ANDROGRAPHOLIDE PRODUCTION AND FUNCTIONAL CHARACTERIZATION OF BIOSYNTHETIC PATHWAYS IN Andrographis paniculata
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Date
2012
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Indira Gandhi Krishi Vishwavidyalaya, Raipur
Abstract
Andrographolide is a diterpene lactone and leading bioactive component of the medicinal plant Andrographis paniculata, which attributes for diverse pharmacological properties. Traditionally andrographolide was extracted from the leaves, stems and other parts of the plant. Leaf contains maximum andrographolide content (2-3%) in comparison to other parts of plant. In the present study, rapid and efficient method for the production of andrographolide from adventitious root culture was established and optimized. Leaf explants of A. paniculata were cultured on different strength Murashige and Skoog (MS) medium supplemented with different concentrations of auxins (0.5 – 5mg/l) for adventitious root induction and elongation. The highest number of adventitious root was initiated from modified MS with 1mg/1 NAA (26.66±1.52) followed by full MS with 1mg/l NAA (24.00±2.64). One month old adventitious roots were dissected from the explants and grown in different strength MS liquid medium with 1mg/l NAA for mass multiplication of adventitious root. The 11.27±0.15 g/l dry adventitious root was recovered in full MS followed by modified MS (10.94±0.60 g/l) at 5 week of culture period. The highest average andrographolide content was recorded (133.28±1.52mg/g DW) from modified MS medium with 1mg/l NAA after 42 days of culture. The produced andrographolide content was 3.5- 5.5 folds higher in compare to natural plant depending on the medium strength.
Callus and cell suspension culture was also established for andrographolide production. Callus was induced by culturing leaf discs on Murashige and Skoog (MS) medium with different concentration of 2,4-D and combination of 2,4-D + Kin, 2,4-D + NAA and BAP + NAA. Best callus induction were obtained at lower concentration of 2,4-D (0.5 and 1.0 mg/l), combination of 2,4-D + NAA (1.0+1.0 mg/l), 2,4-D + Kin (1.0+0.5 mg/l) and BAP + NAA (1.0+1.0mg/l). The best callus was undergone for andrographolide estimation. The high andrographolide content detected in 2,4-D + NAA (1.0+1.0 mg/l) (13.50±2.31 mg/g FW) followed by 2,4-D + Kin (1.0+1.0 mg/l) (12.66±1.13 mg/g FW) Cell suspension culture of A. paniculata was established by inoculating fresh friable fragments of callus on MS medium supplemented with respective callus grown medium. Callus from the 2,4D +NAA (1.0+1.0 mg/l) was friable and best to release cells in suspension culture. Cells were successfully subcultured every 20-21 days on liquid MS media supplemented with respective callus grown medium. The highest amount of andrographolide (32.40 ±2.22 mg/g FW) is found in 2, 4-D + NAA (1.0+1.0 mg/l) cell suspension culture followed by 2,4-D + Kin (1.0+0.5 mg/l) (31.95 ±2.21 mg/g FW). These studies provided an efficient way to produce andrographolide from A. paniculata callus as well as cell suspension culture. The developed method was used for the large quantity production of andrographolide. Methyl jasmonate (MeJA) elicitation study was conducted to elicit andrographolide biosynthesis in cell suspension culture and the resulting experiments demonstrated that MeJA was indeed a potent inducer of andrographolide. The elicited amount of
andrographolide was obtained maximum 5.25-fold (525.18%) higher than the control in 5μM treatment at 24 hour.
The differential expression study was conducted to know MEP and MVA pathways genes regulation on andrographolide biosynthesis in suspension culture of A. paniculata. Lovastatin and fosmidomycin were used to block the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and 1-deoxy-D-xylulose-5-phosphate reducto-isomerase (DXR) in the MVA and MEP pathway respectively. Methyl jasmonate was used to improve the biosynthesis of andrographolide. 100μm Lovastatin inhibit andrographolide 86.75% and 200 μM fosmidomycin inhabit 84.75 % andrographolide at day 4. Also Inhibitors lovastatin and fosmidomycin influence the transcriptional expression of important genes. The results indicated that MEP and MVP pathways have considerable contribution in andrographolide biosynthesis and DXR and HMGR was the important genes of andrographolide biosynthesis pathway. A crosstalk mechanism between the MEP and MVA subnetworks was also established. The fosmidomycin inhibition triggers a flux of precursors (IPP and DMAPP) from the MEP pathway to the MEV pathway, resulting in conserving andrographolide concentration despite the inactivation of MEP pathway vice versa. MeJA elicit the andrographolide biosynthesis by inducing transcriptional expression. The coordination of ISPH, GGPS and HMGS expressions were highly influenced andrographolide biosynthesis. The results clearly showed that andrographolide elicitation process was coordinately regulated.
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Keywords
planting, productivity, biochemical compounds, biosynthesis, vegetative propagation, organic compounds, genes, esters, enzymes, auxins