Isolation of 4-hydroxy 3-methyl 2-butenyl 4-diphosphate reductase (ApHDR) gene of methyl erythritol diphosphate (MEP) pathway, in silico analysis and differential tissue specific ApHDR expression in Andrographis paniculata (Burm. f) Nees.

The full length Andrographis paniculate 4-hydroxy 3-methyl 2-butenyl 4-diphosphate reductase (ApHDR) gene of MEP pathway was isolated for the first time. The ApHDR ORF with 1404 bp flanked by 100 bp 5'UTR and 235 bp 3'UTR encoding 467 amino acids (NCBI accession number: MK503970) and cloned in pET 102, transformed and expressed in E. coli BL21. The ApHDR protein physico-chemical properties, secondary and tertiary structure were analyzed. The Ramachandran plot showed 93.8% amino acids in the allowed regions, suggesting high reliability. The cluster of 16 ligands for binding site in ApHDR involved six amino acid residues having 5-8 ligands. The Fe-S cluster binding site was formed with three conserved residues of cysteine at positions C123, C214, C251 of ApHDR. The substrate HMBPP and inhibitors clomazone, paraquat, benzyl viologen's interactions with ApHDR were also assessed using docking. The affinity of Fe-S cluster binding to the cleft was found similar to HMBPP. The HPLC analysis of different type of tissue (plant parts) revealed highest andrographolide content in young leaves followed by mature leaves, stems and roots. The differential expression profile of ApHDR suggested a significant variation in the expression pattern among different tissues such as mature leaves, young leaves, stem and roots. A 16-fold higher expression of ApHDR was observed in the mature leaves of A. paniculata as compared to roots. The young leaves and stem showed 5.5 fold and fourfold higher expression than roots of A. paniculata. Our result generated new genomic information on ApHDR which may open up prospects of manipulation for enhanced diterpene lactone andrographolide production in A. paniculata. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-00952-0.

Molecular Cloning and Differential Gene Expression Analysis of 1-Deoxy-D-xylulose 5-Phosphate Synthase (DXS) in Andrographis paniculata (Burm. f) Nees.

Andrographis paniculata 1-deoxy-D-xylulose-5-phosphate synthase (ApDXS) gene (GenBank Accession No MG271749.1) was isolated and cloned from leaves for the first time. Expression of ApDXS gene was carried out in Escherichia coli Rosetta cells. Tissue-specific ApDXS gene expression by quantitative RT-PCR (qRT-PCR) revealed maximum fold expression in the leaves followed by stem and roots. Further, the differential gene expression profile of Jasmonic acid (JA)-elicited in vitro adventitious root cultures showed enhanced ApDXS expression compared to untreated control cultures. A. paniculata 3-hydroxy-3-methylglutaryl-coenzyme A reductase (ApHMGR) gene expression was also studied where it was up-regulated by JA elicitation but showed lower expression compared to ApDXS. The highest expression of both genes was found at 25 µm JA elicitation followed by 50 µm. HPLC data indicated that the transcription levels were correlated with increased andrographolide accumulation. The peak level of andrographolide accumulation was recorded at 25 µM JA (9.38-fold) followed by 50 µM JA (7.58-fold) in elicitation treatments. The in silico generated ApDXS 3D model revealed 98% expected amino acid residues in the favored and 2% in the allowed regions of the Ramachandran plot with 92% structural reliability. Further, prediction of conserved domains and essential amino acids [Arg (249, 252, 255), Asn (307) and Ser (247)] involved in ligand/inhibitor binding was carried out by in silico docking studies. Our present findings will generate genomic information and provide a blueprint for future studies of ApDXS and its role in diterpenoid biosynthesis in A. paniculata.

Isolation, characterization and in silico analysis of 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) gene from Andrographis paniculata (Burm. f) Nees.

3-Hydroxy-3-methylglutaryl-coenzymeA reductase (HMGR), the first rate-limiting enzyme of Mevalonate (MVA) pathway was isolated from Andrographis paniculata (ApHMGR) and expressed in bacterial cells. Full length ApHMGR (1937 bp) was submitted to NCBI with accession number MG271748.1. The open reading frame (ORF) was flanked by a 31-bp 5'-UTR, 118-bp 3'-UTR and ApHMGR contained a 1787 bp ORF encoding protein of 595 amino acids. ApHMGR protein was approximately 64 kDa, with isoelectric point of 5.75. Isolated ApHMGR was cloned into pET102 vector and expressed in E. coli BL21 (DE 3) cells, and characterized by SDS-PAGE. HPLC analysis for andrographolide content in leaf, stem and root of A. paniculata revealed highest in leaf tissue. The expression patterns of ApHMGR in different plant tissues using qRT-PCR revealed high in root tissue correlating with HPLC data. Three dimensional (3D) structural model of ApHMGR displayed 90% of the amino acids in most favored regions of the Ramachandran plot with 93% overall quality factor. ApHMGR was highly conserved with plant specific N-terminal membrane domains and C-terminal catalytic regions. Phylogenetic analysis showed A. paniculata sharing common ancestor with Handroanthus impetiginosus. 3D model of ApHMGR was screened for the interaction with substrates NADPH, HMG CoA and inhibitor using Auto Dock Vina. In silico analysis revealed that full length ApHMGR had extensive similarities to other plant HMGRs. The present communication reports the isolation of full length HMGR from A. paniculata, its heterologous expression in bacterial cells and in silico structural and functional characterization providing valuable genomic information for future molecular interventions.

Chemical profiling and anti-psoriatic activity of methanolic extract of Andrographis nallamalayana J.L.Ellis.

Andrographis nallamalayana is being widely used as tribal medicine in the treatment of leucoderma and mouth ulcers. Chemical profiling of methanolic extract of the whole plant (PE), using GC-MS and LC-MS, revealed the presence of compounds viz. α-tocopherol, ß-sitosterol, tetradecanoic acid, monostearin, flavones/flavanones and their glycosides, chromones, etc. Topical application of imiquimod on the dorsal portion of male BALB/C mice resulted in the development of psoriatic symptoms (erythema, scaling, thickening and folding) with a mean disease activity index (DAI) of >7.0. Topical treatment with 100-µL PE (~6.4%/12.8%) formulations, for 12-days, resulted in the alleviation of disease symptoms. Compared to water-based formulations, emu oil-based formulation, PE400EO was found more effective in reducing the mean DAI (>84%), keratinocyte count (>65%) (p < 0.01) and interleukin-22 (~70%) (p < 0.05). We report, for the first time, anti-psoriatic activity of A. nallamalayana having great potential in developing a potent phytomedicine against psoriasis.

Enhanced daidzin production from jasmonic and acetyl salicylic acid elicited hairy root cultures of Psoralea corylifolia L. (Fabaceae).

Daidzin (7-O-glucoside of daidzein) has several pharmacological benefits in herbal remedy, as antioxidant and shown antidipsotropic activity. Hairy root culture of Psoralea corylifolia L. was developed for biomass and enhanced daidzin production using signalling compounds such as jasmonic acid (JA) and acetyl salicylic acid (ASA). Best response of 2.8-fold daidzin (5.09% DW) with 1 µM JA treatment after second week and 7.3-fold (3.43% DW) with 10 µM JA elicitation after 10th week was obtained from hairy roots compared to untreated control. ASA at 10 µM promoted 1.7-fold increase in daidzin (1.49% DW) content after seventh week compared to control (0.83% DW). Addition of 25 µM ASA resulted in 1.44% DW daidzin (1.5-fold increase) with 0.91% DW in control after fifth week and 1.44% DW daidzin (2.3-fold increase) after eighth week when compared to untreated control (0.62% DW). Reduced biomass with increased daidzin content was facilitated by elicited hairy root cultures.