QUANTIFICATION OF METABOLITES OF THE MEVALONIC ACID PATHWAY IN ANDROGRAPHIS PANICULATA (BURM.F.) WALLICH EX NEES TRANSFORMED BY 3-HYDROXY-3-METHYLGLUTARYL COENZYME-A REDUCTASE-1 (HMGR1) GENE

• Main Author: Florencia, Febby
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/69873
• Institution: Institut Teknologi Bandung

Andrographis paniculata (sambiloto) has long been used as a herbal medicine. The major bioactive compound in this plant is andrographolide which has been reported to have anti-cancer, anti-viral, anti-bacterial, anti-diabetic, anti-inflammation, and immunostimulant activities. Andrographolide content in sambiloto was relatively low, ranging from 2-3%. This prompted many studies to increase andrographolide biosynthesis, including research using metabolic engineering by inserting CaMV 35S promoter to overexpress the hmgr1 gene that produces 3-Hydroxy-3-Methylglutaryl Coenzyme-A Reductase-1 (HMGR), a key enzyme in the mevalonic acid (MVA) pathway to increase the rate of andrographolide biosynthesis. In the MVA cascade, there were some intermediate compounds such as mevalonic acid (MVA), geranyl diphosphate (GPP), and geranyl geranyl diphosphate (GGPP) that were suspected to be influenced by overexpression of the hmgr1 gene. This experiment was conducted to quantify the metabolites in the MVA pathway from A. paniculata tissues that have been transformed by the upregulated hmgr1 gene, determine the correlation between MVA, GPP, GGPP, and andrographolide, determine the difference between MVA, GPP, GGPP, and andrographolide content in putative transformed shoots and callus. In this experiment, cotyledonary explants were transformed with Agrobacterium tumefaciens GV3101, which carried a pBI121-hmgr1 plasmid with a super promoter CaMV 35S. The transformation stage includes the agroinfiltration stage, where the cotyledon explants were immersed in A. tumefaciens, and the explants were placed in Murashige and Skoog (MS) medium with 100 ?M acetosyringone—followed by the disinfection stage by washing the explant with 400 ppm of cefotaxime and the selection stage in MS medium containing 20 ppm of kanamycin, continued by shoots and callus regeneration. To quantify compounds in the MVA pathway to the biosynthesis of andrographolide, which includes MVA, GPP, and GGPP, extraction of putative transformed (PT) callus and shoots and non-transformed (NT) callus and shoots were done in methanol: water (7:3). Then the analysis of MVA was conducted in UV ?=280 nm, GPP in ?=214 nm, GGPP in ?=282 nm, and andrographolide in ?=210 nm using Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) column C18 (15 cm × 4,6 mm), the ratio of methanol: water eluent for MVA, GPP, GGPP, and andrographolide analysis respectively (1:1), (9:1), (8:2), and (6:4), with a flow rate of 0.5 ml/minutes, at room temperature. The results showed that cotyledons successfully transformed grew into calli and shoots in a selective medium containing 20 ppm of kanamycin. MVA content in PT and NT calli were 2.83% and 1.14%, whereas MVA content in PT and NT shoots were 21.67% and 5.06%. GPP content in PT and NT calli were 2.76% and 1.08%, while PT and NT shoots were 20.46% and 4.85%. GGPP content in PT and NT calli were 2.13% and 1.06%, while GGPP content in PT and NT shoots were 10.35% and 3.32%. Andrographolide content in callus PT and NT were 2.05% and 0.71%, whereas in PT and NT shoots were 3.52% and 1.17%. These results showed an increase in the amount of compound in the MVA pathway in PT calli and shoots compared to NT samples. It can be concluded that by quantification of metabolites in the MVA pathway from PT samples that have been transformed by the hmgr1 gene with CaMV 35S promoter showed an increased level of metabolites in the MVA pathway, which was characterized by the higher content of MVA, GPP, GGPP, and andrographolide respectively by 2.48; 2.50; 2.00; and 2.88 fold in PT callus over NT callus as well as 4.28; 4.20; 3.10; 3.00 fold higher in PT shoots over NT shoots (control). The Pearson Correlation Coefficient test result showed a positive correlation, indicating that an increased level of MVA led to an increased level of GPP, GGPP, and andrographolide. The MVA, GPP, GGPP, and andrographolide levels from PT shoots were significantly higher than those in PT calli respectively, 7.66; 7.41; 4.86; and 1.72 fold.