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    • 专利摘要:
    PURPOSE: Provided is a method of simply and easily classifying ginsengs by their growing districts, by analyzing RAPD(randomly amplified polymorphic DNA) and PCR-RFLP(PCR-restriction fragment length polymorphism. CONSTITUTION: The method of classifying ginsengs comprises the steps of: extracting DNA from an individual ginseng sample; preparing the reacting solution of the extracted DNA and primers; amplifying the reacting solution; and converting the amplified genomic DNA fingerprints into binary character matrix to determine similarity using SPSS/PC+, in which the amplification is carried out by using RAPD(randomly amplified polymorphic DNA) and PCR-RFLP (PCR-restriction fragment length polymorphism); and the primers have the nucleotide sequences of SEQ ID NO: 1, 3, 5, 6, 9, 13 and 14.
    公开号:KR20020004536A
    申请号:KR1020000038523
    申请日:2000-07-06
    公开日:2002-01-16
    发明作者:김형민
    申请人:김형민；
    IPC主号:
    专利说明:

    Discrimination method of Panax ginseng
    [4] Since 80% of Korean herbal medicines are imported from China, illegally smuggled Chinese ginseng is packaged as Korean ginseng and distributed in large quantities, which is a serious confusion of distribution order. Clearly, effective mountain classification of herbal medicines including ginseng is essential for the healthy development of the pharmaceutical industry.
    [5] Traditionally, morphological, cytological, anatomical and physiological methods have been used to classify herbal medicines, but in many cases it depends not only on the subjective perspective of the discriminator but also on the basis of the fact that most herbal medicines exist as crude drugs. Not only is it processed into powder or cut into pieces, but it is not easy to distinguish. Moreover, in the case of Chinese ginseng, P. ginseng is the same species as Korean, so it is more difficult to distinguish these ginseng by the above method. The chemical analysis of ginsenosides by examining ginsenosides is not appropriate because they are greatly influenced by their growth conditions, storage conditions, and post-harvest processing, and requires a large amount for analysis. : Yip, T., Lau, C., But, P., and Kong, Y. 1985. Am. J. Chin. Med., 13:77; And Lang, Z., Lou, W., and But, P. 1993. J. Chin. Pharm. Sci., 2: 133].
    [6] The genus Panax L. belongs to the Araliaceae family, with 12 species in the genus. However, there are only three medically useful species. P. ginseng C. A. Meyer (Oriental ginseng) and P. quinquefolius L. (American ginseng) are tonics for anti-stress, anti-fatigue and anti-aging. It is used as. P. notoginseng (Burkill) FH Chen (Sanchi) is used as a hemostatic agent in bleeding.
    [7] Conventional reports on the classification of ginseng using RAPD have been directed to the classification of species ( P.ginseng, P. quinquefolius, P. notoginseng, etc.) rather than variations of P. ginseng (Ref. Lim, YP, Shin, SJ, et al. 1993. Korean J. Ginseng Sci. 17 (2), 153-158; Bai, D., Brandle, J., and Recleder, R. 1997. Genome, 40: 111-115; Fritsch , P., Hanson, MA, Spore, CD, Pack, PD and Rieseberg, LH 1993. Mol. Biol. Rep. 11 (1), 10 and Shaw, PC, But, P. 1995. Planta Med. 61: 466 In addition, there is a problem that the random primer used is 10mer, which is short in length and low in reproducibility.
    [8] The present inventors ginseng (P. ginseng) While finding ways to distinguish Chinese ginseng from Korean ginseng from three regions at the molecular level by using interregional variation within the species, the existing 10mer-randomized primer was short in length to make it difficult to obtain reproducible results. When RAPD analysis was performed using random primers, and at the same time or additionally, the PCR-RFLP method was used to analyze the present invention, it was found that distinction between domestic ginseng and Chinese ginseng was possible. It was completed.
    [9] Therefore, the object of the present invention is a ginseng circulating in Korea compared to the conventional report that focused on the classification of ginseng species ( P. ginseng, P. quinquefolius, P. notoginseng, etc.), so as one species ( P. ginseng ), The purpose of this study is to provide a method for the accurate and simple identification of ginseng whose biological activity is expected to be different depending on the origin of P. ginseng species.
    [1] 1 is a RAPD profile generated by primers 1 (A), 3 (B), 5 (C), 6 (D) and 9 (E). M represents the molecular weight marker, lane 1 is Jinan, lane 2 is Geumsan, lane 3 is fortified and lane 4 is Chinese ginseng.
    [2] 2 PCR-RFLP analysis using restriction enzymes DanII (A) and Ddel (B). Lane 1 represents Jin'an, Lane 2 represents Geumsan and Lane 3 represents Ginseng.
    [3] Figure 3 is a RAPD profile from Jinan (A), Chinese (B) ginseng generated by primer 5. M represents a molecular weight marker. Each lane represents a randomized sample.
    [10] The object of the present invention as described above
    [11] a) extracting DNA from each ginseng sample;
    [12] b) preparing a reaction solution of the extracted DNA and the primer;
    [13] c) amplifying the prepared reaction solution; And
    [14] d) converting the amplified genomic DNA fingerprint to a double letter table to determine similarity
    [15] It can be achieved by a method of distinguishing ginseng for each mountain area according to the present invention comprising a.
    [16] In addition to the above, the present invention, as a standard DNA fingerprint of ginseng for each region, compared to the analysis table shown in Figs.
    [17] Provided is a ginseng differentiation kit comprising a standard analysis for comparing ginseng by region according to FIGS. 1, 2 and 3 obtained by RAPD and / or PCR-PFLP analysis and an analysis table analyzing the same.
    [18] Hereinafter, the present invention will be described in more detail.
    [19] P. ginseng and P. quinquefolius belonging to the Araliaceae have a total DNA size of 20Kb, of which 18S rRNA gene is composed of 1809bp and each has a unique base sequence [Fushimi H., Komatsu K. , Isobe M., Namba T. 1997. Biol. Pharm. Bull. 20: 765-769; And Munthali, M., Ford-Lloyd, BV and Newbury, HJ 1992. PCR Methods and Application, 1: 274. Therefore, in the present invention, 18S rRNA gene was amplified in Chinese ginseng and three domestic ginseng, and then cut with restriction enzymes BanII and DdeI to observe their fingerprints.
    [20] The randomly amplified polymorphic DNA (RAPD) assay was developed in the late 1980s and applied to the identification of genetic homogeneity in biomaterials. Can be used for assaying polymorphisms through DNA amplification. Furthermore, the RAPD method is advantageous in that it is inexpensive and can be analyzed with a small amount of sample compared with the isozyme or restriction fragment length polymorphism (RFLP) analysis method. The present inventors attempted to distinguish domestic and foreign ginseng by using randomly amplified polymorphic DNA (RAPD) and PCR-restriction fragment length polymorphism (PCR-RFLP). RAPD analysis using 12 random primers produced reproducible and clear fingerprints from 5 of them. This is similar to the report that some of the primers used by Fritsch et al. Produced clear fingerprints while others did not (Fritsch, et al., Supra). As a result of analyzing the similarity index based on the fingerprint generated as a result of RAPD, the similarity range was from 0.197 to 0.491. In other words, Jinan and Ginseng ginseng showed the highest similarity with 0.491, while Jinan and Chinese ginseng showed 0.266, Geumsan and Chinese ginseng showed 0.231. Ganghwa ginseng and Chinese ginseng showed the lowest similarity with 0.197, showing a clear distinction between domestic ginseng and Chinese ginseng.
    [21] In addition, DNA was extracted from three domestic ginsengs from different regions, and the 18S rRNA gene was amplified and the amplification products were digested with restriction enzymes BanII and DdeI to obtain different fingerprints (PCR-RFLP). In the RAPD analysis, there was no difference between Jinan and Geumsan, which showed a relatively high similarity, but in Ganghwa, which showed low similarity in RAPD analysis, fragments were significantly different from those of Jinan and Geumsan. Therefore, it can be seen that it is possible to discriminate domestic ginseng in the same species by region by RAPD and / or PCR-RFLP method. This band pattern is somewhat different from that reported by Fushimi et al. (Fushimi, et al., Supra), but this is due to several important agronomic characteristics (seed timing, seed germination), even within the same P. ginseng species. It is closely related to the many variations observed in plant height, plant stem strength, number of plant leaves, flowering season, degree of disease resistance, etc., and genetic variation within the same population. diversity).
    [22] In order to prove that the polymorphism of ginseng used for genomic DNA extraction is a reproducible result, a number of samples (n = 10) were randomly selected and examined for each ginseng sample by each region. Reproducible results were obtained.
    [23] Therefore, according to the present invention, it is not only possible to distinguish the origin of ginseng as compared to the analysis table shown in FIGS. 1, 2, and 3 as the standard DNA fingerprint of ginseng by region, but also the RAPD and / or PCR-PFLP of the present invention. It is possible to provide a ginseng differentiation kit comprising a standard analysis diagram for the comparison of ginseng for each region according to FIGS. 1, 2, and 3 obtained by the analysis and an analysis table analyzing the same.
    [24] Hereinafter, the present invention will be described in more detail with respect to the ginseng differentiation method of the present invention by the following experimental examples and examples of the present invention, the present invention is not limited in any way by these examples.
    [25] In the present invention, the Chinese ginseng and the domestic ginseng were discriminated at the molecular level by the RAPD method, and the PCR-RFLP method attempted to discriminate more clearly between the domestic ginseng. The specific method is as follows.
    [26] Ginseng was made from China, Geumsan, Jinan, and Ganghwa, respectively. The instrument for PCR amplification was used by Technie Corp., Taq DNA polymerase was purchased from Takara, and dNTP was purchased from Amersham Pharmacia. Random primers for RAPD were from Promega (UniPrimer Kit, Cat. #: SS4101), and primers for PCR-RFLP were purchased from Bioneer. The base sequence of the random primer is as shown in Figure 4 and the sequence table. Restriction enzyme for PCR-RFLP was purchased from New England Biolab. Agarose used for electrophoresis was purchased from GIBCO-BRL and 100 bp markers from Amersham Pharmacia. For statistical analysis, SPSS / PC + was used.
    [27] Example 1 Genomic DNA Extraction of Ginseng
    [28] Genomic DNA extraction was performed using the method of Hirohsi Fushimi et al. (Fushimi, et al., Supra). After drying the dried ginseng, each powder was washed with PBS-EDTA consisting of 140 mM NaCl, 2.7 mM KCl, 8.1 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , 5.4 mM EDTA, pH 7.0. To the washed precipitate was added 10 times the amount of extraction buffer (100 mM Tris-HCl (pH 8.0), 40 mM EDTA, 250 mM NaCl, 1 volume of 10% sodium N-lauryl sarcosinate and 1 mg of protease K). After the addition, the reaction was carried out at 37 ° C. for 12 hours. 0.1 volume of 10% cetyl trimethylammonium bromide (CTAB) and 0.1 volume of 5M NaCl were added to the clear lysate and extracted with phenol-CHCl 3 (1: 1). Extracted once more with CHCl 3 and precipitated with 0.1 volume of 3M NaOAc and 2.5 volume of anhydrous ethanol. The precipitated DNA was dissolved in TE-buffer (10 mM Tri-HCl, 1 mM EDTA, pH 8.0) The solution was reacted for 30 minutes in 10 ng / ml RNaseA (Sigma, USA) and then Sephadex G-50 (Sigma). , USA).
    [29] Example 2 RAPD Analysis on Ginseng
    [30] A total of twelve 20mer-randomized primers (SRILS Uniprimer ™ ; obtained from Promega, FIG. 4) were screened for DNA extracted from China, Geumsan, Jinan, and Fortified Ginseng. As a result, RAPD fingerprints were generated from eight primers. Of these, five primers (SEQ ID NOS: 1, 3, 5, 6 and 9 of the Sequence Listing) were selected that produced reproducible and clear fingerprints. DNA fragments formed using five primers ranged from 100 base pairs to 2600 base pairs and the average number of bands formed per primer was 11.4 fragments on average (FIG. 1).
    [31] 200 ng of genomic DNA, 50 mM KCl, 10 mM Tris-HCl (pH9.0), 200 μM dNTP, 1.5 mM MgCl 2 , 150 ng random primer, 2.5 U Taq polymerase were included in the total 30 μL reaction solution, 94 After 1 degreeC reaction, the cycle of 94 degreeC 1 minute, 55 degreeC 1 minute, and 72 degreeC 2 minutes was repeated 35 times, and it reacted at 72 degreeC for 7 minutes. After the reaction, the product was electrophoresed on a 2% agarose gel, stained with EtBr, and identified by UV transilluminator.
    [32] The amplified DNA fingerprints are converted to a binary character matrix ("1" for amplified bands present at a specific location and "0" for none) to convert SPSS / PC + (ver 6.0). Similarity was obtained (Bai, D., Brandle, et al., Supra). The test results are shown in Table 1 below.
    [33] Similarity Matrix of Jinan, Ganghwa, Geumsan and Chinese GinsengJaccard's Way JinanreinforceGeumsanChina JinanOne0.3450.4910.266 reinforce0.345One0.2030.197 Geumsan0.4910.203One0.231 China0.2660.1970.231One
    [34] As can be seen from the table, all of the five primers showed a significant difference that can be distinguished from both Jinan, Geumsan, Ganghwa, and Chinese ginseng, especially in the case of Chinese was distinct from domestic. Domestic production was closely related, but Ganghwa Mountain showed a different pattern compared to Jinan and Geumsan Ginseng. The amplified bands are converted into a binary character matrix ("1" for the amplified band present at a specific position and "0" for the nonexistent one), and the similarity is obtained using SPSS / PC + (ver 6.0). Similarity index) showed a similarity range from 0.197 to 0.491 (Table 1). Jinan ginseng and Geumsan ginseng showed the highest similarity with 0.491, while Jinan ginseng and Chinese ginseng showed 0.266, and Geumsan ginseng and Chinese ginseng showed 0.231. Ganghwa ginseng and Chinese ginseng showed the lowest similarity with 0.197, showing the possibility of distinction.
    [35] Example 3: PCR-RFLP Analysis on 18S rRNA Genes
    [36] PCR-RFLP analysis was performed on 18S rRNA genes of Korean ginseng and fortified ginseng.
    [37] 100 ng of the extracted DNA was subjected to PCR using primers prepared for the flanking sequence of the 18S rRNA gene. Each primer sequence is as follows. 18S F is 5 ′ CAA CCT GGT TGA TCC TGC CAG T 3 ′ (SEQ ID NO: 13) and 18S R is 5 ′ CTG ATC CTT CTG CAG GTT CAC CTA C 3 ′ (SEQ ID NO: 14). PCR conditions were 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 200 μM dNTP, 2.5 mM MgCl 2 , 0.25 μM of each primer, 1.5 units of Taq polymerase were included in the total 50 μL reaction solution, 94 ℃ 3 After the reaction for 55 minutes at 55 ° C. for 8 minutes, the cycle of 94 ° C. for 40 seconds and 55 ° C. for 8 minutes was repeated 30 times, followed by reaction at 55 ° C. for 8 minutes. After the reaction, the product was electrophoresed on a 2% agarose gel, stained with EtBr, and identified by UV transilluminator. The resulting 1.8Kb amplification product was purified using PCR-Prep Kit (Promega) and then reacted with restriction enzyme 5 units BanII and DdeI (New England Biolab) for 1.5 hours at 37 ℃. The product was electrophoresed on 6% polyacrylamide gel, stained with EtBr and identified by UV transilluminator.
    [38] Restriction enzyme Ban II recognizes and cleaves '5 GGGCTC 3' and DdeI recognizes 5 'CTCAG 3'. Jinan ginseng and Geumsan ginseng showed the same type of fingerprints ( Bang II and Geumsan Ginseng), which showed a high similarity of SI value of 0.491 with BanII and DdeI , respectively, but fortified ginseng produced different types of fingerprints (Fig. 2). Therefore, PCR-RFLP method using restriction enzymes Ban II and DdeI is also useful for the classification of Korean ginseng.
    [39] Example 4 Review of Reproducibility on Any Sample
    [40] In order to prove that the polymorphism of ginseng used for the extraction of genomic DNA is a reproducible result, a number of samples were randomly selected and examined for each ginseng sample by each region. Reproducible results were also obtained for randomly selected samples (FIG. 3).
    [41] As can be seen from the above experimental results, the method of the present invention is not only capable of authenticating at the molecular level with respect to domestic and foreign ginseng, but also distinguishing ginseng according to mountain regions in domestic cases. Moreover, this method is considered to be useful for the differentiation application of many other herbal medicines by region and morphologically ambiguous.
    [42] The random primer used in the present invention is not only to obtain a reproducible result as 20mer, but also in the reality that the ginseng circulated in Korea is a species of P. ginseng most of the species is of great significance. Moreover, the classification of ginseng by domestic production region using PCR-RFLP is very useful.
    [43] These results show the possibility of differentiation of ginseng by region in domestic and overseas ginseng as well as domestically and internationally. Furthermore, such a method is not only useful for the differentiation of many morphologically ambiguous herbal medicines, but it is also possible to use the analysis results shown in the drawings and the like obtained by the method of the present invention as a standard for differentiating a seal.
    权利要求:
    Claims (5)
    [1" claim-type="Currently amended] a) extracting DNA from each ginseng sample;
    b) preparing a reaction solution of the extracted DNA and the primer;
    c) amplifying the prepared reaction solution; And
    d) converting the amplified genomic DNA fingerprint to a double letter table to determine similarity
    Method of distinguishing ginseng by mountain area, including.
    [2" claim-type="Currently amended] The method of claim 1, wherein said amplification is performed using RAPD and / or PCR-RFLP.
    [3" claim-type="Currently amended] The method of claim 1, wherein the primers are SEQ ID NO: 1, 3, 5, 6, 9 and / or 13, 14 of the Sequence Listing.
    [4" claim-type="Currently amended] The method according to claim 1, wherein as a standard DNA fingerprint of ginseng for each region, the area of ginseng is distinguished from the analysis table shown in FIGS. 1, 2, and 3 below.
    [5" claim-type="Currently amended] Next ginseng differentiation kit comprising a standard analysis for comparing ginseng by region according to Figures 1, 2 and 3 and the analysis table analyzing the same.
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    同族专利:
    公开号 | 公开日
    KR100416765B1|2004-01-31|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    2000-07-06|Application filed by 김형민
    2000-07-06|Priority to KR20000038523A
    2002-01-16|Publication of KR20020004536A
    2004-01-31|Application granted
    2004-01-31|Publication of KR100416765B1
    优先权:
    申请号 | 申请日 | 专利标题
    KR20000038523A|KR100416765B1|2000-07-06|2000-07-06|Discrimination method of Panax ginseng|
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