ENHANCED IN PLANTA AND IN VITRO BLOACCUMULATION OF PHARMACEUTICALLY IMPORTANT TRITERPENOLD SAPONINS IN BACOPA SPECIES

Abstract

The plant Bacopa monnieri (L.) Wettst is commonly referred to as Brahmi and is used extensively in the pharmaceutical industry as an herb. While B. monnieri contains a wide range of bioactive metabolites, its neuroprotective properties are primarily derived from dammarane triterpenoid glycoside saponins (DTGS). Bacosides, bacopasides, and bacosaponins make up most of these DTGS. Bacoside-A is believed to be the main neuroprotective component of Brahmi, among all DTGS. There is widespread knowledge that Bacoside-A has beneficial effects in the treatment of many diseases, but its low abundance in plants makes it difficult to use as a drug. As a result of overexploitation for its high market demand, this plant is considered endangered. Based on the low yield of bacosides in natural plants, this research aims to develop an alternative platform for the sustainable production of bacosides A and related bioactive saponins from B. monnieri. Throughout the thesis, there are five chapters, each of which is briefly described below: The first chapter reviews the literature on medicinal plants. Bacopa monnieri is briefly described in this chapter, including its systematic classification, varieties, herbal-derived medicines, secondary metabolites in Bacopa plants, various plant parts, elicitor treatment to increase secondary metabolites production, statistics of B. monnieri production, consumption and demand of important herbal products, and alternative production platforms. In chapter 2, a high-performance liquid chromatography (HPLC) method for determining bacoside A (mixture of bacoside A3, bacopaside II, bacopaside X, bacopasaponin C, and two flavonoids (apigenin and luteolin) from various parts of B. monnieri plants (leaf, stem, root, and flower) has been developed and validated, along with its mother tincture, as well as commercial formulations. Using three different compositions of water and methanol (100 percent methanol, 50 percent methanol, and only water), the extraction efficacy of bacosides and flavonoids was tested. Further confirmation of the chemical identity of bacosides A was provided by ESI-MS (electrospray ionization mass spectrometry). In addition, the developed method was validated for its sensitivity, range, linearity, accuracy (recovery), reproducibility, limit of detection (LOD), and limit of quantification (LOQ). The developed method was linear (R2 = 0.998) in the 0.05–0.25 μg/μLrange for bacoside A3. Limits of detection (LOD) and limits of quantification (LOQ) were determined to be 0.035 μg/μL and 0.10 μg/μL, respectively. Ayurvedic formulations containing the parts of the B. monnieri plant have also been analysed using this method. Chapter 3 examined the effects of methyl jasmonate (MeJA) on bacoside-A biosynthesis and metabolic profiles in greenhouse-grown plants of B. monnieri. The greenhouse-grown plants were placed inside a closed plastic container and elicited with MeJA vapour. It was found that MeJA treatment (200 μM for 2 h on day zero) followed by harvesting leaves on day three was the optimum treatment, resulting in an increase of 1.8-fold, 2.2-fold, 3.1-fold, and 2.5-fold in bacoside A3, bacopaside II, bacopaside X, and bacopasaponin C in comparison with control plants, respectively. The transcript levels of squalene synthase (SQS), a key gene involved in bacoside-A biosynthesis was transiently induced by MeJA and reached a peak of up to 3.7-fold at 24 h. The relationships between bacosides content and primary metabolites in plants treated with MeJA were analyzed by gas chromatography-mass spectrometry. MeJA enhances glycolysis and TCA cycles, generating acetyl-CoA, which acts as a precursor for bacosides synthesis, as revealed by untargeted metabolomics. MeJA application in planta appears to have a significant impact on bacoside-A accumulation in greenhouse-grown B. monnieri plants. In Chapter 4, monochromatic LED lights are investigated for their impact on bacoside A accumulation and metabolic profiles in in vitro callus cultures of B. monnieri. Among the several tested hormones, media supplemented with dicamba (2 mg/L) showed optimum biomass accumulation fresh weight (FW; 3.28 ± 0.09 g/25 mL) and dry weight (DW; 0.42 ± 0.012 g/25 mL), and among several tested LED, blue light was found to be optimum for biomass accumulation, as well as metabolites accumulation in callus cultures compared to control (white light). The callus culture treated with blue LED light for five days resulted in 4.62-fold, 2.79-fold, 5.77-fold and 1.97-fold increases in bacoside A3, bacopaside II, bacopaside X and bacopasaponin C production, respectively, over the white LED light treatment as control. The bacoside contents were also analyzed in monochromatic LED light. HPLC analysis showed the highest level of bacoside A3 (2.91 ± 0.05 mg/g DW), bacopaside II (3.07 ± 0.02 mg/g DW), bacopaside X (1.50 ± 0.05 mg/g DW) and bacopasaponin C (2.28 ± 0.06 mg/g DW) upon blue LED treatment for five days of callus cultures. It was found that in the presence of blue lights, glycolysis, TCA cycles, and isoprenoid pathways are boosted, resulting in intermediate products squalene and 2,3-oxidosqualene, precursors to all saponins. Blue light is found to have a significant impact on bacoside production in the callus cultures of B. monnieri in the current study. The aim of chapter 5 is to increase bacoside-A accumulation in B. monnieri by overexpressing squalene synthase (SQS), a key regulatory gene involved in the biosynthesis of bacosides via the isoprenoid pathway. First, the complete open reading frame (ORF) of BmSQS (Bacopa monnieri squalene synthase gene) was amplified and cloned into pBI121. Additionally, pBI121:: BmSQS was mobilized to A. tumefaciens GV3101. Afterwards, young in vitro leaves of B. monnieri were transformed with BmSQS gene to improve bacoside A production. Agrobacterium GV3101 strain harbouring BmSQS gene were co-cultivated with in vitro grown leaf explants of B. monnieri. Postco- cultivation, the infected explants were subjected to three rounds of selection in hormonal media supplemented with antibiotics. The putative transgenic calli were obtained after three rounds of selection. BmSQS overexpressing transgenic callus exhibited a 1.8-fold higher bacoside A3, 2.1- fold higher bacopaside II, 1.4-fold higher bacopaside X and 2.9-fold higher bacopasaponin C as compared to normal callus. To summarize, this thesis presents low cost commercially viable strategies for increasing bacoside production in B. monnieri plants.