Comparative analyses of Ajuga bracteosa Wall. ex Benth. plants regenerated from hairy roots

Abstract

Ajuga bracteosa Wall. ex Benth. is a widely used traditional medicinal plant containg a broad spectrum of metabolites with therapeutic properties. The increased demand of A. bracteosa for medicinal purposes and low product yield in wild plants need some alternatives. Tissue culture and genetic transformation techniques offer a promising approach for the conservation and mass-production of plant secondary metabolites. The root oncogenic loci (rol) genes of Agrobacterium rhizogenes enhance the production of medicinally important compounds in plants and provide a first barrier against the overproduction of reactive oxygen species. The main objective of the present study was to investigate and compare biological activities of three independent transgenic lines of A. bracteosa plants, regenerated from hairy roots, with in vitro grown untransformed plants. This study also aimed to explore genes expression, phytochemical composition, antioxidant properties, cytotoxic capabilities, enzyme inhibitory potentials, and in vivo biological activities of A. bracteosa. After successful transformation and regeneration of transformed plants, real-time quantitative PCR confirmed the increased expression (1.94–6.59-fold) of HMGR, HDS, FDS, PAL, and TTG1 genes in all transgenic lines. Furthermore, GC-MS coupled with principal component analysis revealed diverse concentrations of 97 metabolites in A. bracteosa. Essential elements and phytochemicals were significantly enhanced in crude extracts of transgenic plants. Kaempferol was the most abundant polyphenol in transgenic lines (up to 101.26 ± 6 µg/mg). Transgenic line 3 (ABRL3) showed maximum phenolics (13.39 ± 2µg GAE/mg) and flavonoids content (4.75 ± 0.16 µg QE/mg). ABRL3 also demonstrated to have potent total antioxidant capacity (8.16 ± 1 μg AAE/mg), reducing power, (6.60 ± 1.17 μg AAE/mg), DPPH activity (IC50 = 59.5 ± 0.8 μg/mL), hydroxyl ion scavenging (IC50 = 122.5 ± 0.90 μg/mL), and iron-chelating power (IC50 = 154.8 ± 2 μg/mL) among all plants. Administration of 200 mg/kg of transformed plant extracts produced significant analgesic, anti inflammatory, anticoagulant, and antidepressant activities against in vivo mice model. The antifungal resistance of A. bracteosa was assessed against two pathogenic fungal strains. Wild type control plant leaves were completely necrotized by Aspergillus fumigatus (FCBP 66) and Fusarium solani (FCBP 0291), whereas transformed leaves had improved antifungal tolerance. Transgenic lines also showed greater survival under multiple abiotic stresses. This was revealed by significant chlorophyll content ix (8.13–21 µmoles/m2 ), the higher quantum efficiency of PSII (Fv/Fm), and the performance index (PIabs) value in drought and salt stress. Similarly, catalase and peroxidase enzyme activities were enhanced during extreme drought (300–400 mM mannitol) and salinity (150–200 mM NaCl) conditions, compared to untransformed control. Transgenic line ABRL3 produced the highest inhibition of diabetogenic enzymes at all concentrations. ABRL3 exhibited an IC50 of 73.2 ± 3 µg/mL and 69.4 ± 3 µg/mL against α-amylase and α-glucosidase respectively. Transgenic line 2 (ABRL2) showed excellent anti-Alzheimer enzyme activity with an IC50 of 127.5 ± 3 µg/mL against acetylcholinesterase and 215.5 ± 6 µg/mL against butyrylcholinesterase. Oral administration of crude extracts of all transgenic lines (100 mg/kg) reduced blood glucose and HbA1c level and improved body weight of alloxan-induced diabetic mice in a time-dependent manner. Furthermore, plant extracts improved the serum chemistry parameters near to the normal. ABRL3 maximally increased the liver and brain catalase and peroxidase activities and protected both organs from oxidative stress-induced damage. In conclusion, our data suggest that rolABC genes have a significant impact on the synthesis of metabolites involved in enhancing the therapeutic potential and multi-stress tolerance of A. bracteosa. Thus, these plants can be used as an alternative therapeutic regime against chronic life-threatening disorders without damaging natural ecosystems. Further studies can help us understand the underlying mechanisms of these multiple pharmaceutical effects