Volume 2. Issue 1. ISSN 1555-9475 (online) www ... - CIMAP Staff

More documents

Recommendations

Info

96 Natural Product Communications Vol. 2 (1) 2007 Misra et al.Table 1 (Continued)CTG/ACA 157,158,325 108,187,353 104,111,118,129,150,153,163,356, 189372,392CTG/AGG 324,325 - 114,134,152,190,211,245,267,361 268CTG/ACG 106,158,268,302,337 398 287 321,393CTT/ACA 130,189,388 119 235,263,341 223,224,330,331CTT/ACC - 328 299,303 335CTT/AGG 120, 175,347,385 266,317 146,330,331CTT/ACG 189 367 147,148,149,150 329,333,335drug supposedly consisting of a particular population.The frequency of the occurrence of these uniquebands in the analysis of the DNA isolated from thecrude drug preparation could be used as an assay forthe presence of a specific population in it. This couldhelp in quantification of the adulteration of the crudedrug of different species. This will go a long way inestablishing the authenticity and credibility of theayurvedic drug ‘Safed Musli’, which presentlysuffers from the problem of adulteration. Theprincipal component of this drug, A. adscendens, ismainly responsible for the aforementionedtherapeutic properties. However, the presence ofother Chlorophytum species in the crude drug mayalter its efficacy and therapeutic value. Althoughthere has been no study on the deleterious effects ofthe adulterants on human beings, there is a distinctpossibility that they may be harmful for humanusage. The importance of the present study stemsfrom the fact that it provides an authentic tool todetect adulterants in the crude drug ‘Safed Musli’ andvalidates the scientific basis of this drug in Ayurveda.ExperimentalPlant material: The plant material used in this studyconsisted of diverse collections of Asparagusadscendens, Chlorophytum borivilianum, C.arundinaceum and C. tuberosum taken from theCIMAP gene bank. For each of the four samples, leafmaterial pooled from the respective populations wasused for DNA isolation (20 plants in each species).DNA isolation: DNA was isolated using the protocoldescribed by Khanuja et al. [4] and quantified byfluorimetry using a DyNa Quant 200 fluorimeter.AFLP analyses: For AFLP analysis, DNA wasrestricted using two restriction endonucleases, EcoRIand Tru9I (an isoschizomer of MseI), and doublestrandedadapters were ligated to the ends of DNAfragments, generating templates for subsequent PCRamplifications (preselective followed by selective).Restriction and ligation reactions were carried outsimultaneously in a single reaction [5]. To carry outthe reaction, an enzyme master mix for 10 reactionswas prepared containing 1μL 10X T4 DNA ligasebuffer, 1μL 0.5 M NaCl, 0.5μL 1 mg/mL BSA, 1μLTru9I (10 U/μL), 4.25μL EcoRI (12 U/μL), 0.5 μLT4 DNA ligase (20 U/μL, high concentration) and1.75 μL water. The restriction-ligation reactionconsisted of 300 ng of DNA (5.5 μL), 1 μL 10X T4DNA ligase buffer, 1 μL 0.5 M NaCl, 0.5 μL 1mg/mL BSA, 1 μL MseI adaptors (AppliedBiosystems), 1 μL EcoRI adaptors (AppliedBiosystems) and 1 μL enzyme master, as describedabove. The reaction mix was incubated overnight atroom temperature and subsequently diluted 20-foldwith T 10 E 0.1 (10 mM Tris and 0.1 mM EDTA) buffer.The ligated adaptors served as primer binding sitesfor a low-level selection in the preselectiveamplification of restriction fragments. The MseIcomplementary primer had a 3’-C and the EcoRIcomplementary primer a 3’-A. Only the genomicfragments having an adaptor on each end amplifiedexponentially during the PCR. The preselectiveamplification mix was prepared by adding 4 μL of20-fold diluted DNA from the restriction-ligationreaction, 0.5 μL AFLP preselective primer (EcoRI,Applied Biosystems), 0.5 μL AFLP preselectiveprimer (MseI, Applied Biosystems), and 15 μL AFLPcore mix. The preselective amplification was carriedout in a thermal cycler programmed as: 72°C for2 min; 20 cycles of 94°C for 20 sec, 56°C for 30 secand 72°C for 2 min; 60°C for 30 min; and 4°C forinfinity.The preamplified DNA was diluted 20-fold withT 10 E 0.1 buffer and selective amplifications werecarried out using different MseI and EcoRI primercombinations (Applied Biosystems). Primers chosenfor the amplification were from 16 available AFLPselective primers (8 fluorescently tagged EcoRI and8 untagged MseI primers). The EcoRI primerscontain 3 selective nucleotides with the sequence 5’[Dye-Primer-Axx]-3’, while the MseI primers had the3 selective nucleotides starting with C with thesequence 5’ [Primer-Cxx]-3’. Selective amplificationof each sample was done with all the 64 (8x8)-primercombinations (MseI/EcoRI) using multiplex-PCR
AFLP based detection of adulterants in Safed Musli complex Natural Product Communications Vol. 2 (1) 2007 97reactions. For selective amplification the reactionswere set up as follows: 3 μL of 20-fold dilutedpreselective amplification product, 15 μL AFLP coremix, 1 μL MseI primer 5’-[Primer-Cxx]-3’, 1.5 μLEcoRI primers 5’-[Dye-Primer-Axx]-3’{0.5 μL of 3EcoRI primers each were pooled here}. Selectiveamplification was carried out in a thermal cyclerprogrammed as: 94°C for 2 min; 10 cycles of 94°Cfor 20 sec, 66°C (-1°C/cycle) for 30 sec, 72°C for 2min; 20 cycles of 94°C for 20 sec, 56°C for 30 sec,72°C for 2 min; 60°C for 30 min; and 4°C forinfinity. The samples were loaded onto a 5%polyacrylamide gel on an ABI Prism 377 DNASequencer. For gel electrophoresis, 3 μL of theselective amplification reaction product was mixedwith 4 μL of loading buffer {ROX500 size standard(10%), blue dextran (10%), deionised formamide(80%)}, and 1.5 μL of this mix was finally loaded onthe gel. For AFLP reactions the AFLP amplificationmodules and the guidelines supplied by AppliedBiosystems, USA were used.Data analysis: For diversity analysis bands werescored as present (1) or absent (0) to form a raw datamatrix. A square symmetric matrix of similarity wasthen obtained using Jaccard similarity coefficient [6]by SPSS v7.5 software .The average similarity matrixwas used to generate a tree for cluster analyses byUPGMA (Unweighted Pair Group Method withArithmetic average) method using NTSys v2.1.Acknowledgments - The authors gratefullyacknowledge the financial help provided by ICMRand CSIR, India.References[1] Oudhia P. (2000) Problems perceived by safed musli (Chlorophytum borivilianum) growers of Chhattisgarh (India) region: Astudy. Journal of Medicinal and Aromatic Plant Sciences, 23, 396-399.[2] Shariff A, Chennaveeraiah MS. (1972) Karyomorphology of four diploid species of Chlorophytum. Nucleus, 15, 39-45.[3] Bordia PC, Joshi A, Simlot MM. (1995) Safed moosli. In KL Chadha and Rajendra Gupta (eds.), Advances in Horticulture, Vol. II.Medicinal and aromatic plants. Malhotra Publ. House, New Delhi, India, 429-451.[4] Khanuja SPS, Shasany AK, Darokar MP, Kumar S. (1999) Rapid isolation of PCR amplifiable DNA from the dry and fresh sampleof plants producing large amounts of secondary metabolites and essential oils by modified CTAB procedure. Plant MolecularBiology Reporter, 17, 74.[5] Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M, Frijters A, Pot J, Paleman J, Kuiper M, Zabeau M. (1995) AFLPa new technique for DNA fingerprinting. Nucleic Acids Research, 23, 4407-4414[6] Jaccard P. (1908) Nouvelles recherches sur la distribution florale. Bulletin de la Societe Vaudoise des Sciences Naturelles, 44,223-270.[7] Virk PS, Ford-Lloyed BV, Jackson MT, Newburry HJ. (1995) Use of RAPD for the study of diversity within plant germplasmcollections. Heredity, 74, 174-179.[8] Jain N, Shasany AK, Sundaresan V, Rajkumar S, Darokar MP, Bagchi GD, Gupta AK, Kumar S, Khanuja SPS. (2003) Moleculardiversity in Phyllanthus amarus assessed through RAPD analysis. Current Science, 85, 1454-1458.[9] Hansen M, Kraft T, Christiansson M, Nilsson NO. (1999) Evaluation of AFLP in Beta. Theoretical and Applied Genetics, 98,845–852.[10] Jones CJ, Edwards KJ, Castaglione S, Winfield MO, Sala F. (1997) Reproducibility testing of RAPD, AFLP and SSR markers inplants by a network of European laboratories. Molecular Breeding, 3, 381–390.[11] Vuylsteke M, Mank R, Brugmans B, Stam P, Kuiper M. (2000) Further characterization of AFLP data as a tool in genetic diversityassessments among maize (Zea mays L.) inbred lines. Molecular Breeding, 6, 265–276.[12] Hill M, Witsenboer H, Zabeau M, Vos P, Kesseli R, Michelmore RW. (1996) AFLP fingerprinting as a tool for studying geneticrelationships in Lactuca spp. Theoretical and Applied Genetics, 93, 1202–1210.[13] Koopman WJM, Zevenbergen MJ, Van den Berg RG. (2001) Species relationships in Lactuca S.L. (Lactuceae, Asteraceae)inferred from AFLP fingerprints. American Journal of Botany, 88, 1881-1887.[14] Alonso-Blanco C, Peeters AJM, Koornneef M, Lister C, Dean C, van den Bosch N, Pot J, Kuiper MTR. (1998) Development of anAFLP based linkage map of Ler, Col and Cvi Arabidopsis thaliana ecotypes and construction of a Ler/Cvi recombinant inbred linepopulation. Plant Journal, 14, 259–271.[15] Polanco C, Ruiz ML. (2002) AFLP analysis of somaclonal variation in Arabidopsis thaliana regenerated plants. Plant Science, 162,817–824.
Page 1 and 2: Volume 2. Issue 1. Pages 1-116. 200
Page 3 and 4: NPCNatural Product CommunicationsAF
Page 5: AFLP based detection of adulterants
Page 9: Natural Product Communications2007V

Volume 2. Issue 1. ISSN 1555-9475 (online) www ... - CIMAP Staff

Create successful ePaper yourself

Delete template?

Save as template?