A mechanistic insight into hydrogen peroxide-mediated elicitation of bioactive xanthones in Hoppea fastigiata shoot cultures.

MAIN CONCLUSION: Elicitation of xanthones is mediated by ROS where Ca (2+) mediated generation of H 2 O 2 activates the shikimate pathway, a key regulator in early steps of xanthone biosynthesis in H. fastigiata. Shoot cultures of Hoppea fastigiata upon treatment with yeast extract (YE) accumulate an enhanced amount of 1,3,5-trihydroxy-8-methoxy xanthone. We demonstrated that YE treatment was followed by a rapid burst of reactive oxygen species (ROS, O2 (-) and H2O2) and subsequent increase in xanthone contents. The antioxidant enzymes (NADPH oxidase, superoxide dismutase (SOD), peroxidase and catalase) followed a similar kinetics as that of ROS, depending on their role in production or degradation. It was observed that shikimate dehydrogenase (SKDH) and shikimate kinase (SK) activities enhanced after 8 h, benzophenone synthase activity continued to rise after elicitation and peaked at 18 h. Activities of phenylalanine ammonia-lyase and 4-hydroxycinnamoyl-CoA ligase remained suppressed and unaffected, respectively, after elicitation. This suggests a possible phenylalanine-independent biosynthesis of xanthones. Successive treatment of shoots cultures with a NADPH-oxidase inhibitor diphenylene iodide and a ROS-scavenger dihydrolipoic acid showed inhibition in ROS (O2 (-) and H2O2) accumulation. These treatments were also shown to decrease the activities of SKDH and SK, leading to a suppressed amount of xanthones formation. Although O2 (-) showed continuous increase upon treatment with a SOD inhibitor diethyldithiocarbamic acid, the contents of H2O2 and xanthones were decreased, which correlates well with the reduced activities of SKDH and SK. Treatments with calcium antagonists, such as, lanthanum chloride and EGTA were also shown to block the activities of SKDH, SK, NADPH-oxidase and SOD, and consequently leading to suppressed accumulation of ROS (O2 (-) and H2O2) and xanthones.

Shoot cultures of Hoppea fastigiata (Griseb.) C.B. Clarke as potential source of neuroprotective xanthones.

Hoppea fastigiata, an annual medicinal herb belonging to the Gentianaceae, is mostly found in South Asian countries, and is used by local tribes for various brain-related ailments. The genus possesses a unique class of compounds, xanthones, which are known for their potential against Alzheimer's and Parkinson's diseases. Limited availability and the potential pharmacological significance of the plants has led to the establishment of in vitro cultures of H. fastigiata and study of its neuroprotective principles. In vitro plantlets were established from the apical meristem of the plant in Murashige and Skoog medium with a combination of the phytohormones 6-benzylaminopurine (1 mg/L) and kinetin (0.1 mg/L), which was found to be efficacious with a growth index of 0.9 ± 0.01 after 30 days. Four different solvent extracts of in vitro cultures were evaluated for acetylcholinesterase (AChE) and monoamine oxidase A and B (MAO-A and MAO-B) inhibitory activities, amongst which the ethanolic extract showed the lowest IC50 value in all the assays. Three major compounds were isolated from the ethanolic extract and structurally confirmed as 1,5,7-trihydroxy-3-methoxyxanthone (1), 1,5-dihydroxy-3,7-dimethoxyxanthone (2) and 1,3,5-trihydroxy-8-methoxyxanthone (3). Compound 3 showed the strongest AChE inhibitory activity with mixed-type inhibition. Compounds 1 and 2 also showed promising AChE inhibitory properties with mixed and non-competitive types of inhibition, respectively. Compounds 1 and 2 showed inhibition of MAO-A (mixed and competitive, respectively) and compounds 2 and 3 showed inhibition of MAO-B (competitive and mixed, respectively). Extracts and isolated compounds showed good antioxidant capacities. The ethanolic extract and compound 2 showed the strongest antioxidant activities among the other solvent extracts and compounds, respectively.