Ekinezya Ürünlerine Hile Amaçlı Karıştırılan Parthenium integrifolium’un Döngü Aracılı İzotermal Amplifikasyon Yöntemiyle Tespiti
Yıl 2025,
Cilt: 14 Sayı: 2, 392 - 399, 29.12.2025
Muzaffer Arıkan
,
Zelal Zuhal Kaya
Öz
Amaç: Ekinezya, tıbbi amaçlarla dünya çapında yaygın olarak kullanılan bir bitkidir. Ekinezya ürünlerinin önemli bir pazar büyüklüğüne ulaşması, bu ürünlere yönelik hile amaçlı uygulamalarla ilgili endişelerin de artmasına yol açmıştır. Bitkisel ticari ürünlerin test edilmesi için kullanılan geleneksel yöntemler genellikle bitki türlerinin ayırt edici morfolojik özelliklerine dayanmaktadır. Bu nedenle toz haline getirilmiş ya da endüstriyel işlemlere tabi tutulmuş bitki materyallerinde işe yaramamaktadır. Bu çalışmada, Ekinezya ürünlerine hile amaçlı karıştırıldığı bilinen Parthenium integrifolium türünün tespitini sağlayan döngü aracılı izotermal amplifikasyon (LAMP) temelli bir yöntemin geliştirilmesi ve test edilmesi amaçlanmıştır.
Materyal ve Yöntem: Çalışma kapsamında, tıbbi amaçlı kullanılan Echinacea purpurea, Echinacea angustifolia ve Echinacea pallida türleri ve Parthenium integrifolium bitkisi temin edilmiş ve bu bitkilerden DNA izolasyonu yapılmıştır. Veri tabanlarındaki ham Parthenium integrifolium DNA dizileri analiz edilerek hedef gen bölgeleri tespit edilmiştir. Daha sonra bu hedef bölgeleri çoğaltacak spesifik LAMP primer dizileri tasarlanmış ve optimizasyonlar gerçekleştirilmiştir.
Araştırma Bulguları: Yapılan çalışmalar sonucunda, 65 oC sabit sıcaklıkta uygulanan ve hile amaçlı karıştırılan bitki materyali varlığını reaksiyon karışımındaki renk değişimi sayesinde, ek bir analize gerek kalmadan tespit edebilen DNA temelli bir yöntem geliştirilmiştir. Geliştirilen yöntem için P. integrifolium tespit sınırı 0,5 ng, özgüllük ise %100 olarak saptanmıştır. Bu yöntemle İstanbul’da temin edilen 50 ticari Echinacea ürünü test edilmiş ve örneklerin hiçbirinde P. integrifolium varlığına rastlanmamıştır.
Sonuç: Bu çalışmada, Echinacea ürünlerinde Parthenium integrifolium gibi tağşiş unsurlarının varlığını tespit etmeye yönelik, LAMP temelli yeni bir moleküler yöntem geliştirilmiştir.
Destekleyen Kurum
Gıda Tarım ve Hayvancılık Bakanlığı Tarımsal Araştırmalar ve Politikalar Genel Müdürlüğü
Proje Numarası
TAGEM-16/AR-GE/54
Teşekkür
Çalışmamıza, Araştırma-Geliştirme Destek Programı kapsamında sağladıkları maddi destekten (Proje No: TAGEM-16/AR-GE/54) dolayı Gıda Tarım ve Hayvancılık Bakanlığı, Tarımsal Araştırmalar ve Politikalar Genel Müdürlüğü’ne teşekkür ederiz.
Kaynakça
-
Abdel-Latif, A., & Osman, G. (2017). Comparison of three genomic DNA extraction methods to obtain high DNA quality from maize. Plant Methods, 13(1), 1. https://doi.org/10.1186/s13007-016-0152-4
-
Ardjomand-Woelkart, K., & Bauer, R. (2015). Review and Assessment of Medicinal Safety Data of Orally Used Echinacea Preparations. Planta Medica. https://doi.org/10.1055/s-0035-1558096
-
Azizah, N., Halimah, E., Puspitasari, I. M., & Hasanah, A. N. (2021). Simultaneous Use of Herbal Medicines and Antihypertensive Drugs Among Hypertensive Patients in the Community: A Review. Journal of Multidisciplinary Healthcare, Volume 14, 259–270. https://doi.org/10.2147/JMDH.S289156
-
Chabi Sika, K., Kefela, T., Adoukonou-Sagbadja, H., Ahoton, L., Saidou, A., Baba-Moussa, L., Jno Baptiste, L., Kotconi, S. O., & Gachomo, E. W. (2015). A simple and efficient genomic DNA extraction protocol for large scale genetic analyses of plant biological systems. Plant Gene, 1, 43–45. https://doi.org/10.1016/j.plgene.2015.03.001
-
Ganie, S. H., Upadhyay, P., Das, S., & PrasadSharma, M. (2015). Authentication of medicinal plants by DNA markers. Plant Gene. https://doi.org/10.1016/j.plgene.2015.10.002
-
Healey, A., Furtado, A., Cooper, T., & Henry, R. J. (2014). Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species. Plant Methods, 10(21), 1–8. https://doi.org/10.1186/1746-4811-10-21
-
Hollingsworth, P. M., Forrest, L. L., Spouge, J. L., Hajibabaei, M., Ratnasingham, S., van der Bank, M., Chase, M. W., Cowan, R. S., Erickson, D. L., Fazekas, A. J., Graham, S. W., James, K. E., Kim, K.-J., Kress, W. J., Schneider, H., van AlphenStahl, J., Barrett, S. C. H., van den Berg, C., Bogarin, D., … Little, D. P. (2009). A DNA barcode for land plants. Proceedings of the National Academy of Sciences, 106(31), 12794–12797. https://doi.org/10.1073/pnas.0905845106
-
Hollingsworth, P. M., Graham, S. W., & Little, D. P. (2011). Choosing and Using a Plant DNA Barcode. PLoS ONE, 6(5), e19254. https://doi.org/10.1371/journal.pone.0019254
-
Kokkinos, P. a., Ziros, P. G., Bellou, M., & Vantarakis, a. (2014). Loop-Mediated Isothermal Amplification (LAMP) for the Detection of Salmonella in Food. Food Analytical Methods, 7, 512–526. https://doi.org/10.1007/s12161-013-9748-8
-
Kress, W. J., & Erickson, D. L. (2007). A Two-Locus Global DNA Barcode for Land Plants: The Coding rbcL Gene Complements the Non-Coding trnH-psbA Spacer Region. PLoS ONE, 2(6), e508. https://doi.org/10.1371/journal.pone.0000508
-
Kunle, O. F., Egharevba, H. O., & Ahmadu, P. O. (2012). Standardization of herbal medicines - A review. International Journal of Biodiversity and Conservation, 4(3), 101–112. https://doi.org/10.5897/IJBC11.163
-
Laasonen, M., Harmia-Pulkkinen, T., Simard, C. L., Michiels, E., Räsänen, M., & Vuorela, H. (2002). Fast Identification of Echinacea purpurea Dried Roots Using Near-Infrared Spectroscopy. Analytical Chemistry, 74(11), 2493–2499. https://doi.org/10.1021/ac011108f
-
Law, J. W.-F., Ab Mutalib, N.-S., Chan, K.-G., & Lee, L.-H. (2015). Rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations. Frontiers in Microbiology, 5(January), 1–19. https://doi.org/10.3389/fmicb.2014.00770
-
Li, J. J., Xiong, C., Liu, Y., Liang, J. S., & Zhou, X. W. (2016). Loop-mediated isothermal amplification (LAMP): emergence as an alternative technology for herbal medicine identification. Frontiers in plant science, 7, 1956. https://doi.org/10.3389/fpls.2016.01956
-
Li, H., Cui, J., Chen, H., Li, H., Xie, Y., Song, W., & Chen, R. (2024). Rapid colorimetric and fluorescence identification of Pinelliae Rhizoma and adulterate Rhizoma Typhonii Flagelliformis using direct-LAMP assay. Food Chemistry, 437, 137840. https://doi.org/10.1016/j.foodchem.2023.137840
-
Mishra, P., Kumar, A., Nagireddy, A., Mani, D. N., Shukla, A. K., Tiwari, R., & Sundaresan, V. (2016). DNA barcoding: an efficient tool to overcome authentication challenges in the herbal market. Plant biotechnology journal, 14(1), 8-21. https://doi.org/10.1111/pbi.12419
-
Mistríková, I., & Vaverková, Š. (2007). Morphology and anatomy of Echinacea purpurea, E. angustifolia, E. pallida and Parthenium integrifolium. Biologia, 62(1), 2–5. https://doi.org/10.2478/s11756-007-0006-7
-
Mudge, E. (2011). Isolation and Fast Analysis of Phytochemical Constituents in Echinacea species and Rhodiola rosea L. using High-Speed Counter-Current Chromatography and Ultra Fast Liquid Chromatography-Mass Spectrometry. https://doi.org/10.7939/R3WM1H
-
Newmaster, S.G., Fazekas, A. J., & Ragupathy, S. (2006). DNA barcoding in land plants: evaluation of rbcL in a multigene tiered approach. Canadian Journal of Botany, 84(3), 335–341. https://doi.org/10.1139/b06-047
-
Newmaster, Steven G, Grguric, M., Shanmughanandhan, D., Ramalingam, S., & Ragupathy, S. (2013). DNA barcoding detects contamination and substitution in North American herbal products. BMC Medicine, 11(1), 222. https://doi.org/10.1186/1741-7015-11-222
-
Notomi, T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Research, 28(12), 63e – 63. https://doi.org/10.1093/nar/28.12.e63
-
Notomi, T., Mori, Y., Tomita, N., & Kanda, H. (2015). Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects. Journal of Microbiology,53(1), 1–5. https://doi.org/10.1007/s12275-015-4656-9
-
Novak, J., Grausgruber-Gröger, S., & Lukas, B. (2007). DNA-based authentication of plant extracts. Food Research International, 40(3), 388–392. https://doi.org/10.1016/j.foodres.2006.10.015
-
Parveen, I., Gafner, S., Techen, N., Murch, S. J., & Khan, I. A. (2016). DNA Barcoding for the Identification of Botanicals in Herbal Medicine and Dietary Supplements: Strengths and Limitations. Planta Medica, 82(14), 1225–1235. https://doi.org/10.1055/s-0042-111208
-
Rogers, S. O., & Bendich, A. J. (1989). Extraction of DNA from plant tissues. In Plant Molecular Biology Manual (Vol. 10, pp. 73–83). Springer Netherlands. https://doi.org/10.1007/978-94-009-0951-9_6
-
Russi, L., Moretti, C., Raggi, L., Albertini, E., & Falistocco, E. (2009). Identifying commercially relevant Echinacea species by AFLP molecular markers. Genome / National Research Council Canada = Genome / Conseil National de Recherches Canada, 52(11), 912–918. https://doi.org/10.1139/G09-066
-
Sasaki, Y., Komatsu, K., & Nagumo, S. (2008). Rapid Detection of Panax ginseng by Loop-Mediated Isothermal Amplification and Its Application to Authentication of Ginseng. Biological & Pharmaceutical Bulletin, 31(9), 1806–1808. https://doi.org/10.1248/bpb.31.1806
-
Shao, Y., Zhu, S., Jin, C., & Chen, F. (2011). Development of multiplex loop-mediated isothermal amplification-RFLP (mLAMP-RFLP) to detect Salmonella spp. and Shigella spp. in milk. International Journal of Food Microbiology, 148(2), 75–79. https://doi.org/10.1016/j.ijfoodmicro.2011.05.004
-
Sheu, S. C., Wu, Y. C., Lien, Y. Y., & Lee, M. S. (2021). Specific, sensitive and rapid Curcuma longa turmeric powder authentication in commercial food using loop-mediated isothermal nucleic acid amplification. Saudi Journal of Biological Sciences, 28(10), 5931-5936. https://doi.org/10.1016/j.sjbs.2021.06.057
-
Wolf, H. T., Zündorf, I., Winckler, T., Bauer, R., & Dingermann, T. (1999). Characterization of Echinacea species and detection of possible adulterations by RAPD analysis. Planta Medica, 65(08), 773-774. https://doi.org/10.1055/s-2006-960866
-
Xin, Z., & Chen, J. (2012). A high throughput DNA extraction method with high yield and quality. Plant Methods, 8(1), 26. https://doi.org/10.1186/1746-4811-8-26
-
Zhao, X., Lin, C.-W., Wang, J., & Oh, D. H. (2014). Advances in rapid detection methods for foodborne pathogens. Journal of Microbiology and Biotechnology, 24(3), 297–312. https://doi.org/10.4014/jmb.1310.10013
-
Zhao, M., Shi, Y., Wu, L., Guo, L., Liu, W., Xiong, C., ... & Chen, S. (2016). Rapid authentication of the precious herb saffron by loop-mediated isothermal amplification (LAMP) based on internal transcribed spacer 2 (ITS2) sequence. Scientific Reports, 6(1), 25370. https://doi.org/10.1038/srep25370
Detection of Parthenium integrifolium Used for Adulteration in Echinacea Products by a Loop Mediated Isothermal Amplification Method
Yıl 2025,
Cilt: 14 Sayı: 2, 392 - 399, 29.12.2025
Muzaffer Arıkan
,
Zelal Zuhal Kaya
Öz
Objective: Echinacea is a plant widely used worldwide for medical purposes. The fact that Echinacea products have reached a significant market size has also led to increased concerns about adulteration of these products. Traditional methods for testing herbal commercial products are often based on the differential morphological characteristics of the plant species. Therefore, plant materials that have been powdered or subjected to industrial processing cannot be tested using these methods. The aim of this study was to develop and test a loop-mediated isothermal amplification (LAMP)-based method that detects the Parthenium integrifolium species which is known to be used for adulteration of Echinacea products.
Materials and Methods: Echinacea purpurea, Echinacea angustifolia, Echinacea pallida and Parthenium integrifolium plants were obtained and used for DNA isolations. By analysis of raw Parthenium integrifolium DNA sequences in the public databases, specific LAMP primer sequences were designed to amplify targeted gene regions and then reaction optimizations were performed.
Results: As a result of the study, a DNA-based method which is applied at a constant temperature of 65 °C and detects the presence of plant material mixed for adulteration purposes, by means of the color change in the reaction mixture, without the need for an additional analysis has been developed. The method had a detection limit of 0.5 ng for Parthenium integrifolium and a specificity of 100%. 50 commercial Echinacea products from Istanbul were tested and the results showed no presence of Parthenium integrifolium in the products.
Conclusion: In this study, a new molecular method based on LAMP was developed to detect the presence of adulterants such as Parthenium integrifolium in Echinacea products.
Proje Numarası
TAGEM-16/AR-GE/54
Kaynakça
-
Abdel-Latif, A., & Osman, G. (2017). Comparison of three genomic DNA extraction methods to obtain high DNA quality from maize. Plant Methods, 13(1), 1. https://doi.org/10.1186/s13007-016-0152-4
-
Ardjomand-Woelkart, K., & Bauer, R. (2015). Review and Assessment of Medicinal Safety Data of Orally Used Echinacea Preparations. Planta Medica. https://doi.org/10.1055/s-0035-1558096
-
Azizah, N., Halimah, E., Puspitasari, I. M., & Hasanah, A. N. (2021). Simultaneous Use of Herbal Medicines and Antihypertensive Drugs Among Hypertensive Patients in the Community: A Review. Journal of Multidisciplinary Healthcare, Volume 14, 259–270. https://doi.org/10.2147/JMDH.S289156
-
Chabi Sika, K., Kefela, T., Adoukonou-Sagbadja, H., Ahoton, L., Saidou, A., Baba-Moussa, L., Jno Baptiste, L., Kotconi, S. O., & Gachomo, E. W. (2015). A simple and efficient genomic DNA extraction protocol for large scale genetic analyses of plant biological systems. Plant Gene, 1, 43–45. https://doi.org/10.1016/j.plgene.2015.03.001
-
Ganie, S. H., Upadhyay, P., Das, S., & PrasadSharma, M. (2015). Authentication of medicinal plants by DNA markers. Plant Gene. https://doi.org/10.1016/j.plgene.2015.10.002
-
Healey, A., Furtado, A., Cooper, T., & Henry, R. J. (2014). Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species. Plant Methods, 10(21), 1–8. https://doi.org/10.1186/1746-4811-10-21
-
Hollingsworth, P. M., Forrest, L. L., Spouge, J. L., Hajibabaei, M., Ratnasingham, S., van der Bank, M., Chase, M. W., Cowan, R. S., Erickson, D. L., Fazekas, A. J., Graham, S. W., James, K. E., Kim, K.-J., Kress, W. J., Schneider, H., van AlphenStahl, J., Barrett, S. C. H., van den Berg, C., Bogarin, D., … Little, D. P. (2009). A DNA barcode for land plants. Proceedings of the National Academy of Sciences, 106(31), 12794–12797. https://doi.org/10.1073/pnas.0905845106
-
Hollingsworth, P. M., Graham, S. W., & Little, D. P. (2011). Choosing and Using a Plant DNA Barcode. PLoS ONE, 6(5), e19254. https://doi.org/10.1371/journal.pone.0019254
-
Kokkinos, P. a., Ziros, P. G., Bellou, M., & Vantarakis, a. (2014). Loop-Mediated Isothermal Amplification (LAMP) for the Detection of Salmonella in Food. Food Analytical Methods, 7, 512–526. https://doi.org/10.1007/s12161-013-9748-8
-
Kress, W. J., & Erickson, D. L. (2007). A Two-Locus Global DNA Barcode for Land Plants: The Coding rbcL Gene Complements the Non-Coding trnH-psbA Spacer Region. PLoS ONE, 2(6), e508. https://doi.org/10.1371/journal.pone.0000508
-
Kunle, O. F., Egharevba, H. O., & Ahmadu, P. O. (2012). Standardization of herbal medicines - A review. International Journal of Biodiversity and Conservation, 4(3), 101–112. https://doi.org/10.5897/IJBC11.163
-
Laasonen, M., Harmia-Pulkkinen, T., Simard, C. L., Michiels, E., Räsänen, M., & Vuorela, H. (2002). Fast Identification of Echinacea purpurea Dried Roots Using Near-Infrared Spectroscopy. Analytical Chemistry, 74(11), 2493–2499. https://doi.org/10.1021/ac011108f
-
Law, J. W.-F., Ab Mutalib, N.-S., Chan, K.-G., & Lee, L.-H. (2015). Rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations. Frontiers in Microbiology, 5(January), 1–19. https://doi.org/10.3389/fmicb.2014.00770
-
Li, J. J., Xiong, C., Liu, Y., Liang, J. S., & Zhou, X. W. (2016). Loop-mediated isothermal amplification (LAMP): emergence as an alternative technology for herbal medicine identification. Frontiers in plant science, 7, 1956. https://doi.org/10.3389/fpls.2016.01956
-
Li, H., Cui, J., Chen, H., Li, H., Xie, Y., Song, W., & Chen, R. (2024). Rapid colorimetric and fluorescence identification of Pinelliae Rhizoma and adulterate Rhizoma Typhonii Flagelliformis using direct-LAMP assay. Food Chemistry, 437, 137840. https://doi.org/10.1016/j.foodchem.2023.137840
-
Mishra, P., Kumar, A., Nagireddy, A., Mani, D. N., Shukla, A. K., Tiwari, R., & Sundaresan, V. (2016). DNA barcoding: an efficient tool to overcome authentication challenges in the herbal market. Plant biotechnology journal, 14(1), 8-21. https://doi.org/10.1111/pbi.12419
-
Mistríková, I., & Vaverková, Š. (2007). Morphology and anatomy of Echinacea purpurea, E. angustifolia, E. pallida and Parthenium integrifolium. Biologia, 62(1), 2–5. https://doi.org/10.2478/s11756-007-0006-7
-
Mudge, E. (2011). Isolation and Fast Analysis of Phytochemical Constituents in Echinacea species and Rhodiola rosea L. using High-Speed Counter-Current Chromatography and Ultra Fast Liquid Chromatography-Mass Spectrometry. https://doi.org/10.7939/R3WM1H
-
Newmaster, S.G., Fazekas, A. J., & Ragupathy, S. (2006). DNA barcoding in land plants: evaluation of rbcL in a multigene tiered approach. Canadian Journal of Botany, 84(3), 335–341. https://doi.org/10.1139/b06-047
-
Newmaster, Steven G, Grguric, M., Shanmughanandhan, D., Ramalingam, S., & Ragupathy, S. (2013). DNA barcoding detects contamination and substitution in North American herbal products. BMC Medicine, 11(1), 222. https://doi.org/10.1186/1741-7015-11-222
-
Notomi, T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Research, 28(12), 63e – 63. https://doi.org/10.1093/nar/28.12.e63
-
Notomi, T., Mori, Y., Tomita, N., & Kanda, H. (2015). Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects. Journal of Microbiology,53(1), 1–5. https://doi.org/10.1007/s12275-015-4656-9
-
Novak, J., Grausgruber-Gröger, S., & Lukas, B. (2007). DNA-based authentication of plant extracts. Food Research International, 40(3), 388–392. https://doi.org/10.1016/j.foodres.2006.10.015
-
Parveen, I., Gafner, S., Techen, N., Murch, S. J., & Khan, I. A. (2016). DNA Barcoding for the Identification of Botanicals in Herbal Medicine and Dietary Supplements: Strengths and Limitations. Planta Medica, 82(14), 1225–1235. https://doi.org/10.1055/s-0042-111208
-
Rogers, S. O., & Bendich, A. J. (1989). Extraction of DNA from plant tissues. In Plant Molecular Biology Manual (Vol. 10, pp. 73–83). Springer Netherlands. https://doi.org/10.1007/978-94-009-0951-9_6
-
Russi, L., Moretti, C., Raggi, L., Albertini, E., & Falistocco, E. (2009). Identifying commercially relevant Echinacea species by AFLP molecular markers. Genome / National Research Council Canada = Genome / Conseil National de Recherches Canada, 52(11), 912–918. https://doi.org/10.1139/G09-066
-
Sasaki, Y., Komatsu, K., & Nagumo, S. (2008). Rapid Detection of Panax ginseng by Loop-Mediated Isothermal Amplification and Its Application to Authentication of Ginseng. Biological & Pharmaceutical Bulletin, 31(9), 1806–1808. https://doi.org/10.1248/bpb.31.1806
-
Shao, Y., Zhu, S., Jin, C., & Chen, F. (2011). Development of multiplex loop-mediated isothermal amplification-RFLP (mLAMP-RFLP) to detect Salmonella spp. and Shigella spp. in milk. International Journal of Food Microbiology, 148(2), 75–79. https://doi.org/10.1016/j.ijfoodmicro.2011.05.004
-
Sheu, S. C., Wu, Y. C., Lien, Y. Y., & Lee, M. S. (2021). Specific, sensitive and rapid Curcuma longa turmeric powder authentication in commercial food using loop-mediated isothermal nucleic acid amplification. Saudi Journal of Biological Sciences, 28(10), 5931-5936. https://doi.org/10.1016/j.sjbs.2021.06.057
-
Wolf, H. T., Zündorf, I., Winckler, T., Bauer, R., & Dingermann, T. (1999). Characterization of Echinacea species and detection of possible adulterations by RAPD analysis. Planta Medica, 65(08), 773-774. https://doi.org/10.1055/s-2006-960866
-
Xin, Z., & Chen, J. (2012). A high throughput DNA extraction method with high yield and quality. Plant Methods, 8(1), 26. https://doi.org/10.1186/1746-4811-8-26
-
Zhao, X., Lin, C.-W., Wang, J., & Oh, D. H. (2014). Advances in rapid detection methods for foodborne pathogens. Journal of Microbiology and Biotechnology, 24(3), 297–312. https://doi.org/10.4014/jmb.1310.10013
-
Zhao, M., Shi, Y., Wu, L., Guo, L., Liu, W., Xiong, C., ... & Chen, S. (2016). Rapid authentication of the precious herb saffron by loop-mediated isothermal amplification (LAMP) based on internal transcribed spacer 2 (ITS2) sequence. Scientific Reports, 6(1), 25370. https://doi.org/10.1038/srep25370