KR101629570B1 - A method distinguishing the location of cultivation of Panax ginseng using a QTOF-MS coupled RRLC - Google Patents

Abstract

The present invention relates to a method for distinguishing a cultivation location of Korean ginseng roots using rapid-resolution liquid chromatography (RRLC) coupled quadrupole time-of-flight (QTOF) mass spectrometry (MS) and multivariable statistical analysis. The method of the present invention can distinguish a cultivation location within a short time, thereby preventing Korean ginseng cultivated in China from being distributed to a medical herb market in a state in which the Korean ginseng cultivated in China is disguised as Korean ginseng cultivated in Korea or Japan.

Description

The present invention relates to a method and apparatus for determining the location of cultivation of Korean ginseng using a high-resolution liquid chromatography coupled with quadrupole time-of-flight mass spectrometry

The present invention relates to a method for identifying a cultivation area of Korean ginseng using a high-resolution liquid chromatography, and more particularly, to a method for identifying a cultivation area of Chinese ginseng, Korean ginseng and Japanese ginseng, and a marker therefor.

Korean ginseng (Panax ginseng C.A. Meyer) is a herb that is widely used in traditional oriental medicine and widely used in the world. Recent phytochemical and pharmacological studies have found that ginseng extracts and bioactive components include saponins, polyacetylenes, sesquiterpenes and polysaccharides. However, the therapeutic effect of ginseng roots is mainly related to triterpenoids and ginseng saponins. The qualitative and quantitative characteristics of ginseng saponin, known as ginsenoside, may be significantly different depending on the cultivation area even if the same ginseng is the same. The efficacy and bioactive components of ginseng roots can also vary depending on the cultivation area. Therefore, it is necessary to distinguish the cultivation areas of ginseng, and ingredients present in Korean ginseng cultivated in Korea, China and Japan may be misrepresented or confused in the Korean medicinal product market due to complicated plant sources. Therefore, there is a need to establish a reliable method of distinguishing these herbal medicines.

Korean Patent Registration No. 10-0215084 discloses a method of identifying a mountain area by a marking through random amplified polymorphic DNA analysis (RAPD). Specifically, the DNA of ginseng to be the target of the mountain region is isolated and purified; Purified ginseng DNA was amplified by PCR; Electrophoresing the amplified DNA on an agarose gel containing ethidium bromide; And selecting the RAPD label by comparing the pattern of the DNA band on the UV transilluminator with the agarose gel in which the electrophoretic ginseng DNA is present. The above method requires a complicated process of separating DNA from the sample to be analyzed and amplifying and analyzing the DNA, so that much time and effort are required.

In recent years, chromatographic performance has been improved by the introduction of rapid-resolution liquid chromatography (RRLC), which utilizes the technique of passing through a short column filled with small particles at sub-2 μm level at a relatively high flow rate. In this technique, the advantages of the chromatographic principle are utilized to achieve a good theoretical number of steps and ultra-high resolution separation in a short time using a small amount of solvent. Continuous development of RRLCs coupled to quadrupole time-of-flight (QTOF-MS) mass spectrometry (QTOF-MS) is a new method for performing composition and structure analysis of complex constituents in complex mixtures . Rapid acquisition rates and detection in the broad mass range as well as qualitative and quantitative analysis are achieved by QTOF-MS. Furthermore, the RRLC-QTOF-MS technique has an absolute advantage over conventional assays in sensitivity and selectivity.

The inventors of the present invention have made efforts to find a method of identifying the cultivation area of Korean ginseng which is rapidly distributed through a single process which does not require any additional process such as DNA isolation and amplification. As a result, The present inventors have confirmed that the above problems can be achieved by analyzing the secondary metabolite change by applying a fingerprint, thereby completing the present invention.

In order to achieve the above object, the present invention provides a method of mass spectrometric analysis of Korean ginseng root samples by quadrupole time-of-flight (QTOF) mass spectrometry combined with rapid-resolution liquid chromatography (RRLC) A method for determining the cultivation area of Korean ginseng roots, which comprises performing a multivariate statistical analysis on the kind and amount of the ginsenoside compound obtained after separation into a major ginsenoside compound, wherein the cultivation area is from China, Korea or Japan, Ginsenoside Rc; A compound selected from the group consisting of ginsenoside Rf, ginsenoside F1, notogensenoside R2 and 20- O -glucosinenoside Rf (20-glc-Rf); And a compound selected from the group consisting of ginsenoside Rh1, ginsenoside Rk3, and ginsenoside Rd according to the amount of the compound selected from the group consisting of Chinese ginseng, Chinese ginseng, and Japanese ginseng. to provide.

The present invention relates to a method for identification of unique markers that can discriminate homologous Korean ginseng samples cultivated in China, Korea and Japan at once, and a method capable of quickly and easily identifying the markers by RRLC-QTOF-MS using multivariate statistical analysis using the same .

Preferably, when the main ginsenoside compound of the Korean ginseng root sample is ginsenoside Rc, the ginseng root of the Korean ginseng root is considered to be Chinese, and the main ginsenoside compound of the ginseng root sample of the Korean ginseng root is ginsenoside Rf, In case of Side F1, Notoginsenoside R2 or 20- O -Glucosynesenoside Rf (20-glc-Rf), the Korean ginseng roots are determined to be Korean, and the main ginsenoside compounds of the Korean ginseng root samples In the case of ginsenoside Rh1, ginsenoside Rk3 or ginsenoside Rd, the corresponding ginseng root may be considered to be Japanese.

Preferably, the multivariate statistical analysis may be a multivariate statistical analysis using principal component analysis (PCA) data, but is not limited thereto.

As a matter of fact, the ginseng root of Chinese ginseng plant in which the cultivation area is Chinese can be ginsenoside Rc as the main ginsenoside compound. In addition, the Korean ginseng roots of which the cultivation area is Korean can be selected from the group consisting of ginsenoside Rf, ginsenoside F1, notoginsenoside R2 or 20- O -glucosinenoside Rf (20-glc-Rf) And ginsenoside Rh1, ginsenoside Rk3 or ginsenoside Rd may be a major ginsenoside compound in the Korean ginseng roots of which the cultivation area is Japanese.

In a specific embodiment of the present invention, differences in Korean ginseng cultivated in three countries were identified through PCA loading plots and the markers for each were identified (Fig. 6). As a result, Korean ginseng was found to contain ginsenoside Rf, ginsenoside F1, norotinosenoside R2 or 20- O -glucosinenoside Rf (20-glc-Rf), Chinese ginsenoside Rc, It was confirmed that the acid contained ginsenoside Rh1, ginsenoside Rk3 or ginsenoside Rd as main components. Furthermore, it was confirmed that the PCA score plot derived from this was clearly distinguished by the top two components of Korean ginseng produced in the three provinces (Fig. 5).

The Korean ginseng root sample can be prepared by extracting with C _{1 -4} alcohol. Preferably, the C _{1 -4} alcohol extract may be an extract of methanol, but is not limited thereto. At this time, the extract can be extracted at a temperature of 35 to 55 ° C, but is not limited thereto. For example, Korean ginseng root samples are dried and finely ground to prepare fine powders, dispersed in 70% (v / v) methanol, extracted with ultrasonic waves at 50 ° C for 1 hour, and the extracts are evaporated under reduced pressure to obtain the residue The sample for analysis can be prepared by dissolving in 70% methanol.

Preferably, the high-resolution liquid chromatography can be performed by gradient elution. Since most of the ginsenosides have similar polarities and structures, it is difficult to separate them clearly with isocratic elution but better separation can be achieved using gradient elution. That is, by using the gradient elution, individual peaks corresponding to several tens to several hundred species of components can be separated and displayed without overlapping each other, thereby achieving higher separation efficiency.

At this time, the eluent used for the gradient elution may further include 0.07 to 0.15% by volume of formic acid. If the eluent contains less than 0.07% by volume of formic acid, it may be difficult to achieve the desired improvement in separation efficiency. If the eluent contains more than 0.15% by volume, it may be difficult to calculate an accurate mass value. Can be shortened.

Preferably, the mass spectrometry can be performed using an electron spraying method of anion mode. For example, it is preferable to perform analysis using only data from the negative ion mode since mass spectrometric analysis using a cation-mode electron atomization method may exhibit poor ionic stability. For example, by performing analysis using an electron spraying method in an anion mode, target components and unknown samples with a very small concentration can be accurately qualitatively and quantitatively analyzed.

Further, the present invention provides a marker for discriminating a mountain region used in discriminating Korean ginseng cultivated in Japan selected from ginsenoside Rh1, ginsenoside Rk3 and ginsenoside Rd.

As described above, the Korean ginseng cultivated in Japan contains ginsenoside Rh1, ginsenoside Rk3 and / or ginsenoside Rd as main components, which is distinguished from main components detected in Korean or Chinese acids, Can be used as a marker for discriminating Korean ginseng cultivated in Korea.

The method of the present invention can identify the plantation area of Korean ginseng within a short period of time, and can prevent the misrepresentation of origin in the herbal medicines market or sale of Chinese products from Japanese or domestic market.

FIG. 1 is a graph showing the results of a comparison between Korean ginseng ( Panax ginseng ) root.
Figure 2 shows the TIC of ginsenosides obtained from QTOF / MS analysis in the 100-1,500 m / z range.
FIG. 3 shows RRLC-QTOF / MS and ESI ^- base peak intensity (BPI) chromatograms of Korean ginseng roots.
FIG. 4 is a chart showing chromatograms of Chinese ginseng extract (A, Cn), Japanese acid (B, Jp) and Korean acid (C, Kr) Korean ginseng extract and ginsenoside standard material (D).
5 shows a PCA score plot showing the analysis results derived from the ESI negative ion mode. Segregation was confirmed by PCA score plot showing significant impact on the plantation (ie, Korea, China and Japan) in the difference determination.
Figure 6 shows a PCA loading plot using Pareto (Pareto scaling with mean centering) adjusted to the mean center.
FIG. 7 shows the PLS-induced relationship between the observed origin and the estimated origin of 5-year-old Korean ginseng samples. 29 training samples were used (Korea: Kr-5, Japan: Jp-5, China: Cn-5).
8 is a diagram showing the relationship between the ginseng origin of the Korean ginseng observed and estimated by the PLS model. The training chef and test set are shown in gray and blue, respectively.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for describing the present invention more specifically, and the scope of the present invention is not limited by these examples.

Example  1: Korean ginseng sample

In 2009, we received Korean Ginseng Korean Ginseng from Korea Punggi Ginseng Cooperative Association in Punggi area, Gyeongbuk province. Five-year-old Chinese ginseng was collected from three regions of Anto, Dunhua and Wangcheng in 2009 in Jinlin Province. The 5 year old Korean ginseng from Japan was provided in 2009 from the traditional medicinal market in Fukushima, Japan (Fig. 1). Sample specimens (Voucher specimens) were deposited in Kyunghee University Natural Product Chemical Laboratory. The number, weight, length, collection place and parts of each sample are shown in Table 1.

Sample No. Weight (g) Major diameter (cm) Length (cm) Number of Entourage (ea) Collection place Kr-5-1
Kr-5-2
Kr-5-3 30.9
26.8
33.8 2.2
2.0
2.3 9.3
10.7
11.3 3
2
One
Seongheung Kr-5-4
Kr-5-5
Kr-5-6 30.5
20.9
33.9 2.2
2.1
2.3 8.2
6.5
11.8 3
2
2
stability Kr-5-7
Kr-5-8
Kr-5-9
Kr-5-10 26.0
33.2
36.4
27.9 1.9
2.6
2.3
2.5 7.1
5.0
9.0
1.5 5
6
3
6
Trilogy Cn-5-1
Cn-5-2
Cn-5-3 16.3
16.0
14.1 3.5
3.5
3.9 1.5
2.2
2.1 5
3
3
Anto Cn-5-4
Cn-5-5
Cn-5-6 25.4
25.5
23.1 6.5
6.5
6.4 2.0
2.1
2.0 3
2
5
Slowing Cn-5-7
Cn-5-8
Cn-5-9 22.0
19.6
19.4 6.3
6.5
6.7 2.0
2.3
1.7 2
2
3
Wangcheng Jp-5-1
Jp-5-2
Jp-5-3
Jp-5-4
Jp-5-5
Jp-5-6
Jp-5-7
Jp-5-8
Jp-5-9
Jp-5-10 9.7
7.6
7.7
8.3
8.1
6.9
6.1
11.7
7.2
11.4 1.5
1.5
1.4
1.8
1.2
1.5
1.4
1.6
1.2
1.4 4.0
4.7
6.7
2.5
5.5
4.7
3.4
6.3
8.2
5.8 2
2
One
2
2
2
2
2
2
One



Fukushima

Example  2: Standard components and reagents

HPLC-grade acetonitrile, methanol and water were purchased from Merck, Darmstadt, Germany. Formic acid was purchased from Sigma-Aldrich (St. Louis, Mo., USA). The ginsenosides were separated and purified from Korean ginseng and red ginseng through a series of chromatographic procedures and their structures were confirmed by comparison of spectroscopic data (MS, ¹ H-NMR and ¹³ C-NMR) with the literature data: Ginsenoside Rb1 (Cho, JG et al ., J. Ginseng Res . , 2010, 34: 113-121), Ra2 (Zhu, GY et al, Chem Biodiver, 2011, 8:... 1853-1863), Ra3 (Zhu, GY et al . , Chem . Biodiver. , 2011, 8: 1853-1863), Rc (Cho, JG et al ., J. Ginseng Res . , 2010, 34: 113-121), Ra1 (Zhu, GY et al al . , Chem . Biodiver . , 2011, 8: 1853-1863), Rb2 (Cho, JG et al ., J. Ginseng Res . , 2010, 34: 113-121), Rb3 (Sanada, S. et al, Chem Pharm Bull, 1978, 26:.... 1694-1697), Rd (Cho, JG et al ., J. Ginseng Res . , 2010, 34: 113-121), F2 (Ko, SR et al ., Chem . Pharm . Bull . , 2007, 55: 1522-1527), Rg3 (Ko, SR et al ., Chem . Pharm . Bull . , 2007, 55: 1522-1527), Rh2 (Baek, NI et al ., J. Korean Soc . Appl . Biol . Chem . , 1995, 38: 184-189), compound K (Ko, SR et al ., Chem . Pharm . Bull . , 2007, 55: 1522-1527), 20-O-glucogensenoside Rf (Sanada, S. et al ., Chem . Pharm . Bull . , 1978, 26: 1694-1697), ginsenoside Rg1 (Teng, R. et al ., Magn . Reson . Chem . , 2002, 40: 483-488), Rg2 (Teng, R. et al ., Magn . Reson . Chem . , 2002, 40: 483-488), Re (Teng, R. et al ., Magn . Reson . Chem . , 2002, 40: 483-488), Rf (Zhou, J. et al, Chem Pharm Bull, 1981, 29:.... 2844-2850), Rh1 (Teng, R. et al ., Magn . Reson. Chem . , 2002, 40: 483-488), notoginsenoside R2 (Teng, R. et al ., Magn . Reson. Chem . , 2002, 40: 483-488), ginsenoside F1 (Ko, SR et al ., Chem . Pharm. Bull . , 2003, 51: 404-408), Rg6 (Liu, GY et al ., J. Asian Nat . Prod . Res. , 2010, 12: 865-873.), Rk1 (Park, IH et al ., Arch . Pharm . Res . , 2002, 25: 428-432), Ro (Wang, Z. et al ., Acta Bot . Sin . , 1985, 27: 618-621.), Rk3 (Park, IH et al ., Arch . Pharm . Res . , 2002, 25: 428-432), ginsenoside F4 (Zhang, S. et al ., Planta Med . , 1990, 56: 298-300), Rh4 (Teng, R. et al ., Magn. Reson . Chem . , 2002, 40: 483-488), and Rg5 (Kim, SI et al ., Arch . Pharm. Res . , 1996,19: 551-553). The purity of the isolated compound was confirmed to be greater than 98% by normalization of the peak area detected by HPLC analysis. The chemical structures of the subject ginsenosides are shown in Table 2.

In the table, Glc represents? -D-glucopyranosyl, Ara (p) represents? -D-arabinopyranosyl, Ara (f) represents? -D- arabinofuranosyl and Rha represents? -Aminopyranosyl, Xyl represents? -D-xylopyranosyl, and glcU represents? -D-glucuronic acid.

Example  3: Sample preparation

After washing, each sample was dried and weighed at 40 DEG C for 48 hours in a forced convection drying oven. After removing rhizomes and fine roots, main and lateral roots were used in each experiment. The roots were ground (<0.5 mm) using a mixer (Hanil, Seoul, Korea), mixed thoroughly, and subsamples were further analyzed using a Retsch MM400 mixer mill (Retsch GmbH, Haan, Germany) Homogenized. The fine powder was weighed (100 mg) and dispersed in 1.0 mL of 70% (v / v) methanol and extracted with ultrasonic at 50 ° C for 1 hour. The extract was evaporated under reduced pressure and the residue was dissolved in 70% methanol. The solution was filtered through a syringe filter (0.22 μm) and injected directly into the RRLC system. A cross-talking effect of the samples was demonstrated by injecting a blank (3 μL) consisting of 70% methanol between the selected assays.

Example  4: RRLC - QTOF - MS  analysis

RRLC was performed with a 1200 RRLC SL + system (Agilent, Santa Clara, Calif., USA) equipped with a dual solvent delivery system and an automatic sampler. Chromatographic separation was performed on a 2.1 x 100 mm, 1.8 μm RRHT C ₁₈ chromatography column. The column oven was maintained at 40 ° C and the mobile phase consisted of solvent A (0.1% aqueous formic acid, v / v) and solvent B (0.1% acetonitrile in formic acid solution, v / v). The optimized LC elution conditions are as follows: 0-1 min, 25% B; 1-5 minutes, 30% B; 5-15 min, 75% B; 15-18 min, 90% B; 18-20 minutes, 100% B and 20-23 minutes, 25% B. The flow rate was 0.60 mL / min. The 3 μL aliquot was loaded into the column using an automatic sampler. MS analysis was performed using 6530 QTOF-MS (Agilent) operating in anion electron spray mode. The driving parameters are as follows: Dry gas (N ₂ ) flow rate, 5.0 L / min; Drying gas temperature, 350 캜; A nebulizer, 45 psig; Sheath gas temperature, 400 캜; Blocking gas flow, 12 L / min; Capillary, 3500 V; Skimmer, 65 V; OCT RF V, 750 V; And a fragmentor voltage of 175 V. For MS / MS experiments, the collision energy was adjusted from 20 V to 50 V to optimize the signal from the ion of interest and obtain maximum structural information. The data scan time was set to 0.5 s with 0.02 s scan delay. The total number of scans automatically combined is set to two. The number of cycles was set to 5. Thus, a total of 10 scans were performed and the mass range was set at m / z 100-1500.

Rapid chromatographic separation of ginsenosides was performed using RRLC to avoid excessive system pressure. The most efficient way to achieve high column efficiency and resolution while simultaneously achieving fast assay times is to use small particle size columns. Thus, chromatographic analysis was performed using a 2.1 x 100 mm, 1.8 μm particle size RRHT C ₁₈ column. Within a short time (20 minutes), higher separation peaks with narrower widths are formed to achieve higher sensitivities, making the RRLC analysis system more intensive. In the present invention, the LC-QTOF / MS spectrum per sample was recorded three times. The high resolution and speed of RRLC combined with accurate mass measurement of QTOF / MS allowed for the simultaneous separation of 27 standard ginsenosides within 20 minutes. BuOH solvent system including n-BuOH extraction and CHCl ₃ -MeOH-H ₂ O mixture as an extraction solvent in partitioning with CHCl ₃ for solvent extraction, The chromatogram could not be obtained. However, when 70% methanol was used, ginseng roots were well extracted. Different extraction temperatures (30 ° C, 40 ° C and 50 ° C) were tested, and at 50 ° C, good separation rates with only a few peaks and slight decreases in peak signals were obtained. The column temperature was set at 45 캜 so as to alleviate the additional column pressure caused by higher flow rates and to improve chromatographic separation and peak shape. Since most of the ginsenosides have similar polarities and structures, it is difficult to separate two organic solvents by isocratic elution by mixing two organic solvents, while the two organic solvents are poured through different pumps Gradient elution allowed better separation. In addition, the effect of adding formic acid to elution solvents at different concentrations (0.1%, 0.05% and 0.01%) was confirmed. As a result, it was confirmed that when formic acid was added at a concentration of 0.1%, the best resolution with only a slight peak signal reduction could be achieved. Both cation and anion modes were tested in an electrospray ionization (ESI) device. However, chromatographic data from the cationic mode were not well ionized and the number of signals of the peaks was significantly reduced. Therefore, the experiment was conducted in an ion mode with relatively good ionization and multivariate analysis was performed with the data from the measured values. Accurate mass measurement was achieved by means of an automated calibration delivery system containing an internal reference mass at m / z 112.9856, 1033.9881, and 1933.9306 in anion mode. The data measured in the negative ion mode were collected in the range of 100 to 1,500 m / z. Molecular ions were matched in the mass spectra with those in the standard and internal libraries to identify each ginsenoside peak in total ion chromatogram (FIG. 2). The basic peak intensity (BPI) chromatogram for Korean ginseng obtained in anion mode is shown in FIG.

Example  5: Data processing and multivariate analysis ( Multivariate Analysis )

Data processing was performed on both the Mass Hunter workstation (software version B.02.04 provided by Agilent, USA) and MZmine. The mass spectra were analyzed using a mass hunter, separated from the search peak. Peak area was assigned to the identity of m / z and retention time used in the fingerprint. For MZmine, raw chromatography data was converted to NetCDF format (.cdf) prior to application to software. A peak corresponding to the compound was found by peak detection. Alignment aims at matching corresponding peaks through multiple sample runs. Spectral filtering aims at reducing data complexity and eliminating noise. The role of normalization is to reduce the systematic error by controlling the intensity within each sample run.

Principal component analysis (PCA) using SIMCA-P + version 12.0 (Umetrics, Umea, Sweden) was performed initially to understand the relationship represented by similarities or differences between groups of multivariate data. The projections are continuously performed on latent structures using partial least-squares (PLS) using SIMCA-P + version 12.0 to obtain a quality-prediction model I have created.

Metabolite fingerprinting of 5-year-old ginseng samples (Kr-, Cn-, and Jp-5) was performed using PCA, an unsupervised multivariate pattern recording method, to efficiently visualize differences in chromatographic patterns 4). Mean-centered and reference-proportional (proportional to the square root of the standard deviation) repair procedure was performed to pre-process the data sets from the samples using SIMCA-P + 12.0 software. PCA results showed a distinct mode of separation in all three types of ginseng samples (Kr-, Cn-, and Jp-5) (Fig. 5). The PCA score plot showing an analysis derived from the negative ion mode depicted a total variance of 65.3%. At this time, the optimal segregation was achieved between PC 1 (36.5%) and PC 2 (28.8%), the main components of sample separation. Cn-5 and others were clearly separated by the principal component (PC) 1. Jp-5 was easily distinguished from the others by Principle 2 (PC 2). The values of the corresponding loading plot obtained from the data values of the negative ion mode were in agreement with the results of the respective score plots and the apparent intensity of the specific RT-m / z parameters for the correlative clusters clearly showed the change in the metabolite fingerprint (Fig. 6). The preferential distribution of marker ions in the first quadrant of the loading plot (m / z 799.4835: Rf, 683.4375: F1, 769.4731: R2, 961.5360: 20-glc- 5). Similarly, the distributions in the second and third quadrants of the loading plot are respectively shown in Chinese (Cn-5; m / z 987.5467 [MH] ^- , 1149.6025 [MH] ^- , 1077.5835: Rc) / z 683.4275: Rh1, 665.4270: Rk3, 945.5416: Rd). This is a marker for simultaneously identifying Korean Ginseng roots samples cultivated in Korea, China, and Japan. The Ginsenoside Rc was selected for Chinese, Ginsenoside Rf, Ginsenosides F1, Ginsenoside R2 or 20- O -glucosinenoside Rf (20-glc-Rf), and for Japanese acid, ginsenoside Rh1, ginsenoside Rk3 or ginsenoside Rd. An experimentally determined m / z of the selected marker ion was used to calculate the possible calculated mass (mDa) and mass accuracy (ppm). MassHunter workstation data acquisition software (B.03.00) was used to generate and study elemental compositions related to the measured mass of marker ions. The major fraction ions and retention times of the marker ions were determined by comparing ginsenosides with the values for the available standards and accurate mass measurements (Table 3).

Example  6: PLS Method of Predicting Ginseng Origin Using

As an algorithm for establishing a quality prediction model for ginseng, we considered PLS, which is a metabolic fingerprint projected on a bivariate structure. The relationship between the metabolism profiling (X variable) of the sample and its origin (Y variable) was observed. Samples were classified into training set and test set group. The PLS-induced relationship between the observed origin and the estimated origin of Korean ginseng samples from Korean (Kr-5), Chinese (Cn-5) and Japanese (Jp-5) Respectively. The original Y variables were randomly selected for each sample (1: Cn-5; 2: Jp-5; 3: Kr-5). The training set included individual samples of Kr-5, Cn-5 and Jp-5 ginseng roots and two random samples were used as a test set for the model (FIG. 8). After projecting the test set to the model, the prediction is based on the Root-Mean-Square Error of Prediction (RMSEE) = 0.124532, which is in good agreement with the model- (RMSEP) = 0.115468].

Developed in combination with metabolomics, RRLC-QTOF / MS provides markers or candidates for fast, reliable and accurate quality assays. By observing PCA plots, ginseng samples could be categorized on the area where the roots were grown. Roots cultivated in Korea, China and Japan were classified to be consistent with each other, and variations in the fingerprints of metabolites from different samples reflected the geographic impact. Specifically, it was possible to perform specific classification according to the geographical influence even within the roots of Korean phosphoric acid for 5 years. These results indicate that the cultivation area is the main influential factor for quality discrimination. Techniques such as RRLC-QTOF / MS-based metabolism profiling may be useful in identifying and describing metabolic outcomes as a result of geographic and seasonal changes as well as different cultivation methods used.

Claims (10)

After separation into major ginsenoside compounds of Korean ginseng root samples by quadrupole time-of-flight (QTOF) mass spectrometry combined with rapid-resolution liquid chromatography (RRLC) In the method for determining the cultivation area of Korean ginseng roots in which multivariate statistical analysis is performed on the kind and amount of the ginsenoside thus obtained,
The plantation is made in China, Korea or Japan,
Ginsenoside Rc; A compound selected from the group consisting of ginsenoside Rf, ginsenoside F1, notogensenoside R2 and 20- O -glucosinenoside Rf (20-glc-Rf); And a compound selected from the group consisting of ginsenoside Rh1, ginsenoside Rk3, and ginsenoside Rd, and determining whether the cultivation area of Korean ginseng is Chinese, Korean or Japanese,
When the main ginsenoside compound of the Korean ginseng root sample is ginsenoside Rc, the ginseng root of the Korean ginseng root is considered to be Chinese,
When the main ginsenoside compound of the Korean ginseng roots sample is ginsenoside Rf, ginsenoside F1, notogensenoside R2 or 20- O -glucosinenoside Rf (20-glc-Rf) , It is judged that the cultivation area is Korea,
Wherein the ginseng root is the Japanese ginseng root when the main ginsenoside compound of the Korean ginseng root sample is ginsenoside Rh1, ginsenoside Rk3 or ginsenoside Rd.
delete The method according to claim 1,
Wherein the multivariate statistical analysis method is multivariate statistical analysis using principal component analysis (PCA) data.
The method according to claim 1,
Wherein the Korean ginseng roots sample is a C _{1 -4} alcohol extract.
5. The method of claim 4,
_Wherein the C _{1 -4} alcohol extract is a methanol extract.
6. The method of claim 5,
Wherein the extract is extracted at a temperature of 35 to 55 캜.
The method according to claim 1,
Wherein said high-resolution liquid chromatography is performed by gradient elution.
The method according to claim 1,
Wherein the high-resolution liquid chromatography is carried out using an eluent containing 0.07 to 0.15% by volume of formic acid.
The method according to claim 1,
Wherein the mass spectrometry is carried out using an electron spraying method in an anion mode.
A marker composition for distinguishing a mountain ginseng used in discriminating Korean ginseng cultivated in Japan selected from ginsenoside Rh1, ginsenoside Rk3 and ginsenoside Rd.

Images