component decreased significantly.

In particular, in FIG. 4 As confirmed, the extracted seed component and resveratrol component were expressed according to seasonal average, monthly average, previous month average, previous month average precipitation data and analysis of correlation between changes in secondary metabolites and seasonal average and monthly average precipitation changes. Volume showed a significant difference.

Therefore, the cause of death of coniferous trees, including spherical trees, is due to the increase of moisture stress in trees due to climate change, which is estimated to be due to lack of moisture and humidity due to the degradation of photosynthesis.

This is in line with the result of the survey of dead tree in the azalea field of Hallasan National Park and Banyabong of Jirisan National Park, and the selection of the nobular tree growing area according to the presence of seed or resveratrol component of secondary tree metabolites As it is possible to judge and grow trees, it can be useful for the protection and management of evergreen coniferous forests in national parks that are vulnerable to climate change.

Hereinafter, the present invention will be described in more detail with reference to production examples.

This manufacture example is for demonstrating this invention more concretely, The scope of the present invention is not limited to these manufacture examples.

<Example 1>

Spring 2017 (April), Summer (July), Autumn (10) in Yeongsil (Horse Spring, altitude 1500-1650m), Witteoorum (altitude 1650-1700m), Azalea field (altitude 1500-1650m) at Hallasan National Park The leaves of the globular tree were collected in the month.

In addition, the leaves of spherical trees were collected in the spring (April), summer (July), and autumn (October) in Seseok (1500-1600m altitude) and Banyabong (1400-1500m altitude) areas in Jirisan National Park. .

Five spherical spherical leaves collected in each region were naturally dried and chopped to fill the chopped samples in a Soxhlet reactor including a cylindrical filter, and hexane was added to obtain a hexane extract, followed by ethyl acetate. Then, ethyl acetate extract was obtained, and then methanol was sequentially added, and then methanol extract was obtained, and spherical tree extract was obtained from samples collected by region.

Experimental Example 1 Component Analysis of Secondary Metabolites

For the component analysis of the spherical tree extract obtained in Example 1, the component was analyzed using HPLC (Dionex U-3000 UHPLC).

At this time, the reference material was selected from the secondary metabolite analysis data distributed in known Abies spp. At this time, as a standard material, two kinds of taxifolin, keempferol-3-glucoside as flavonoid components, para-coumaric acid as a ferrolic component, Three types of 4-hydroxybenzoic acid and Protocatechuic acid, and one type of dehydroabietic acid were selected as steroid components.

HPLC analysis conditions:

Column: YMC-Triart C18, 5 μm, 250 × 4.6 mm

LC gradient: 100% A Buffer 2 minutes / 0 to 100% B Buffer 33 minutes / 100% B Buffer 8 minutes

Flow rate: 1 ml / min

Wavelength: 210nm, 254nm, 280nm, 340nm

Temperature: 35 ℃

Injection volume: sample (10 μl)

LC buffer solution: ① A Buffer: H ₂ O, B Buffer: MeOH

② A Buffer: H ₂ O + 0.04% (Trifluoroacetic acid, TFA), B Buffer: MeOH + 0.04% TFA

As a result of the HPLC component analysis, six secondary metabolites of the selected standard materials were identified, and in addition to six, three analytes were found, and mass spectrometry and nuclear magnetic resonance analysis were performed.

Thus, the product was separated and purified by a trans- Piceid component represented by Chemical Formula 1 [ESIMS m / z 391.4 [M + H] ⁺ , UV λ _max (nm): 216.85, 318.62; ¹ H-NMR (600 MHz, CD ₃ OD-d ₄ ), δ = 7.36 (2H, d, J = 8.4 Hz, H-2 ′, 6 ′), 7.02 (1H, d. J = 16.2 Hz, H -8), 6.84 (1H, d, J = 16.2 Hz, H-7), 6.77 (1H, t, J = 1.2 Hz, H-2), 6.76 (2H, d, J = 9.0 Hz, H-3 ′, 5 ′), 6.62 (1H, t, J = 1.2 Hz, H-6), 6.45 (1H, t, J = 1.8 Hz, H-4), 4.90 (1H, d, J = 7.8 Hz, H -1 ″), 3.94 (1H, dd, J = 1.8, 12.0 Hz, H-6a ″), 3.72 (1H, dd, J = 6.0, 12.0 Hz, H-6b ″), 3.46 (3H, m, H -2 ", 5", 3 "), 3.39 (1H, m, H-4"), ¹³ C-NMR (150 MHz, CD ₃ OD-d ₄ ) δ = 160.5 (C-3), 159.9 (C -5), 158.9 (C-4 ′), 141.5 (C-1), 130.3 (C-1 ′), 130.0 (C-8), 128.9 (C-2 ′, 6 ′), 126.7 (C-7 ), 116.7 (C-3 ′, 5 ′), 108.5 (C-6), 107.0 (C-2), 104.2 (C-4), 102.5 (C-1 ″), 78.3 (C-5 ″), 78.1 (C-3 ″), 75.0 (C-2 ″), 71.6 (C-4 ″), 62.7 (C-6 ″)].

Formula 1

In addition, the residue was purified by the Resveratrol component represented by the formula (ESIMS m / z 229.3 [M + H] ⁺ , UV λ _max (nm): 217.35, 309.38); ¹ H-NMR (600 MHz, CD ₃ OD-d ₄ ), δ = 7.34 (2H, d, J = 8.4 Hz, H-2 ′, 6 ′), 6.94 (1H, d, J = 16.8 Hz, H -8), 6.78 (1H, d, J = 16.8 Hz, H-7), 6.76 (2H, d, J = 9.0 Hz, 3 ', 5'), 6.44 (2H, d, J = 1.8 Hz, H -2, 6), 6.16 (1H, d, J = 0.6 Hz, H-4)

¹³ C-NMR (150 MHz, CD ₃ OD-d4) δ = 159.8 (C-3), 159.7 (C-5), 158.7 (C-4 ′), 141.4 (C-1), 130.4 (C-1 ′), 129.5 (C-8), 128.9 (C-2 ′, 6 ′), 127.0 (C-7), 116.7 (C-3 ′, 5 ′), 105.8 (C-2, 6), 102.8 ( C-4)].

Formula 2

Experimental Example 2 Quantitative Analysis of Expression of Secondary Metabolites

The assay graph was prepared by quantifying the expression level of each of the nine secondary metabolites separated and purified from the spherical tree extract identified in Experimental Example 1. In this case, the nine secondary metabolites are taxoxyline, camphorol-3-glycoside, para-coumaric acid, 4-hydroxybenzoic acid, protocatechuic acid, dihydroabietic acid, maltol, and pisid. It was resveratrol.

Especially for the seed component and the resveratrol component, which have been separated and purified, FIG. Figure 3 shows the results of quantifying the expression amount according to time and region for the pisid component, Figure 3 shows the results of quantifying the expression amount according to time and region for the resveratrol component. As a result, as shown in FIG. 2, the purified seed components separated from spherical tree extracts collected from Yeongsil, Witseum and Azalea fields of Hallasan National Park in April and July 2017 were Youngsil, Witseoreum and Hallasan National Park. The azalea field showed a high expression level, but in the samples collected in October, the components of Yeongsil and Upseoreum increased significantly, whereas the aphids did not express the seed component.

In addition, the phidicide component separated from spherical tree extracts collected from Jirisan National Park in April and July 2017 was extremely low in both samples collected at Banyabong in April and July, In addition, the trend increased in April and July.

In addition, through the results of FIG. 3, the resveratrol component purified from spherical tree extracts collected from Yeongsil, Witseum and Rhododendron fields of Hallasan National Park in April 2017 is more resveratrol than the samples collected in July and October. Resveratrol component was not expressed at all, especially when it was collected from azalea fields in October.

On the other hand, the resveratrol component, which has been purified from spherical tree extracts collected from Jirisan National Park in April and July, 2017, shows a sharp decrease in the amount of expression in Banyabong, but the resveratrol component in cesite is 7 times lower than in April. The expression level improved in the month.

From the above, it was confirmed that the seed component and the resveratrol component, the secondary metabolite purified from the spherical tree extract of the present invention, showed a difference in expression level due to non-biological environmental factors such as average temperature, precipitation, and wind speed. . In particular, as a result of the survey on plant growth, as a result of dead wood in azalea field in Hallasan National Park and Banyabong in Jirisan National Park, and as a result, the seed and resveratrol components, which are secondary tree metabolites, are not expressed in the area. In accordance with the present invention, the spherical tree secondary metabolites separated and purified in the present invention may be used as an indicator material for diagnosing the growth or dead environment of the spherical tree depending on whether the spherical tree secondary metabolites are expressed by time and region. .

Therefore, it is possible to judge the selection of spherical tree growth area according to the presence of seed or resveratrol component among the secondary metabolites of spherical tree, and it is possible to diagnose the growth of trees. And it can be usefully used for management.

Although the present invention has been described in detail only with respect to the described embodiments, it will be apparent to those skilled in the art that various modifications and variations are possible within the technical scope of the present invention, and such modifications and modifications are within the scope of the appended claims.

Claims (6)

Spruce tree ( Aies Koreana ) Using a purified spherical tree secondary metabolites separated from the extract, characterized in that the tree for the photogenic step material, characterized in that the trans- Piceid component represented by the following formula (1).
Formula 1

The spherical tree ( Aies Koreana ) using a spherical tree secondary metabolite purified from the extract, characterized in that the resveratrol component represented by the following formula (2) indicators for the photomontage of trees.
Formula 2

1) a sample preparation step of washing and drying the spherical tree collected by the habitat and cutting it;
2) filling the prepared sample in a test tube divided by habitat and extracting by sequential solvent circulation to obtain a spherical tree extract for each habitat; and
3) HPLC analysis of the spherical tree extracts by habitats, in the process of analysis to determine whether or not the dead tree of the tree according to the presence of the indicator material using the indicator material for the photo of the tree of claim 1 or 2 as a reference material. Growth diagnosis method of the tree which consists of steps. 4. The method for diagnosing growth of trees according to claim 3, wherein the spherical trees collected by the habitat in step 1) were collected in July to October. The method for diagnosing growth of trees according to claim 3, wherein the tree leaf extract is obtained in step 2). 4. The method for diagnosing growth of trees according to claim 3, wherein in the sequential solvent circulation method, a single organic solvent is sequentially added from a non-polar solvent to a polar solvent to obtain a spherical tree extract.

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