Priyanka Mishra

DNA barcoding: an efficient tool to overcome authentication challenges in the herbal market

The past couple of decades have witnessed global resurgence of herbal-based health care. As a result, the trade of raw drugs has surged globally. Accurate and fast scientific identification of the plant(s) is the key to success for the herbal drug industry. The conventional approach is to engage an expert taxonomist, who uses a mix of traditional and modern techniques for precise plant identification. However, for bulk identification at industrial scale, the process is protracted and time-consuming. DNA barcoding, on the other hand, offers an alternative and feasible taxonomic tool box for rapid and robust species identification. For the success of DNA barcode, the barcode loci must have sufficient information to differentiate unambiguously between closely related plant species and discover new cryptic species. For herbal plant identification, matK, rbcL, trnH-psbA, ITS, trnL-F, 5S-rRNA and 18S-rRNA have been used as successful DNA barcodes. Emerging advances in DNA barcoding coupled with next-generation sequencing and high-resolution melting curve analysis have paved the way for successful species-level resolution recovered from finished herbal products. Further, development of multilocus strategy and its application has provided new vistas to the DNA barcode-based plant identification for herbal drug industry. For successful and acceptable identification of herbal ingredients and a holistic quality control of the drug, DNA barcoding needs to work harmoniously with other components of the systems biology approach. We suggest that for effectively resolving authentication challenges associated with the herbal market, DNA barcoding must be used in conjunction with metabolomics along with need-based transcriptomics and proteomics.

1,4-Dioxane and ergosterol derivatives from Withania somnifera roots

The chemical investigation on the n-hexane extract of Withania somnifera roots has yielded octacosane, oleic and stearic fatty acids, stigmasterone, stigmasterol, sitostanone, oleanolic acid along with the ergosterol and 1,4-dioxane derivatives as new compounds. The isolation of alkenyl-1,4-dioxane compound is rare, whereas the ergosterol derivative may have biogenetic significance in the lactone formation in the E ring of withanolides. The presence of a 1,4-dioxane derivative in the nonpolar extract of roots assumes importance as this type of compound has not been reported earlier from W. somnifera. The structures of new compounds were elucidated by spectroscopic methods and chemical transformations.

Higher efficiency of ISSR markers over plastid psbA‐trnH region in resolving taxonomical status of genus Ocimum L.

Abstract High level of morphological as well as chemical variability exists within the genus Ocimum, and its taxonomy and phylogenetic relationships are still doubtful. For evaluating interspecific genetic relationships among the Ocimum species, genotyping with intersimple sequence repeat (ISSR) markers and sequence analyses of noncoding psbA‐trnH intergenic region belonging to chloroplast DNA were carried out. Although ISSR markers are highly efficient and reproducible, they have not been used extensively in phylogenetic studies. The use of the plastidial barcode candidate was expected to provide more variable and informative insight into evolutionary rates, and was thus employed as a phylogenetic marker to assess interspecific relationships. This study revealed that the ISSR markers were more efficient than psbA‐trnH sequences in resolving the current status of Ocimum L. genus. Distance‐ and character‐based methodological approaches applied on the molecular data with biparental and maternal inheritance were used for deducing the phylogenetic relationships among Ocimum species. Average polymorphic information content (0.344) and resolving power (6.285) depicted through ISSR markers proved to be efficient in discriminating the studied species of Ocimum. The primers used in this study revealed 99.585% polymorphism across the species demonstrating the polymorphic nature of ISSR markers.

Chemical composition of root aroma of Decalepisarayalpathra (J. Joseph and V. Chandras.) Venter, an endemic and endangered ethnomedicinal plant from Western Ghats, India

Decalepisarayalpathra (J. Joseph and V. Chandras.) Venter, which belongs to the family Apocynaceae, is a perennial under shrub, endemic to southern Western Ghats, India. The highly aromatic tuberous roots of the D. arayalpathra are used as an effective remedy for peptic ulcer, cancer-like afflictions and as rejuvenating tonic by native tribes. The objective of this study was to characterise the root aroma of D. arayalpathra for possible industrial applications. Hydrodistilled volatile oil of the roots was analysed by gas chromatography-flame ionisation detector and gas chromatography–mass spectrometry. The volatile oil was characterised by the presence of higher amount of an industrially important flavour molecule, 2-hydroxy-4-methoxybenzaldehyde (96.8%) along with some other minor or trace constituents. Owing to characteristic vanillin-like flavour, the root oil of the D. arayalpathra can be explored as a potential substitute of vanillin-aroma in the flavour industry.

Essential oil composition of four Ocimum spp. from the Peninsular India

Abstract Compositional characteristics of the essential oils of four Ocimum spp., namely Ocimum adscendens Willd., Ocimum gratissimum L., Ocimum tenuiflorum L. and Ocimum americanum L. were examined using GC-FID, GC-MS, and hierarchical cluster analyses from peninsular India. The essential oil content varied from 1.0% to 2.0% in different Ocimum spp. Altogether eighty-four constituents, corresponding to 89.9−96.5% of the total oil compositions were identified. Major constituents of O. adscendens oil were eugenol (47.6%), (E)-caryophyllene (15.7%) and β-elemene (11.3%). O. gratissimum oil was characterized by the presence of higher amounts of eugenol (52.9%), caryophyllene oxide (7.2%) and (Z)-β-ocimene (3.5%). Major constituents of O. tenuiflorum oil were methyl eugenol (50.9%), caryophyllene oxide (7.5%) and (E)-caryophyllene (5.5%). Moreover, the oil of O. americanum was dominated by camphor (41.8%), limonene (7.1%), α-pinene (6.2%), β-selinene (5.6%) and camphene (5.0%). To the best of our knowledge, a detailed essential oil profile of O. adscendens is being reported for the first time.

Evaluation of single and multilocus DNA barcodes towards species delineation in complex tree genus Terminalia

DNA barcoding is used as a universal tool for delimiting species boundaries in taxonomically challenging groups, with different plastid and nuclear regions (rbcL, matK, ITS and psbA-trnH) being recommended as primary DNA barcodes for plants. We evaluated the feasibility of using these regions in the species-rich genus Terminalia, which exhibits various overlapping morphotypes with pantropical distribution, owing to its complex taxonomy. Terminalia bellerica and T. chebula are ingredients of the famous Ayurvedic Rasayana formulation Triphala, used for detoxification and rejuvenation. High demand for extracted phytochemicals as well as the high trade value of several species renders mandatory the need for the correct identification of traded plant material. Three different analytical methods with single and multilocus barcoding regions were tested to develop a DNA barcode reference library from 222 individuals representing 41 Terminalia species. All the single barcodes tested had a lower discriminatory power than the multilocus regions, and the combination of matK+ITS had the highest resolution rate (94.44%). The average intra-specific variations (0.0188±0.0019) were less than the distance to the nearest neighbour (0.106±0.009) with matK and ITS. Distance-based Neighbour Joining analysis outperformed the character-based Maximum Parsimony method in the identification of traded species such as T. arjuna, T. chebula and T. tomentosa, which are prone to adulteration. rbcL was shown to be a highly conservative region with only 3.45% variability between all of the sequences. The recommended barcode combination, rbcL+matK, failed to perform in the genus Terminalia. Considering the complexity of resolution observed with single regions, the present study proposes the combination of matK+ITS as the most successful barcode in Terminalia.

Feasibility of nuclear ribosomal region ITS1 over ITS2 in barcoding taxonomically challenging genera of subtribe Cassiinae (Fabaceae)

Premise of the Study The internal transcribed spacer (ITS) region is situated between 18S and 26S in a polycistronic rRNA precursor transcript. It had been proved to be the most commonly sequenced region across plant species to resolve phylogenetic relationships ranging from shallow to deep taxonomic levels. Despite several taxonomical revisions in Cassiinae, a stable phylogeny remains elusive at the molecular level, particularly concerning the delineation of species in the genera Cassia, Senna and Chamaecrista. This study addresses the comparative potential of ITS datasets (ITS1, ITS2 and concatenated) in resolving the underlying morphological disparity in the highly complex genera, to assess their discriminatory power as potential barcode candidates in Cassiinae. Methodology A combination of experimental data and an in-silico approach based on threshold genetic distances, sequence similarity based and hierarchical tree-based methods was performed to decipher the discriminating power of ITS datasets on 18 different species of Cassiinae complex. Lab-generated sequences were compared against those available in the GenBank using BLAST and were aligned through MUSCLE 3.8.31 and analysed in PAUP 4.0 and BEAST1.8 using parsimony ratchet, maximum likelihood and Bayesian inference (BI) methods of gene and species tree reconciliation with bootstrapping. DNA barcoding gap was realized based on the Kimura two-parameter distance model (K2P) in TaxonDNA and MEGA. Principal Findings Based on the K2P distance, significant divergences between the inter- and intra-specific genetic distances were observed, while the presence of a DNA barcoding gap was obvious. The ITS1 region efficiently identified 81.63% and 90% of species using TaxonDNA and BI methods, respectively. The PWG-distance method based on simple pairwise matching indicated the significance of ITS1 whereby highest number of variable (210) and informative sites (206) were obtained. The BI tree-based methods outperformed the similarity-based methods producing well-resolved phylogenetic trees with many nodes well supported by bootstrap analyses. Conclusion The reticulated phylogenetic hypothesis using the ITS1 region mainly supported the relationship between the species of Cassiinae established by traditional morphological methods. The ITS1 region showed a higher discrimination power and desirable characteristics as compared to ITS2 and ITS1 + 2, thereby concluding to be the locus of choice. Considering the complexity of the group and the underlying biological ambiguities, the results presented here are encouraging for developing DNA barcoding as a useful tool for resolving taxonomical challenges in corroboration with morphological framework.

Character-based DNA barcoding for authentication and conservation of IUCN Red listed threatened species of genus Decalepis (Apocynaceae)

The steno-endemic species of genus Decalepis are highly threatened by destructive wild harvesting. The medicinally important fleshy tuberous roots of Decalepis hamiltonii are traded as substitute, to meet the international market demand of Hemidesmus indicus. In addition, the tuberous roots of all three species of Decalepis possess similar exudates and texture, which challenges the ability of conventional techniques alone to perform accurate species authentication. This study was undertaken to generate DNA barcodes that could be utilized in monitoring and curtailing the illegal trade of these endangered species. The DNA barcode reference library was developed in BOLD database platform for candidate barcodes rbcL, matK, psbA-trnH, ITS and ITS2. The average intra-specific variations (0–0.27%) were less than the distance to nearest neighbour (0.4–11.67%) with matK and ITS. Anchoring the coding region rbcL in multigene tiered approach, the combination rbcL + matK + ITS yielded 100% species resolution, using the least number of loci combinations either with PAUP or BLOG methods to support a character-based approach. Species-specific SNP position (230 bp) in the matK region that is characteristic of D. hamiltonii could be used to design specific assays, enhancing its applicability for direct use in CITES enforcement for distinguishing it from H. indicus.

Molecular and chemotypic variability of forskolin in Coleus forskohlii Briq., a high value industrial crop collected from Western Himalayas (India)

C. forskohlii (willd.) Briq. is an industrially viable medicinal crop and is widely exploited for the therapeutic potential of its bioactive metabolite, forskolin. The present investigation aimed to explore the chemotypic variability of forskolin content and existing molecular diversity in the wild population of C. forskohlii from the Western Himalayan region of India. Twelve germplasm(s) from different populations were assessed for molecular fingerprinting (ISSR marker) and densitometeric quantification of forskolin. Two elite germplasms viz. NBC-24 (0.728%) and NBC-16 (0.641%) were obtained as the highest accumulator of forskolin with high genetic variability (92%). The UPGMA hierarchical clustering patterns revealed strong genetic grouping between the individuals corresponding to their geographical ranges. Mantel tests showed positive correlation (r = 0.354, p = 0.003) between molecular and chemical fingerprints that reflects the feasibility of the ISSR markers in analyzing genome information related to forskolin biosynthesis from varied phytogeography. Pearson correlation coefficient (0.102) between forskolin content with altitude gradient also denoted a positive correlation. However, the association of both genetic and chemical fingerprinting data with the geographic distance matrix was apparently negative (r = −0.234, p = 0.054; r = −0.067, p = 0.584) which meant that distance might be a predictor of population differentiation. Our study signifies the utility of metabolic and molecular fingerprints for identification of elite accessions and provides a lead to industry for commercial exploitability of Coleus species including its location specific commercial cultivation.

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

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