Narendra A. Gajbhiye

Next Generation Sequencing and Transcriptome Analysis Predicts Biosynthetic Pathway of Sennosides from Senna (Cassia angustifolia Vahl.), a Non-Model Plant with Potent Laxative Properties

Senna (Cassia angustifolia Vahl.) is a world’s natural laxative medicinal plant. Laxative properties are due to sennosides (anthraquinone glycosides) natural products. However, little genetic information is available for this species, especially concerning the biosynthetic pathways of sennosides. We present here the transcriptome sequencing of young and mature leaf tissue of Cassia angustifolia using Illumina MiSeq platform that resulted in a total of 6.34 Gb of raw nucleotide sequence. The sequence assembly resulted in 42230 and 37174 transcripts with an average length of 1119 bp and 1467 bp for young and mature leaf, respectively. The transcripts were annotated using NCBI BLAST with ‘green plant database (txid 33090)’, Swiss Prot, Kyoto Encylcopedia of Genes & Genomes (KEGG), Cluster of Orthologous Gene (COG) and Gene Ontology (GO). Out of the total transcripts, 40138 (95.0%) and 36349 (97.7%) from young and mature leaf, respectively, were annotated by BLASTX against green plant database of NCBI. We used InterProscan to see protein similarity at domain level, a total of 34031 (young leaf) and 32077 (mature leaf) transcripts were annotated against the Pfam domains. All transcripts from young and mature leaf were assigned to 191 KEGG pathways. There were 166 and 159 CDS, respectively, from young and mature leaf involved in metabolism of terpenoids and polyketides. Many CDS encoding enzymes leading to biosynthesis of sennosides were identified. A total of 10,763 CDS differentially expressing in both young and mature leaf libraries of which 2,343 (21.7%) CDS were up-regulated in young compared to mature leaf. Several differentially expressed genes found functionally associated with sennoside biosynthesis. CDS encoding for many CYPs and TF families were identified having probable roles in metabolism of primary as well as secondary metabolites. We developed SSR markers for molecular breeding of senna. We have identified a set of putative genes involved in various secondary metabolite pathways, especially those related to the synthesis of sennosides which will serve as an important platform for public information about gene expression, genomics, and functional genomics in senna.

Accumulation of Three Important Bioactive Compounds in Different Plant Parts of Withania somnifera and its Determination by the LC–ESI-MS-MS (MRM) Method

A comprehensive experiment was conducted to study the accumulation pattern and determination of three important bioactive compounds namely withaferin-A (WA), 12-deoxywithastramonolide (WO) and withanolide-A (WD) and its determination by the liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) method in root, stem, fruits and leaves of Withania somnifera. A rapid and sensitive LC-ESI-MS-MS method was developed and validated for the determination of these three important bioactive compounds, having same molecular weight. The multiple reaction monitoring method was established by two transitions for each analyte and intense transition used for quantification. Separation of the three analytes was achieved within a run time of 5 min on an RP-18 column using a mobile phase consisting of acetonitrile and 0.1% acetic acid in water in an isocratic condition. The developed method was validated as per the ICH guidelines. The developed method was found to be suitable for identification and quantification of WA, WO and WD in different plant parts such as roots, stems, fruits and leaves of W. somnifera. The accumulation of WA was highest in leaves samples (8.84 ± 0.37 mg/g) and it was 2.23, 5.85 and 27.26 times higher than its concentration in fruits, stems and roots, respectively. WO and WD contents were highest (0.44 ± 0.016 and 0.72 ± 0.016 mg/g, respectively) in root.

Extraction Optimization for Phenolic- and Withanolide-Rich Fractions from Withania somnifera Roots: Identification and Quantification of Withaferin A, 12-Deoxywithastromonolide, and Withanolide A in Plant Materials and Marketed Formulations Using a Reversed-Phase HPLC–Photodiode Array Detection Method

Background: Both the roots and leaves of Withania somnifera are products of commerce. They contain active compounds of therapeutic value and mostly different withanolides. Several pharmacological activities of W. somnifera have links to one or more withanolides. The presence of phenolic compounds in extracts could play a vital role in the reduction of blood glucose levels in diabetic subjects. Objective: The present study was carried out for the selection of a solvent to prepare extracts rich in phenolics, withaferin A (WA), 12-deoxywithastromonolide (12WD), and withanolide A (WDA). A simple, rapid HPLC method was also developed for the identification and quantification of WA, 12WD, and WDA. Methods: The extraction efficiency of aqueous alcoholic solvents including hexane, chloroform, ethyl acetate, and methanol were compared for three selected withanolides and total phenolic content. The contents of WA, 12WD, and WDA and total phenolics were determined in the extracts. The quality of nine formulations containing W. sominfera were also compared in terms of the content of WA, 12WD, and WDA and total phenolics. Results: The maximum extract yield and the total withanolide and phenolic content were obtained from aqueous alcoholic compositions at 50:50 (v/v), 70:30 (v/v), and 100:0 (v/v), respectively. In the case of organic solvents, chloroform and ethyl acetate yielded the highest concentrations of phenolics and three withanolides, respectively. The total phenolic content in formulations was in the range of 1.84-3.13%, and total withanolide content showed wide variability. Conclusions: The outcome of the present investigation could be utilized for the selection of extraction solvents to prepare W. somnifera-enriched extracts and their quality monitoring by using the developed and validated HPLC-Photodiode array detection method. Highlights: A process for preparation of phenolics and withanolides (withaferin A, 12-deoxywithastramonolide and withanolide A) enriched extracts of Withania somnifera. Simple and rapid HPLC method was also developed and validated as per the ICH guidelines for identification and quantification of three major withanolides. The developed HPLC method was applied to analyze the quality of extracts and marketed herbal products (mono, as well as poly constituents). Optimized extraction process could be utilized for upscaling process development in preparation of enriched extracts from Withania somnifera, crop improvement, bio-prospection studies and quality control.

High-performance liquid chromatography tandem mass spectrometry for simultaneous detection of aflatoxins B1, B2, G1 and G2 in Indian medicinal herbs using QuEChERS-based extraction procedure

ABSTRACT Since the discovery of aflatoxins (AFs) in the 1960s, much research has focused on detecting the toxins in contaminated food and feedstuffs. But the quality determination in medicinal plant matrices with respect to AFs is scare. Hence, a simple, accurate and sensitive high-performance liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method was developed for simultaneous determination of AFs AFB1, AFB2, AFG1 and AFG2 in two Indian popular medicinal herbs i.e. senna (Cassia angustifolia) and kalmegh (Andrographis paniculata). AFs have been extracted from herb matrix using a QuEChERS (quick, easy, cheap, effective, rugged and safe)-based extraction procedure followed by applying primary secondary amine and C18 for further clean-up step and then were quantified under the multiple reaction monitoring together with positive ionisation modes. Matrix-matched calibration was used for quantification in order to reduce the matrix effect. Validation of the method was carried out in herbs by recovery experiments. Recoveries of the spiked samples were in the range of 61.9–111.5% with an inter-day and intraday relative standard deviation lower than 20.0%. Limits of detection and quantification ranged from 0.41 to 0.95 ng mL−1 and 1.2 to 3.8 μg kg−1, respectively. The expanded uncertainty of the method was <21% for all the toxins in both the herbs. Finally, the proposed method was successfully applied to determine AF residues in real field samples of senna and kalmegh obtained from different locations in India.

Foliar Application of Signal Molecules can Augment Quality in Andrographis paniculata under Prolonged Water Deficit Stress

The quality of a medicinal plant is decided by the content of specific secondary metabolite respective to the plant species. Therefore, an attempt was made to assess the effect of water deficit (WD) stress and signal molecules on physiological and biochemical responses of Andrographis paniculata under polyhouse condition. To keep the soil at field capacity (FC) in control (well-watered) treatment, a pre-measured quantity of water was applied to the pots once in a week. The signal molecules (i) ascorbic acid @200 mg l−1, (ii) glutamic acid @150 mg l−1 and salicylic acid @200 mg l−1 applied foliar at 10, 25, 40 and 55 days after WD treatment. Total chlorophyll, total carotenoid and proline content increased due to WD stress at 50 days after treatment (DAT) whereas, total chlorophyll and total carotenoid content decreased but, proline content increased at 80 DAT. Ascorbic acid enhanced the ascorbate peroxidase activity and glutamic acid enhanced guiacol peroxidase activity of leaves under WD stress. WD stress reduced actual efficiency of photosynthesis (Fv’/Fm’) and energy utilized for photochemistry (¦PSII) at 80 DAT. WD stress during 20–50 DAT did not improve the andrographolide content in leaves of A. paniculata. However, a prolonged WD stress till 80 DAT hastened andrographolide content in leaves. Foliar application of salicylic acid under polyhouse condition indicated its scope to augment the quality of Andrographis paniculata under prolonged WD stress.

Development and validation of LC—ESI—MS/MS method for simultaneous determination of four coumarin derivatives and an alkaloid from root and stem bark of Aegle marmelos Correa

Aegle marmelos Correa (Bael tree) is a medicinal fruit tree, widely used for healing purposes in various systems of medicines. Coumarins and alkaloids present in various parts of bael tree including roots and fruit pulp are the primary active constituents implicated for its biological activities. An efficient liquid chromatography–electrospray ionization—tandem mass spectrometry (LC—ESI—MS/MS) method was developed for identification and simultaneous determination of four coumarin derivatives, namely, umbelliferone, psoralene, marmin, and imperatorin, and an alkaloid, skimmianine, in root and stem bark of A. marmelos. The chromatographic separation of analytes was performed on Altima C18 (50 × 4.6 mm, 3 μm) column using methanol and 0.1% acetic acid in water (54:46, v/v) as the mobile phase under isocratic conditions. The LC–MS/MS parameters were optimized in the positive ionization mode using electrospray ionization source. The quantification of the analytes was performed using multiple reaction monitorin...

Simultaneous determination of marmin, skimmianine, umbelliferone, psoralene, and imperatorin in the root bark of Aegle marmelos by high-performance thin-layer chromatography

Aegle marmelos (Rutaceae) has gained traditional therapeutic importance owing to their coumarins (umbelliferone, psoralene, marmin, and imperatorin) and alkaloid (skimmianine). Presently, there is no appropriate thin-layer chromatography (TLC) based method available for simultaneous determination important coumarins and alkaloids of A. marmelos. A simple, sensitive, and accurate high-performance thin-layer chromatographic (HPTLC) method has been developed for the determination of marmin, skimmianine, umbelliferone, psoralene, and imperatorin for in the methanolic extract of root bark of A. marmelos. Analysis was performed by using TLC precoated silica gel 60 F 254 aluminium plates (20 × 10 cm) with toluene-ethyl acetate-formic acid (6:4:0.1, v/v) as mobile phase. Quantitative evaluation of the plate was performed in the absorption mode at 304 nm. The calibration curves were prepared in the concentration range of 40–500 ng spot−1 for marmin, skimmianine, and umbelliferone, for psoralene in the range of 16–200 ng spot−1 and for imperatorin in the range of 20–250 ng spot−1. The method was validated for precision, repeatability, and accuracy. The technique has been applied, for the first time, for the simultaneous estimation of marmin, skimmianine, umbelliferone, psoralene, and imperatorin. The proposed method was found to be robust, precise, and accurate; it therefore holds potential for detection, monitoring, and quantification of marmin, skimmianine, umbelliferone, psoralene, and imperatorin in A. marmelos raw root drug materials and chemical screening of germplasms.