Variability in starch granular morphology of Tinospora cordifolia: an important species of Indian Systems of Medicine (ISM).

Tinospora cordifolia is an important medicinal plant species known for therapeutic action of starch along with other medicinal ingredients. The starch prepared from the aqueous extract of fresh stems is used in the Indian Systems of Medicines. The plant extract prepared from T. cordifolia is a  promising source for the treatment of COVID-19. This investigation explores for the first time, the morphological details of the starch granules and its accumulation pattern along with its variability among the germplasm of T. cordifolia collected from different parts of India. Starch content was 39.80% on dry weight basis and moisture content was about 28.21%. Starch granule recovery based on stem dry weight and starch content ranged from 14.70 to 20.28% and 52.02 to 71.76%, respectively in different starch settling methods. Starch accumulation pattern in the stem was also studied in the species. Even though wide variability in starch granule shapes was observed among the germplasm, majority of the genotypes had starch granules of round or oval shape. Similarly, starch granule size also varied greatly (38.32-88.03 µm) within and among the genotypes. Significantly small sized starch granules (p = 0.05) were present in the genotype, IC 283650 and biggest (p = 0.05) starch granules were present in the genotype, IC 310610. The information generated in the present study will have application in starch industry for the inclusion of T. cordifolia as an alternative source of starch in addition to its use in Traditional Systems of Medicine. Supplementary Information: The online version contains supplementary material available at 10.1007/s42535-021-00286-y.

Altered biomass allocation and quality improvement in roots of Indian ginseng Withania somnifera Dunal. Linn. through physiological interventions.

Ashwagandha (W. somnifera Dunal. Linn.), known as Indian ginseng, contains three major bioactive compounds, withaferin-A (WA), 12-deoxywithastramonolide (WO) and withanoloide A (WD). In a field experiment, the impacts of foliar application of growth retardants/promoters was assessed with respect to biomass allocation pattern and major withanoloide content at different phenological stages in W. somnifera. Biomass accumulation pattern showed that foliar application of 500 mg l-1ethrel at 50, 65, 85, 105, and 120 days after sowing (DAS) restricted phenological progression and reduced berry weight by 61% as comparted to the control at 160 DAS. 500 mg l-1 succinic acid foliar application resulted in maximum plant height (56.4 cm), maximum dry stem weight (DWS) and dry root weight (DRW) whereas 500 mg l-1 ethrel had resulted in minimum plant height and DRW at 180 DAS. During last 50 days of crop growth, the accumulation pattern drastically changed with more than 60% of the biomass allotment to the reproductive part, the berries. The WD in roots ranged between 0.325 mg g-1and 0.342 mg g-1 during all growth stages. WA content decreased with increase in progression of crop growth and reached the lowest at 180-190 DAS. In a pot experiment, ethrel application up regulated DWF-5 by 2.44, SQE by 3.79 and CYP450s by 1.17 log2fold in roots 8 h after treatment and succinic acid had up regulated the expression of all these genes by nearly 3 log2fold change. This is in accordance with the withanoloide accumulation pattern in field condition under foliar application of these molecules.

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.

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.

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.