Identification of a Herbal Powder by Deoxyribonucleic Acid Barcoding and Structural Analyses.

BACKGROUND: Authentic identification of plants is essential for exploiting their medicinal properties as well as to stop the adulteration and malpractices with the trade of the same.
OBJECTIVE: To identify a herbal powder obtained from a herbalist in the local vicinity of Rajkot, Gujarat, using deoxyribonucleic acid (DNA) barcoding and molecular tools.
MATERIALS AND METHODS: The DNA was extracted from a herbal powder and selected Cassia species, followed by the polymerase chain reaction (PCR) and sequencing of the rbcL barcode locus. Thereafter the sequences were subjected to National Center for Biotechnology Information (NCBI) basic local alignment search tool (BLAST) analysis, followed by the protein three-dimension structure determination of the rbcL protein from the herbal powder and Cassia species namely Cassia fistula, Cassia tora and Cassia javanica (sequences obtained in the present study), Cassia Roxburghii, and Cassia abbreviata (sequences retrieved from Genbank). Further, the multiple and pairwise structural alignment were carried out in order to identify the herbal powder.
RESULTS: The nucleotide sequences obtained from the selected species of Cassia were submitted to Genbank (Accession No. JX141397, JX141405, JX141420). The NCBI BLAST analysis of the rbcL protein from the herbal powder showed an equal sequence similarity (with reference to different parameters like E value, maximum identity, total score, query coverage) to C. javanica and C. roxburghii. In order to solve the ambiguities of the BLAST result, a protein structural approach was implemented. The protein homology models obtained in the present study were submitted to the protein model database (PM0079748-PM0079753). The pairwise structural alignment of the herbal powder (as template) and C. javanica and C. roxburghii (as targets individually) revealed a close similarity of the herbal powder with C. javanica.
CONCLUSION: A strategy as used here, incorporating the integrated use of DNA barcoding and protein structural analyses could be adopted, as a novel rapid and economic procedure, especially in cases when protein coding loci are considered.
SUMMARY: Authentic identification of plants is essential for exploiting their medicinal properties as well as to stop the adulteration and malpractices with the trade of the same. A herbal powder was obtained from a herbalist in the local vicinity of Rajkot, Gujarat. An integrated approach using DNA barcoding and structural analyses was carried out to identify the herbal powder. The herbal powder was identified as Cassia javanica L.

INTRODUCTION

Medicinal plants have been used by mankind since ages to cure common as well as serious health ailments. Indian flora comprises of an affluent florisitic diversity of medicinal plants. Medicinal values of these plants are exploited in major modes of therapy such as homeopathy, aroma therapy, traditional ayurvedic medicine, Unani, herbal therapy, etc. The process of herbal drug development often experiences (a) deliberate or indeliberate adulteration, and (b) unintentional substitution due to lack of authentic information.[1] Hence, the identification of the plant products are of prime importance in preventing the adulteration and malpractices with the trade of the same.[2] Unambiguous identification and authentication of the plants used for the production is therefore an elementary and critical step at the beginning of an extensive quality assurance process. Many different methods have been used since long for the identification of medicinal plants such as traditional pharmacognostic analysis,[34] chromatographic and spectroscopic,[567] physical techniques,[89] and molecular markers.[10] The latter are more suitable for identification as deoxyribonucleic acid (DNA), in contrast to ribonucleic acid, is a stable macromolecule that is found in all tissues, including the dead ones.

Among these, molecular marker techniques provide valuable data on diversity through their ability to detect variation at the DNA level, and have been routinely employed as they are easy, rapid and reliable to perform. Many DNA based methods have been used previously for detecting and differentiating plant materials from their contaminants viz. Random Amplified Polymorphic DNA,[1112] polymerase chain reaction (PCR)-Restriction Fragment Length Polymorphism,[1314] high-resolution melting analysis,[1516] DNA barcoding,[1718192021] etc. Among these, DNA barcoding is a comparatively recent technique, which uses DNA sequences from a small fragment of the genome to identify organisms.[22] Furthermore, bioinformatics approaches have been of immense help for the in-depth understanding of the plant systems in various ways.[2324]

The rbcL gene codes for the large subunit of Rubisco enzyme in plants, is exclusively chloroplastic in origin and is a very useful molecule in phylogenetic analysis[25] as well as designated as a core barcode in plants.[26] It has been used in identification of medicinal materials earlier.[182728] In this study, a herbal powder was obtained from a herbalist in the local vicinity of Rajkot, Gujarat. Thereafter, the DNA was extracted, followed by the PCR and sequencing of the rbcL barcode. The basic local alignment search tool (BLAST) results, showed an equal sequence similarity to Cassia roxburghii and Cassia javanica. Hence, the comparative protein structural analysis was carried out to solve the ambiguity observed in the BLAST results.

MATERIALS AND METHODS

In this study, an easy, inexpensive, and rapid approach was used for the identification of a herbal powder, using the molecular and bioinformatics tools.

Deoxyribonucleic acid isolation from the medicinal extract and Cassia species

The DNA was isolated from the unknown medicinal extract using the cetyltrimethylammonium bromide method.[29] The same was done for the Cassia sp. available in the local vicinity viz. Cassia tora, Cassia fistula and C. javanica.

Polymerase chain reaction amplification, nucleotide sequencing of the rbcL gene and basic local alignment search tool analysis

The rbcL gene was amplified from the herbal powder as well as the selected species of Cassia (mentioned earlier) with the primer pair: rbcLa_F (5’- ATG TCA CCA CAA ACA GAG ACT AAA GC-3’)[30] and rbcLa_R (5’- GTA AAA TCA AGT CCA CCR CG-3’).[31] The PCR amplification was conducted using 25 μL of reaction mixture containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 25 mM MgCl2, 10 mM of each dNTPs, 10 pmol each of forward and reverse primers, 2 μL of template DNA and 1 units of Taq DNA polymerase (Genei, India) (without proof-reading activity) on a Veriti (96 Well Fast Thermal Cycler), Applied Biosystems, USA. 5 μl of each PCR product was electrophoresed through 2% agarose gel to determine the size of fragment. Positive products were then purified using the Exo-Sap-IT reagent (USB, Cleveland OH, USA) using the manufacturer's recommended protocol (2 μL reagent per 5 μL amplified DNA product). Thereafter, the purified samples were subjected to nucleotide sequencing using Big Dye Terminator V3.1 Cycle Sequencing Kit (Applied Biosystems, NY, USA) as per manufacturer's protocol. After purification, the reaction products were analyzed on an ABI PRISM Genetic Analyzer 3130 (Applied Biosystems, NY, USA). Sequence editing was performed using Bioedit.[32] The nucleotide sequences from the samples of: C. tora, C. fistula, C. javanica were submitted to the National Center for Biotechnology Information (NCBI) Genbank database using the standalone submission tool Sequin. The obtained rbcL sequence of the herbal power was compared with sequences in the NCBI-Genbank using the BLAST. In order to increase the taxonomic coverage for the further bioinformatics’analyses, the rbcL sequences of Cassia abbreviata and C. roxburghii were retrieved from the GenBank and used in addition to those plants for which the wetlab studies were carried out. All the nucleotide sequences were translated computationally using Bioedit[32] in order to obtain the protein sequences, which were further used for the three-dimension (3D) structural analyses.

Homology modeling of the rbcL proteins

The protein 3D structures of rbcL proteins of different Cassia species along with that from the herbal powder were elucidated using the homology modeling approach. The 3D models were checked for their validity using the Ramchandran plot analysis. The models were also stereochemically checked with the help of Verify 3D server.[33] Furthermore, the protein 3D models were submitted to the protein model database (PMDB)[34] with the PMDBIDs: PM0079748-PM0079753 [Table 1].

Table 1

Ramchandran plot analyses of the homology models (using Rampage)

Structural analyses using bioinformatics’ tools

The multiple structural alignment was carried out using PROMALS3D server.[35] Further in order to clear ambiguity of the previously obtained BLAST results, the pair-wise structural alignment analyses was also carried using herbal rbcL protein as template and that of C. javanica and C. roxburghii as targets.

RESULTS AND DISCUSSION

In the present investigation, a herbal powder, procured from a local herbalist, was tested with the plant DNA barcoding approach. rbcL is a core barcode locus[26] and otherwise has been used in the phylogenetic analysis of plants since decades.[363738394041] The DNA was successfully isolated from the herbal powder evidenced in the form of sharp bands on the 1% agarose gel [Figure 1]. The rbcL barcode locus was amplified (~550 bp) [Figure 2] and sequenced on the Applied Biosystems 3130 Genetic Analyzer. The sequences obtained in this study were submitted to NCBI (Accession No. JX141397, JX141405, JX141420). The sequence was then compared with other sequences in the database using the NCBI-BLAST tool [Figure 3]. The query sequence showed an identity of 89% and query coverage of 96% (with the same maximum score, total score and e-value) to two species of the Cassia genus: C. javanica and Cassia roxburghii. In addition, the nucleotide sequences of Cassia roxbughii (with accession no. JQ301861) and C. abbreviata (with accession no. JF265329.1) were retrieved from Genbank and used for the further bioinformatics’ analyses.

Figure 1

Agarose gel electrophoresis of the genomic deoxyribonucleic acid from the herbal powder and other Cassia species (Lane 1: Herbal powder; Lane 2: Cassia fistula; Lane 3: Cassia javanica; Lane 4: Cassia tora)

Figure 2

Agarose gel electrophoresis of the amplified rbcL gene from the selected samples (Lane 1: Herbal powder; Lane 2: Cassia fistula; Lane 3: Cassia javanica; Lane 4: Cassia tora)

Figure 3

Ambiguity observed in the National Center for Biotechnology Information basic local alignment search tool output

The nucleotide sequences were computationally translated to amino acid sequences and the homology modeling of the protein sequences was carried out for the herbal powder as well as the selected Cassia species [Figure 4]. The hypothetical protein 3D structures were validated using bioinformatics tools. To assess stereochemical quality and structural integrity of the model, RAMPAGE[42] and verify 3D[33] servers were used.

Figure 4

Homology modelling of the rbcL protein from selected samples

The homology models obtained so were checked for the stereochemical viability using RAMPAGE server[42] (http://mordred.bioc.cam.ac.uk/rapper/rampage.php) [Table 1]. The Rampage server assesses the accuracy of the generated hypothetical models based on the Ramchandran plot analyses of the same. The Ramachandran diagram plots phi versus psi dihedral angles for each residue in the protein. The predicted models were found to be acceptable since all had more than 90% of the residues in the favored region. Verify 3D analyzes the compatibility of an atomic model (3D) with its own amino acid sequence (1D) and hence tests the accuracy of the model. The models were also checked for the 3D-1D profiles using Verify 3D server[33] [Table 2]. Residues with a score over 0.2 are considered reliable. In the present study, the profiles for all the predicted models were >0.2 indicating the correctness of the predicted models. This indicates the correctness of the models predicted in this study. Hence, the predicted models were used for the further bioinformatics analysis. The multiple structural alignment of the predicted homology models was carried out using PROMALS3D server[35] using the default parameters in order to find the structural distance between the rbcL proteins of the herbal powder with that of the Cassia species. The promals 3D server analyses the sequence-structure relationships in form of multiple structural alignment and generates a phylogenetic tree. In this study, the phylogenetic tree [Figure 5] shows, the structural similarities between the rbcL protein of the medicinal extract with that of C. javanica, C. roxburghii and C. abbreviata. Furthermore, in order to clear ambiguity of the NCBI-BLAST results, the pairwise structural alignment analyses was carried out using the RCSB Protein Databank Protein Comparison Tool.[43] In this study, the medicinal extract rbcL protein was used as a template and the rbcL proteins of C. javanica and C. roxburghii were used as targets. A lower root mean square deviation (RMSD) value was observed in the pairwise rbcL protein structural alignment of the herbal powder with C. javanica, than that with C. roxburghii [Table 3]. A lower RMSD value implies, a closer relationship between the molecules under study. Hence, the herbal powder had a closer relationship with the C. javanica, which is also evidenced in the phylogenetic tree obtained from the multiple structural alignment. Hence, the herbal powder was identified as C. javanica.

Table 2

Three-dimension - one-dimension profile of the homology models (using verify three-dimension)

Figure 5

Phylogenetic tree based on the multiple structural alignment between herbal powder (medicinal extract) and Cassia species

Table 3

Pairwise structural alignment of rbcL protein from the medicinal extract with that of Cassia spp

There has been quite a discussion about the use of the sequence databases and similarity searching in the literature.[44454647] Little and Stevenson[48] found that BLAST can give accurate identification at the genus, but not the species, when DNA barcode sequences are considered. Similarly, there have been identification related problems, in doing similarity searching with BLAST.[49] Hence, a strategy to solve such cases, is imperative, especially when protein coding loci are considered. The sequence similarity results, can be further confirmed by the protein structure data, as in this study; particularly when ambiguous BLAST results are obtained. Thus, the combined use of DNA barcoding and protein 3D structural analyses can help the identification of samples. The present study can therefore be used as an example of this new approach in conjunction with routine approaches of species’ identification.

CONCLUSION

Hence this strategy, incorporating the integrated use of DNA barcoding and protein structural analyses could be adopted, as a novel, rapid and economic procedure for identification of plant species, especially in cases when protein coding loci are considered.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Miss. Bhavisha P. Sheth

ABOUT AUTHORS

Miss. Bhavisha P. Sheth, is currently working as a Ph. D student at the UGC-CAS Department of Biosciences, Saurashtra University, Rajkot. She is a recipient of the Senior Research Fellowship from the University Grants Commission, New Delhi, India. Her research interests include plant biotechnology and systems biology. She has authored nine publications in peer reviewed national and international reputed journals.

Dr. Vrinda S. Thaker

Dr. Vrinda S. Thaker, is a senior Professor at the Department of Biosciences, Saurashtra University, Rajkot. Her research interests include plant physiology and molecular biology. She has authored more than 100 publications in peer reviewed journals of international repute. She has guided 13 Ph. D and 24 M. Phil students till date.