American Journal of Life Sciences
Volume 4, Issue 5, October 2016, Pages: 118-128
Received: Sep. 15, 2016;
Accepted: Sep. 28, 2016;
Published: Oct. 19, 2016
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Sudhir S. P., Department of Life Science, Jaipur National University, Jaipur, India
Alagappan Kumarappan, Department of Microbiology, Marine Biology, and Virology, University of Modern Sciences, Dubai, UAE
Lalit K. Vyas, Vidyabahrti Mahavidyalaya, Department of Cosmetic Technology, Amravati, India
Divya Shrivastava, Department of Life Science, Jaipur National University, Jaipur, India
Padma Deshmukh, Department of Microbiology, Smt. C.H.M. College of Arts, Commerce and Science, Ulhasnagar, Mumbai, India
H. N. Verma, Department of Life Science, Jaipur National University, Jaipur, India
Adulteration, misidentification, and substitution are the biggest challenges in maintaining safety and therapeutic efficacy of medicinal herbs. Nigella sativa seed, which is well known medicinal herb susceptible to adulteration or substitution due to its great therapeutic value. Adulteration and substitution by morphologically similar seeds are the primary concern in commercially available Nigella sativa seed. In this study, we have used DNA barcode marker to find out adulteration, misidentification, and substitution of Nigella sativa seed sold in various markets. We collected 10 samples, which were labelled as Black seed/Nigella sativa seed from open markets in India (1 No.), Pakistan (1 No.), Saudi Arabia(1 No.), Egypt (2 No.), Turkey (1 No.), Syria (1 No.), Tunisia(2 No.) and Oman (1 No.). All samples collected from different geographies were studied morphologically. Although few samples were quickly identified as Nigella sativa seeds, few were tough to detect and differentiate accurately. This is where DNA barcode marker proved to be useful. Plant DNA were obtained from seed coat cells of samples, was amplified by PCR with forward and reverse rbcl and matK primers as recommended by CBOL (The Consortium for the Barcode of Life). PCR amplification of plastid genome with matK was not very successful, while PCR amplification with rbcl primers was quite successful. We used rbcl sequences for alignment and further analysis. PCR products obtained were subjected to electrophoresis on 1.5% agarose plate. PCR products were sent to Macrogen (Seoul, South Korea) for DNA sequencing. DNA reads obtained with rbcl sequences were aligned and analyzed for nucleotide composition, conserved sites, variable sites, singleton sites and parsimony-informative sites, genetic distance and phylogenetic tree using MEGA 7. The phylogenetic tree was constructed using UPGMA method. NCBI Blast along with phylogenic tree and nucleotide characteristic were used to identify Nigella sativa seeds from different geographies and discriminate two adulterants as Allium cepa seed and Clitoria guianensis seed. Both of these adulterants are different regarding their active medicinal contents and therapeutic utility from Nigella sativa seed. This study proved the utility of DNA marker, especially rbcl loci in accurately identifying medicinal herb and its adulterants.
Sudhir S. P.,
Lalit K. Vyas,
H. N. Verma,
Identification of Nigella sativa Seed and Its Adulterants Using DNA Barcode Marker, American Journal of Life Sciences.
Vol. 4, No. 5,
2016, pp. 118-128.
Showkat Hussain Ganie, Priti Upadhyay, Sandip Das, Maheshwer Prasad Sharma, Authentication of medicinal plants by DNA markers, Plant Gene, Volume 4, December 2015, Pages 83-99, ISSN 2352-4073, http://dx.doi.org/10.1016/j.plgene.2015.10.002.(http://www.sciencedirect.com/science/article/pii/S235240731500030X)
Al-Bukhari. MI. Division (71) on medicine. In Sahi Al-Bukhari, the collection of authentic sayings of Prophet Mohammad (peace be upon him). 2nd ed. Hilal Yayinlari, Ankara, Turkey, 1976.
Padmaa, M. and Paarakh (2010). Nigella sativa. Linn.A comprehensive review. Indian Journal of Natural Products and Resources 1(4): 409-429.
Dermatological effects of Nigella sativa, Salih H. M. Aljabrea, Omar M. Alakloby a, Mohammad A. Randhaw, Journal of Dermatology & Dermatologic Surgery 19 (2015) 92–98.
Handbook of herbs and spices, Volume 2,2004, Edited by,K. V. Peter, CRC Press Boca Raton Boston New York Washington, DC. Page 225-226.
Rania Mubarak AwadObaid, Anti-Mycetoma, anti-oxidant and Phytochemical Screening of Nigella sativa seeds, A thesis for Master Degree in pharmacy (pharmacognosy),The National Ribat University Faculty of Graduate studies & Scientific Research, May 2015.
Anjoo Kamboj (2012). Analytical Evaluation of Herbal Drugs, Drug Discovery Research in Pharmacognosy, Prof. OmboonVallisuta (Ed.), ISBN: 978-953-51-0213-7, InTech, Available from: http://www.intechopen.com/books/drug-discovery-research-in-pharmacognosy/analytical-evaluation-of-herbaldrugs.
Xie PS, Leung AY. 2009. Understanding the traditional aspect of Chinese medicine in order to achieve meaningful quality control of Chinese materiamedica. Journal of Chromatography A. 1216: 1933- 1940.
Yi L, Liang Y, Wu H, Yuan D. 2009. The analysis of Radix Angelicae Sinensis (Danggui). Journal of Chromatography A. 1216: 1991-2001.
Hebert PDN, Cywinska A, Ball SL, deWaard JR. Biological identifications through DNA barcodes. Proceedings of the Royal Society B: Biological Sciences. 2003;270 (1512): 313-321. doi: 10.1098/rspb.2002.2218.
Kress WJ, Erickson DL (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. doi: 10.1371/journal.pone.0000508
Caterina Villa, Joana Costa, Liliana Meira, M. Beatriz P.P. Oliveira, Isabel Mafra, Exploiting DNA mini-barcodes as molecular markers to authenticate saffron (Crocus sativus L.), Food Control, Volume 65, July 2016, Pages 21-31, ISSN 0956-7135, http://dx.doi.org/10.1016/j.foodcont.2016.01.008.http://www.sciencedirect.com/science/article/pii/S0956713516300093)
Hong-liang Ma, Zai-biao Zhu, Xiao-ming Zhang, Yuan-yuan Miao, Qiao- sheng Guo, Species identification of the medicinal plant Tulipa edulis (Liliaceae) by DNA barcode marker, Biochemical Systematics and Ecology, Volume 55, August 2014, Pages 362-368, ISSN 0305-1978, http://dx.doi.org/10.1016/j.bse.2014.03.038.http://www.sciencedirect.com/science/article/pii/S0305197814001240)
Runglawan Sudmoon, Arunrat Chaveerach, Arisa Sanubol, Pansa Monkheang, Nantiya Kwanda, Sarocha Aungkapattamagul, TawatchaiTanee, KowitNoikotr, Chattong Chuachan and Napaporn Kaewdoungdee [e], Identifying Efficiency in Herbal medicine Cinnamomum Species (Lauraceae) Using Banding Patterns and Sequence Alignments of rpoB, rbcL and matK Regions, Chiang Mai J. Sci. 2014; 41 (5.1): 1094-1108 http://epg.science.cmu.ac.th/ejournal/Contributed Paper
Mohamed Enan1, 2, Nael Fawzi1, 3, Mohammad Al-Deeb1, Khaled Amiri1, DNA Barcoding of Ricinuscommunis from Different Geographical Origin by Using Chloroplast matK and Internal Transcribed Spacers,American Journal of Plant Sciences, 2012, 3, 1304-1310 http://dx.doi.org/10.4236/ajps.2012.39157 Published Online September 2012 (http://www.SciRP.org/journal/ajps)
COBOL Plant Working Group (2009) A DNA barcode for land plants. Proc Nat AcadSci 106: 12794–12797.
Edgar, Robert C. (2004), MUSCLE: multiple sequence alignment with high accuracy and high throughput, Nucleic Acids Research 32(5), 1792-97.
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance,and maximum parsimony methods. MolBiolEvol. 2011 Oct; 28(10): 2731-9. doi: 10.1093/molbev/msr121. Epub 2011 May 4. PubMed PMID: 21546353; PubMed Central PMCID: PMC3203626.
Kimura M. (1980). A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16.
Sneath P. H. A. and Sokal R.R. (1973).Numerical Taxonomy. Freeman, San Francisco.
Kumar S., Stecher G., and Tamura K. (2016). MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets.Molecular Biology and Evolution 33: 1870-1874.
Felsenstein J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: 783-791.
Kimura M. (1980). A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16: 111-120.
Hou DY, Wang GP, Zhi LH, Xu 1 HW, Liang 1 HL, Yang 1 MM, Shi 1 GA. Molecular.
Vassou SL, Nithaniyal S, Raju B, Parani M. Creation of reference DNA barcode library and authentication of medicinal plant raw drugs used in Ayurvedic medicine. BMC Complement Altern Med. 2016 Jul 18;16Suppl 1: 186. doi: 10.1186/s12906-016-1086-0. PubMed PMID: 27454470; PubMed Central PMCID: PMC4959393.
Feng S, Jiang M, Shi Y, Jiao K, Shen C, Lu J, Ying Q, Wang H. Application of the Ribosomal DNA ITS2 Region of Physalis (Solanaceae): DNA Barcoding and Phylogenetic Study. Front Plant Sci. 2016 Jul 19;7: 1047. doi: 10.3389/fpls.2016.01047. eCollection 2016. PubMed PMID: 27486467; PubMed Central PMCID: PMC4949264.
Parveen I, Gafner S, Techen N, Murch SJ, Khan IA. DNA Barcoding for the Identification of Botanicals in Herbal Medicine and Dietary Supplements: Strengths and Limitations. Planta Med. 2016 Jul 8. [Epub ahead of print] PubMed PMID: 27392246.
U Xiong B, Zhao ZL, Ni LH, Gaawe D, Mi M. [DNA-based identification of Gentianarobusta and related species]. ZhongguoZhong Yao ZaZhi. 2015 Dec;40 (23): 4680-5.Chinese. PubMed PMID: 27141683.
Yancy, H. F., Zemlak, T. S., Mason, J. A., Washington, J. D., Tenge, B. J. and Nguen, N. T. (2008) Potential Use of DNA Barcodes in Regulatory Science: Applications of the Regulatory Fish Encyclopedia. Journal of Food Protection, 71, 210-217.
Fazekas AJ, et al. (2008) Multiple multilocus DNA barcodes from the plastid genome discriminate plant species equally well. PLoS ONE 3: e2802.
Kress WJ, Erickson DL (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: e508.
Renaud Lahaye, Michelle van der Bank, Diego Bogarin, Jorge Warner, Franco Pupulin, Guillaume Gigot, Olivier Maurin, Sylvie Duthoit, Timothy G. Barraclough, Vincent Savolainen (2008) DNA barcoding the floras of biodiversity hotspots. Proc Natl AcadSci USA 105: 2923–2928.