Genome sequencing and characterization of microsatellite markers of Pterocarpus santalinus L.f.: an economically important endangered tree of Eastern Ghats, India.

Pterocarpus santalinus L.f. (red sanders) is an endemic, endangered and economically important tree species distributed in the Eastern Ghats of Andhra Pradesh, India. This tree is well known for its blood-red coloured timber which has a high value in the international market. Due to its high timber demand, illegally logging of red sanders has resulted in fragmentation and depletion of its natural populations. Assessing the genetic diversity is a prerequisite for the identification of distinct populations of red sanders in the natural habitat for prioritizing conservation efforts. The present study has focussed on genome sequencing, identification and validation of microsatellite markers of P. santalinus. A total of 282,918 simple sequence repeat (SSR) loci were identified using whole genome sequence from P. santalinus leaf tissue. A total of 28 SSRs were selected for polymorphism analysis across the 52 individuals belonging to three populations of P. santalinus and identified a sum of 502 alleles with polymorphic information content of 0.83; observed heteozygosity (Ho) 0.42 and expected heterozygosity (He) 0.69. Genetic differentiation coefficient (FST) of 0.19 (FST˂0.25) which is indicating moderate genetic differentiation among the populations. Six SSRs from P. indicus Willd. and P. erinaceus Poir. were successfully amplified in P. santalinus and produced 131 alleles. These newly identified SSRs are useful in detecting genetic diversity and further developing conservation strategies for P. santalinus.

Rohitukine content across the geographical distribution of Dysoxylum binectariferum Hook F. and its natural derivatives as potential sources of CDK inhibitors.

Dysoxylum binectariferum is an important medicinal plant distributed in the Western Ghats of India. The species has gained international importance for its anticancer component, rohitukine, a chromone alkaloid. Flavopiridol, P-276-00 and IIIM-290 are the derivatives of rohitukine in clinical trials against a wide range of cancers. Flavopiridol was recently approved as an orphan drug for chronic lymphocytic leukemia treatment. In this study, we report the isolation and characterization of rohitukine from the bark of D. binectariferum. Further, rohitukine was estimated across the Western-Ghats and the North-East regions of India. Additionally, D. binectariferum is also reported (∼45 compounds) to produce many natural derivatives of rohitukine and terpenoids, which were investigated in-silico to reveal promising CDK inhibitors. The metabolite fingerprinting of tissues of D. binectariferum was studied using HPTLC and FTIR. The distribution of major chromone alkaloid rohitukine was estimated by HPLC. Further, the pharmacological potential of D. binectariferum compounds was evaluated in-silico by discovering the potential protein targets, molecular docking, ADMET analysis and MD simulation. The isolation of rohitukine has yielded 0.6% from the bark of D. binectariferum. A higher percent of rohitukine was found in the Jog populations (0.58% & 1.28%: leaf & bark), whereas least was observed in the Phasighat population (∼0.06%: both leaf & bark). Across the geographic regions, a higher percent of rohitukine was found in the Central-southern Western Ghats, whereas lower in the northern parts of the Western Ghats and Northeast regions. The leaves produce a considerably higher percent of rohitukine and could be used as a sustainable source of rohitukine. The rohitukine analogues, along with other chromone alkaloids of D. binecatariferum were found to be more interactive with the "kinases" family of proteins, majorly "Serine/threonine-protein kinase PFTAIRE-2" (CDK15) with high confidence level (0.94-0.98). The molecular docking of these chromone alkaloids found a strong binding energy with six CDKs (-3.1 to -10.6 kcal/mol) along with a promising ADMET profile. In addition, molecular dynamic simulation found that the rohitukine complexes are virtually constant with CDK-1, 2, 9 and 15, which is substantiated with MM-PBSA free energy calculations. The chromone alkaloids, majorly rohitukine and its analogues were closely clustered with flavopiridol, P-276-00 and IIIM-290 along with other chrotacumines in the chemical phylogeny. In conclusion, D. binectariferum is a rich source of chromone alkaloids, which could lead to the discovery of more potential scaffolding for CDK inhibitors as anticancer drugs.

UPLC and ESI-MS analysis of metabolites of Rauvolfia tetraphylla L. and their spatial localization using desorption electrospray ionization (DESI) mass spectrometric imaging.

Rauvolfia tetraphylla L. (family Apocynaceae), often referred to as the wild snakeroot plant, is an important medicinal plant and produces a number of indole alkaloids in its seeds and roots. The plant is often used as a substitute for Ravuolfia serpentine (L.) Benth. ex Kurz known commonly as the Indian snakeroot plant or sarphagandha in the preparation of Ayurvedic formulations for a range of diseases including hypertension. In this study, we examine the spatial localization of the various indole alkaloids in developing fruits and plants of R. tetraphylla using desorption electrospray ionization mass spectrometry imaging (DESI-MSI). A semi-quantitative analysis of the various indole alkaloids was performed using UPLC-ESI/MS. DESI-MS images showed that the distribution of ajmalcine, yohimbine, demethyl serpentine and mitoridine are largely localized in the fruit coat while that for ajmaline is restricted to mesocarp of the fruit. At a whole plant level, the ESI-MS intensities of many of the ions were highest in the roots and lesser in the shoot region. Within the root tissue, except sarpagine and ajmalcine, all other indole alkaloids occurred in the epidermal and cortex tissues. In leaves, only serpentine, ajmalcine, reserpiline and yohimbine were present. Serpentine was restricted to the petiolar region of leaves. Principal component analysis based on the presence of the indole alkaloids, clearly separated the four tissues (stem, leaves, root and fruits) into distinct clusters. In summary, the DESI-MSI results indicated a clear tissue localization of the various indole alkaloids, in fruits, leaves and roots of R. tetraphylla. While it is not clear of how such localization is attained, we discuss the possible pathways of indole alkaloid biosynthesis and translocation during fruit and seedling development in R. tetraphylla. We also briefly discuss the functional significance of the spatial patterns in distribution of metabolites.

Camptothecin-producing endophytic bacteria from Pyrenacantha volubilis Hook. (Icacinaceae): A possible role of a plasmid in the production of camptothecin.

BACKGROUND: Camptothecin (CPT), a quinoline alkaloid, is a potent inhibitor of eukaryotic topoisomerase I. Because of this property, several derivatives of CPT are used as chemotherapeutic agents. CPT is produced by several plant species belonging to the Asterid clade as well as by a number of endophytic fungal associates of these plants. In this study, we report the production of CPT by four bacterial endophytes and show the possible role of a plasmid in the biosynthesis of CPT. METHODS: Endophytic bacteria were isolated from leaves, stems and fruits of Pyrenacantha volubilis Hook. (Icacinanceae). The bacterial isolates were purified and analyzed for production of CPT by ESI-MS/MS and NMR analysis. Bacterial identity was established based on the morphology and 16s rRNA sequence analysis. Crude extracts of the bacterial endophytes were evaluated for their cytotoxicity using colon cancer cell lines. The role of plasmid in the production of CPT was studied by purging the plasmid, using acriflavine, as well as reconstituting the bacteria with the plasmid. RESULTS: Four bacterial isolates, Bacillus sp. (KP125955 and KP125956), Bacillus subtilis (KY741853) and Bacillus amyloliquefaciens (KY741854) were found to produce CPT in culture. Both based on ESI-MS/MS and NMR analysis, the identity of CPT was found to be similar to that produced by the host plant. The CPT was biologically active as evident by its cytotoxicity against colon cancer cell line. The production of CPT by the endophyte (Bacillus subtilis, KY741853) attenuated with sub-culture. A likely role of a plasmid in the production of CPT was established. A 5 kbp plasmid was recovered from the bacteria. Bacterial isolate cured of plasmid failed to produce CPT. CONCLUSION: Our study implies a possible role of a plasmid in the production of CPT by the endophytic bacteria and opens up further work to unravel the exact mechanisms that might be involved.

Ambient ionization mass spectrometry imaging of rohitukine, a chromone anti-cancer alkaloid, during seed development in Dysoxylum binectariferum Hook.f (Meliaceae).

Rohitukine, a chromone alkaloid, possesses anti-inflammatory, anti-cancer and immuno-modulatory properties. It has been reported from four species, belonging to the families, Meliaceae and Rubiaceae. Stem bark of Dysoxylum binectariferum (Meliaceae) accumulates the highest amount of rohitukine (3-7% by dry weight). In this study, we examine the spatial and temporal distribution of rohitukine and related compounds during various stages of seed development in D. binectariferum using desorption electrospray ionization mass spectrometry imaging (DESI MSI). Rohitukine (m/z 306.2) accumulation increased from early seed development to seed maturity stage. The spatial distribution of rohitukine was largely restricted to the cotyledonary tissue followed by the embryo and least in the seed coat. Besides rohitukine, rohitukine acetate (m/z 348.2) and glycosylated rohitukine (m/z 468.2) were also detected, both through mass fragmentation and exact mass analysis through Orbitrap mass spectrometry. These results indicate a dynamic pattern of chromane alkaloid accumulation through seed development in D. binectariferum.