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
Highlights
IntroductionThe plant is cultivated all over the subtropical tracts of India and is concentrated in the semi-arid parts of Tamil Nadu, Gujarat, and Rajasthan and exported under the brand name “Tirunelveli Senna” (Rama Reddy et al, 2015)
Seven potential species of the genus Senna representing a total of 21 individuals were successfully amplified and sequenced using five DNA barcodes, viz. rbcL, matK, psbA-trnH, ITS1, and ITS2 with 100% PCR and sequencing success rate (Table 1)
This study is the first attempt to derive high-resolution melting (HRM) assays based on ITS1 barcodes toward detection of species composition of S. alexandrina raw drug samples currently in the market
Summary
The plant is cultivated all over the subtropical tracts of India and is concentrated in the semi-arid parts of Tamil Nadu, Gujarat, and Rajasthan and exported under the brand name “Tirunelveli Senna” (Rama Reddy et al, 2015). The dried leaves and pods are the potent drug parts and contain anthraquinone glycosides known as Senna glycosides or sennosides (four types: A, B, C, and D). The leaves and pods of the plant have been globally investigated for various therapeutic effects such as antimutagenic, anti-genotoxic, and anti-fungal properties (Lewis et al, 2005; Sultana et al, 2012; Cirillo and Capasso, 2015). India is presently the main source of cultivated Senna (recorded in over 10,000 ha) directed to the world market (Balasankar et al, 2013). An export volume of 15,975 metric tons, valued at USD 10 million, was achieved in 2012–2013, which has been growing steadily since (The Hindu, Tuticorin Edn. dated 15.10.13)
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