Candidate DNA Barcode Tags Combined With High Resolution Melting (Bar-HRM) Curve Analysis for Authentication of Senna alexandrina Mill. With Validation in Crude Drugs

Abstract

Senna alexandrina (Fabaceae) is a globally recognized medicinal plant for its laxative properties as well as the only source of sennosides, and is highly exported bulk herb from India. Its major procurement is exclusively from limited cultivation, which leads to risks of deliberate or unintended adulteration. The market raw materials are in powdered or finished product form, which lead to difficulties in authentication. Here, DNA barcode tags based on chloroplast genes (rbcL and matK) and intergenic spacers (psbA-trnH and ITS) were developed for S. alexandrina along with the allied species. The ability and performance of the ITS1 region to discriminate among the Senna species resulted in the present proposal of the ITS1 tags as successful barcode. Further, these tags were coupled with high-resolution melting (HRM) curve analysis in a real-time PCR genotyping method to derive Bar-HRM (Barcoding-HRM) assays. Suitable HRM primer sets were designed through SNP detection and mutation scanning in genomic signatures of Senna species. The melting profiles of S. alexandrina and S. italica subsp. micrantha were almost identical and the remaining five species were clearly separated so that they can be differentiated by HRM method. The sensitivity of the method was utilized to authenticate market samples [Herbal Sample Assays (HSAs)]. HSA01 (S. alexandrina crude drug sample from Bangalore) and HSA06 (S. alexandrina crude drug sample from Tuticorin, Tamil Nadu, India) were found to be highly contaminated with S. italica subsp. micrantha. Species admixture samples mixed in varying percentage was identified sensitively with detection of contamination as low as 1%. The melting profiles of PCR amplicons are clearly distinct, which enables the authentic differentiation of species by the HRM method. This study reveals that DNA barcoding coupled with HRM is an efficient molecular tool to authenticate Senna herbal products in the market for quality control in the drug supply chain.CIMAP Communication Number: CIMAP/PUB/2017/31

In vitro chondroprotective potential of Senna alata and Senna tora in porcine cartilage explants and their species differentiation by DNA barcoding-high resolution melting (Bar-HRM) analysis

“…In order to prevent such scenarios, taxonomic evaluation must be performed based on plant morphology for species identification. In fact, in the case of dried and ground raw materials of medical plants, morphological observations for species identification has proven difficult or even impossible [25–28]. Bar-HRM can be used as an alternative aid for taxonomic evaluation.…”

“…However, their anthraquinones are significantly different in several Senna species [17], which are likely to influence their biological activities. In addition, the raw materials produced by these plants are usually processed and/or modified into various products, such as powder capsules and herbal infusions, which can further contribute to difficulty in the species differentiation [25–28]. Accurate species identification during the manufacturing process and data generation to support the use of herbal plants as complementary and alternative medicine (CAM) options is necessary for the safety, quality, and efficacy of medicinal plant ingredients [25–28].…”

“…alata and S . tora given that we expect the sequence of internal transcribed sequence 2 (ITS2) of each species to be different [28, 29]. Thereby, when disassociated from double strand to single strand, DNA from the different species will give a different melting temperature that is a unique characteristic to that organism dependent on base sequence, length, and neighboring DNA [30, 31].…”

Sequencing and Analysis of Chrysanthemum carinatum Schousb and Kalimeris indica. The Complete Chloroplast Genomes Reveal Two Inversions and rbcL as Barcoding of the Vegetable

“…More importantly, it also could differentiate different family species, which illustrate that the rbcL gene is not only highly distinguished within the Asteraceae family, but also highly distinguished within most of vegetables. So, rbcL would be one of the best choices for DNA barcoding to distinguish between vegetables [ 71 , 72 , 73 ].…”

Botanical origin authentication of dietary supplements by DNA‐based approaches

“…Mishra, Shukla, and Sundaresan (2018) evaluated several barcode regions, namely rbcL , matK , psbA‐trnH , ITS, ITS1, and ITS2, aiming at developing an HRM approach for Senna authentication. The authors concluded that ITS1 region gave the best results and proposed its use as a biomarker to discriminate six tested Senna species by HRM.…”