KR20240000120A - Biomarker for the discriminating geographical origins of codonopsis lanceolata and method for discriminating geographical origin using the same - Google Patents

Abstract

The present invention relates to a metabolite marker for determining the origin of deodeok and a method for determining the origin using the same. Specifically, metabolites showing differences in expression levels in domestic and Chinese deodeok are analyzed to select metabolite markers suitable for determining the origin, and these are used to determine the origin. This is about how to determine the country of origin of domestic and Chinese commercial deodeok.

Description

Biomarker for determining the origin of deodeok and method for determining the origin using the same

The present invention relates to a metabolite marker for determining the origin of deodeok and a method for determining the origin using the same. Specifically, the present invention relates to a metabolite marker for determining the origin of deodeok that shows a difference in expression level in domestic and Chinese deodeok, and a metabolite marker for determining the origin of deodeok produced in Korea and China using the same. It's about how to determine.

Country of origin refers to the country, region, or sea area where agricultural or marine products were produced, collected, or caught (Article 2, Paragraph 4 of the Act on Labeling of Origin of Agricultural and Fishery Products, etc.). In accordance with the Foreign Trade Act, Korea introduced the country of origin labeling system for imported and exported goods in 1991, and subsequently enacted and implemented the Act on Country of Origin Labeling for Agricultural and Fishery Products in 2010. The expansion of free trade agreements (FTAs) around the world has increased the possibility that agricultural products can freely cross the borders of each country, but there are cases where agricultural products are sold with forged origins, disrupting market order and causing consumer distrust. Accordingly, a method to scientifically and objectively determine the origin of agricultural products is needed.

To date, the country of origin of agricultural products has been determined mainly by isotope-ratio mass spectrometry (IRMS), HPLC, visible micro-Raman spectroscopy, and ultraviolet-visible absorption spectroscopy (ultraviolet). -visible absorption spectroscopy, UV-vis), and elemental analysis-like inductively coupled plasma-atomic emission spectrometry (ICP-AES). However, these research methods all have limitations in that they are targeted approaches that focus on specific ingredients or groups.

Meanwhile, metabolomics uses various analytical instruments such as nuclear magnetic resonance (NMR), near-infrared radiation (NIR) spectrometer, and capillary electrophoresis (CE). It is a study that extensively analyzes and compares metabolites existing in living organisms through statistical algorithms. Existing metabolome research has been mainly limited to analyzing metabolites for the treatment of diseases and extension of human life. However, by analyzing metabolites, it is possible to efficiently identify genetic differences in agricultural products or differences in environmental changes, so recently, Applications are also increasing in fields such as determining the origin and quality of agricultural products and medicinal plants.

1. Republic of Korea Patent Publication No. 10-2020-0136669

In the above situation, the present inventors studied a method to distinguish between domestic and Chinese deodeok and discovered 15 types of metabolites that showed significant differences in levels depending on the place of origin and completed the present invention.

Therefore, the purpose of the present invention is to provide a metabolite marker composition for determining the origin of Deodeok containing the above 15 metabolites, a kit containing the same, and a method for determining the origin of Deodeok.

In order to achieve the above object, one aspect of the present invention is adenosine, alpha-ketoglutarate, ascorbic acid, aspartate, dimethylarginine, and gallic acid. (gallic acid), gentisic acid, sinapic acid, tangshenoside II, 7-hydroxycoumarin, acetylcholine, caffeoylquinic acid ( Provided is a metabolite marker composition for determining the origin of Deodeok, which includes one or more metabolites selected from the group consisting of caffeoylquinic acid, quinic acid, raffinose, and tyrosine.

The metabolite marker composition for determining the origin of deodeok of the present invention is characterized by being able to determine the origin of deodeok using the difference in the level of deodeok metabolites depending on the origin. The expression level of the above metabolite means that it is highly or low expressed in imported deodeok compared to domestically produced deodeok.

Codonopsis lanceolata Trautv. is a perennial vascular plant belonging to the Campanulaceae family and is widely distributed in East Asia, including Korea and China. The young leaves are boiled and eaten as a vegetable or wrap, but the roots are mainly used for edible and medicinal purposes.

Therefore, the deodeok may refer to the root part of the deodeok.

The term "country of origin" used in this specification refers to the country, region or sea area where agricultural or marine products were produced, collected or caught (Article 2, Paragraph 4 of the Act on Labeling of Origin of Agricultural and Fishery Products, etc.). Korea's country-of-origin labeling system has been in effect since July 1, 1991, and the Foreign Trade Act stipulates regulations on “origin determination standards,” “goods subject to country-of-origin labeling,” and “penalties for violations.”

The term “identification” used in this specification means determining and distinguishing the origin of deodeok. According to one embodiment of the present invention, the discrimination may be to distinguish domestically produced deodeok from Chinese deodeok.

As used herein, the term “level difference” means that the level of a specific metabolite is high or low in the deodeok being compared.

According to one embodiment of the present invention, the metabolite marker composition for determining the origin of deodeok includes adenosine, alpha-ketoglutaric acid, ascorbic acid, aspartic acid, dimethylarginine, gallic acid, gentis acid, sinapic acid, and tangsenoic acid. It contains one or more metabolites selected from the group consisting of seed II, 7-hydroxycoumarin, acetylcholine, caffeoylquinic acid, quinic acid, raffinose and tyrosine.

In the present invention, in order to determine the origin of deodeok, the level of metabolites isolated from deodeok must be measured, so the metabolite marker composition for determining the origin of deodeok of the present invention adds one or more agents capable of detecting the metabolite markers. It can be included as .

According to one embodiment of the present invention, among the metabolite markers, the levels of adenosine, alpha-ketoglutaric acid, ascorbic acid, aspartic acid, dimethylarginine, gallic acid, gentis acid, sinapic acid, and tangsenoside II are found in Korean. Compared to deodeok, it is characterized by a decrease in Chinese deodeok.

In addition, among the above metabolite markers, the levels of 7-hydroxycoumarin, acetylcholine, caffeoylquinic acid, quinic acid, raffinose, and tyrosine are characteristically increased in Chinese deodeok compared to Korean deodeok.

Another aspect of the present invention provides a kit for distinguishing between domestic and Chinese deodeok, including a metabolite marker composition for determining the country of origin of the deodeok.

The kit for distinguishing between domestic and Chinese deodeok of the present invention can be used to determine the country of origin of a deodeok sample by detecting or quantitatively analyzing the presence of the above metabolites in a deodeok sample of suspected origin, but is not particularly limited thereto.

In addition, the kit for distinguishing between domestic and Chinese deodeok may include not only direct means for detecting and quantifying metabolites, but also other components, solutions or devices used in the analysis method. Examples may include test tubes, containers, reaction buffers, enzymes for analysis, sterilized water, etc.

The kit of the present invention may include a conventional well-shaped microtiter plate capable of carrying a sample. The well may contain a porous support capable of absorbing a sample and one or more metabolite markers. The support contains internal biomarkers at a predetermined concentration in preparation for the addition of Deodeok metabolites, and can also fix each sample. Additionally, the porosity of the support allows maximum exposure to the extraction solvent added during sample analysis.

Accordingly, the support may be any support having a high level of porosity, such supports being known in the art or commercially available.

Specifically, it may be a solid support. More specifically, it is made of a material absorbent for liquid. The absorbent material may be an adsorbent or an adsorbent material.

The liquid absorbent material allows the solution of the metabolite marker and subsequent samples for analysis to be constantly adsorbed or absorbed through the pores.

The absorbent material may include one or more of carbohydrate materials such as cellulose, glass fiber, glass beads, polyacrylamide gel, porous plastic inert polymer, and porous graphite. More specifically, it may include carbohydrate substances such as agarose, agar, cellulose, dextran, chitosan, or konjac, or their derivatives, cannaginan, gellan, or alginate. Most specifically, it may be cellulose or glass fiber.

The metabolite marker included in the kit of the present invention can be understood as an absolute amount of a comparative substance of known amount that is used as a comparison with similar or identical analogues to quantify the amount of metabolites present in a Deodeok sample. The metabolite marker may be isotopically labeled for proper differentiation from the metabolite of the sample.

The types of metabolite markers are as described above.

In addition, samples that can be used in the kit of the present invention are metabolite extracts by origin or part of Deodeok, and can be provided in the kit in the form of a liquid sample.

The metabolite extract can be obtained by homogenizing Deodeok in a mixed solvent of ethanol and water, but is not particularly limited thereto.

In addition, the microtiter plate may additionally include a filter and outlet for discharging the analyte, and their arrangement, discharge method, etc. can be adjusted within a range that can be understood by those skilled in the art.

In addition, the kit of the present invention may further include reagents, solvents, software systems, etc. included in a device that can prepare various metabolites for quantitative and targeted analysis of a range of metabolites in combination with an analysis device.

Another aspect of the present invention provides a method for distinguishing domestically produced deodeok from Chinese deodeok comprising the following steps:

(a) extracting metabolites from deodeok that has been confirmed to be domestic or Chinese and a test deodeok whose origin is unknown;

(b) Among the metabolites extracted in step (a), adenosine, alpha-ketoglutaric acid, ascorbic acid, aspartic acid, dimethylarginine, gallic acid, gentis acid, sinapic acid, tangsenoside II, 7-hydride Analyzing the level of one or more metabolites selected from the group consisting of roxicoumarin, acetylcholine, caffeoylquinic acid, quinic acid, raffinose, and tyrosine;

(c) Comparing the metabolite levels of the deodeok tested with the deodeok confirmed to be domestic or Chinese; and

(d) Step of determining the origin of the deodeok to be tested showing a level of metabolites similar to that of the deodeok to be tested.

The method for extracting the metabolites is not particularly limited, but solvent extraction is preferred. The solvent includes water; Lower alcohols such as methanol, ethanol, propanol, and butanol; acetone; trifluoroethanol; tetrahydrofuran; dichloromethane; phosphate; and mixed solvents thereof can be used.

The step of analyzing the level of the metabolite can be performed by chromatography/mass spectrometry, specifically liquid chromatography-quadrupole time-of-flight mass spectrometry (LC Q-TOF MS).

Since the composition for determining the origin of deodeok of the present invention can detect 15 types of metabolite markers, using the composition, the origin of domestic and Chinese deodeok can be determined using the difference in metabolite levels of deodeok. The metabolite marker can be used as an important tool to crack down on the practice of gaining unfair profits by lying about the origin used in actual crackdowns, and this can contribute to establishing market order for agricultural products distributed domestically and securing consumer trust.

Figure 1 shows the schematic results of metabolites isolated from Deodeok using principal component analysis.
Figure 2 shows the results of partial least squares discriminant analysis of metabolites isolated from deodeok to confirm the distribution of metabolites according to the origin of deodeok.
Figure 3 shows the results of selecting metabolites that can determine the origin of Deodeok with a VIP score of 1.2 or more through the variable importance scale list of the partial least squares discriminant analysis results and showing the relative abundances.
Figure 4 shows the results of confirming the ability of the metabolite biomarkers discovered in the present invention to determine the origin of Deodeok.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for illustrative purposes of one or more specific examples and the scope of the present invention is not limited to these examples.

Example 1: Pretreatment and metabolite extraction of Deodeok samples

Domestic deodeok was purchased from three main producing areas: Jeongseon, Hoengseong, and Jeju in Gangwon-do, and Chinese deodeok was purchased. Each purchased deodeok sample was washed with water, the skin was removed, and freeze-dried. Dried deodeok samples were homogenized into powder form and stored frozen at -78°C until metabolite extraction. For sample extraction and analysis, ethanol (Thermo Fisher; USA) and organic solvent and water (Thermo Fisher; USA) were used, respectively.

After accurately weighing 50 mg of each sample, 1 ml of 70% ethanol was added and extracted for 20 minutes using an ultrasonic treatment device (POWERSONIC 605; Korea). The extract was centrifuged, the supernatant was filtered, and then used as a sample for analysis.

Example 2: Metabolite detection and analysis

2-1. Liquid chromatography analysis

To detect metabolites in the Deodeok sample, 5 μl of the sample for analysis obtained in Example 1 was injected into liquid chromatography and separated. The liquid chromatography equipment used was Exion LC ^TM AD (AB ACIEX, Concord, ON, Canada) and Waters Acquity UPLC HSS T3 Column (2.1 ㎜x100 ㎜, 1.8 ㎛). DW (solvent A) and acetonitrile (solvent B) containing 0.1% formic acid were used as mobile phases, and the analysis was performed using the following solvent gradient at a flow rate of 300 μl/min; 0 to 1 minute, 1% to 5% solvent B; 1 to 3 minutes, 5% to 25% solvent B; 3 to 4.8 minutes, 25% to 35% solvent B; 4.8 to 5.8 minutes, 35% solvent B; 5.8 to 6.8 minutes, 35% to 45% solvent B; 6.8 to 7.8 minutes, 45% solvent B; 7.8 to 8.8 minutes, 45% to 60% solvent B; 8.8 to 9.3 minutes, 60% to 100% solvent B; 9.3 to 10 minutes, 100% to 1% Solvent B; 10 to 13 minutes, 1% solvent B.

2-2 Mass spectrometry analysis

To detect metabolites in deodeok samples, mass spectrometry was performed using a mass spectrometer. For mass analysis, an X500R QTOF (AB ACIEX) mass spectrometer was used, and mass spectra of metabolites were obtained in positive and negative ionization modes in the mass range of 50-1100 m/z.

2-3 Initial identification of metabolites

For the initial identification of the metabolites analyzed in 2-1 and 2-2 above, comparative analysis was performed through spectral concordance using online databases such as HMDB (the Human Metabolome Database) and METLIN and references. Through this process, a total of 97 metabolites were detected in deodeok samples (Table 1).

metabolites metabolites metabolites metabolites metabolites 4-Methylnicotinic acid Caffeic acid Glucose/Fructose Lobetolin Shikimic acid 6-Methylcoumarin Capillene Glutamate Lobetyolinin Sinapic acid Dihydrojasmonic acid Carnitine Glutamine Lobetolinin sulfate Succinate foetidissimoside A Chlorogenic acid / Caffeoylquinic acid Glutathione Lysine Syringic acid (E)-2-hexenyl-α-L-arabinopyranosyl-(1→2)-β-D-glucopyranoside Choline Hexyl-b-Dglucopyranosyl-(1/2)-b-Dglucopyranoside Malate Syringin 2-Methylindole Cinnamaldehyde Hexyl-b-D-glucoside sulfate Methionine Tangshenoside I 3,4-Dihydroxybenzoic acid (Protocatechuic acid) Cinnamic acid Histidine Myristicin Tangshenoside II 3-Hydroxybenzoic acid cis-Aconitic acid / trans-Aconitic acid Indole Ornithine Tangshenoside IV 6-Methylquinoline Citrate Indoleacrylic acid Pantothenic acid Tangshenoside VIII 7-hydroxycoumarin Codonolaside I Indolelactic acid Phenyl acetate Threonine Acetylcholine Codonopyrrolidium A Kaempferol Phenylalanin Tianshi acid Adenosine Coumaric acid Kaempferol-O-glucoside Phosphocholine Tryptophan alpha-ketoglutarate Dimethylarginine lancemaside A Proline Tyrosine Arginine D-Maltose/Sucrose/Lactose lancemaside B Pyroglutamate Valine Ascorbic acid Echinocystic acid lancemaside C Quinic acid Vanillic acid Aspartate Ferulic acid lancemaside D Raffinose Woodorien Aster saponin Hb Fumarate lancemaside E Riboflavin Azelaic acid Gallic acid lancemaside G Salicylic acid Benzaldehyde Geniposide Leucine/Isoleucine Scopoletin Betaine Gentisic acid Lobetyol Serine

Example 3: Metabolite Biomarkers

3-1. Principal component analysis (PCA)

As an analysis for biomarker selection among a total of 98 metabolites detected through the processes of Examples 1 and 2, the metabolites were first schematized through principal component analysis (PCA), which is shown in Figure 1. .

3-2. Partial least squares-discriminant analysis (PLS-DA)

In addition, partial least squares-discriminant analysis (PLS-DA) was performed to confirm the distribution of metabolites according to the origin of Deodeok, and the results are shown in Figure 2. As shown in Figure 2, it was confirmed that samples were gathered by country of origin according to the metabolite level, and it was found that there was a difference in the level (expression) of metabolites in deodeok samples by country of origin.

3-3. Metabolite biomarker screening

Metabolites with a VIP score of 1.2 or higher were selected through the variable importance in projection (VIP) list of the partial least squares discriminant analysis results. Afterwards, 15 statistically identifiable metabolites were selected and their relative abundances (peak area value of the metabolite, meaning the relative amount present in Deodeok) were analyzed, which is shown in Figure 3 shown in Among the 15 selected metabolites, the ones with high expression levels in Korean deodeok are as follows: adenosine, alpha-ketoglutarate, ascorbic acid, and aspartate. ), dimethylarginine, gallic acid, gentisic acid, sinapic acid and tangshenoside II. In addition, among the 15 types of metabolites selected, the metabolites with low expression levels in Korean deodeok were 7-hydroxycoumarin, acetylcholine, caffeoylquinic acid, and quinic acid. ), raffinose and tyrosine.

Through the results of this example, it was confirmed that there were differences in the distribution of 15 metabolites by country of origin. Therefore, the above 15 metabolites were selected as metabolite biomarkers that can distinguish between Korean and Chinese deodeok.

Example 4: Metabolite biomarker validation and establishment

In order to verify and establish the metabolite biomarkers selected in Example 3, a model using the selected metabolite biomarkers was applied, and the ability to discriminate the origin of deodeok was expressed as a ROC (receiver operating characteristic) curve, This is shown in Figure 4.

As shown in Figure 4, all of the selected metabolites had AUC (aurea under the ROC curve) values of 0.8 or higher, confirming that the discovered markers had a very high ability to determine the origin of Deodeok.

Claims (7)

Adenosine, alpha-ketoglutarate, ascorbic acid, aspartate, dimethylarginine, gallic acid, gentisic acid. , sinapic acid, tangshenoside II, 7-hydroxycoumarin, acetylcholine, caffeoylquinic acid, quinic acid, raffinose A metabolite marker composition for determining the origin of Deodeok, comprising at least one metabolite selected from the group consisting of raffinose and tyrosine.
The metabolite marker composition for determining the origin of deodeok according to claim 1, wherein the composition is used to distinguish between Korean deodeok and Chinese deodeok.
According to paragraph 1,
The levels of adenosine, alpha-ketoglutarate, ascorbic acid, aspartic acid, dimethylarginine, gallic acid, gentis acid, sinapic acid, and Tangsenoside II are decreased in Chinese deodeok compared to Korean deodeok. Metabolite marker composition for determining the origin of.
According to paragraph 1,
A metabolite marker composition for determining the origin of deodeok, wherein the levels of 7-hydroxycoumarin, acetylcholine, caffeoylquinic acid, quinic acid, raffinose, and tyrosine increase in Chinese deodeok compared to Korean deodeok.
A kit for discriminating between domestic and Chinese deodeok, comprising the metabolite marker composition for determining the origin of deodeok of claim 1.
Method for distinguishing between domestic and Chinese deodeok including the following steps:
(a) extracting metabolites from deodeok that has been confirmed to be domestically produced or produced in China and a test deodeok whose country of origin is unknown;
(b) Among the metabolites extracted in step (a), adenosine, alpha-ketoglutaric acid, ascorbic acid, aspartic acid, dimethylarginine, gallic acid, gentis acid, sinapic acid, tangsenoside II, 7-hydride Analyzing the level of one or more metabolites selected from the group consisting of roxicoumarin, acetylcholine, caffeoylquinic acid, quinic acid, raffinose, and tyrosine;
(c) Comparing the metabolite levels of the deodeok tested with the deodeok confirmed to be domestic or Chinese; and
(d) Step of determining the origin of the deodeok that shows a level of metabolite similar to that of the deodeok to be tested as the origin of the deodeok to be tested.
The method of claim 6, wherein the step of analyzing the level of metabolites in (b) is performed by chromatography/mass spectrometry.