Phytochemical Profiling and Investigation of Bioactivities of a New Zealand Grown Chemotype of Centella asiatica (GOTUKOLA) : A Potential Neuroprotective Herb

Abstract

Natural ethnic foods with traditionally known health benefits offer a substantive foundation for the discovery of functional foods with therapeutic potentials. Centella asiatica (Gotu Kola) is a green leafy vegetable and a medicinal herb in a few countries of the world. Complementing its reputation in traditional medicine as a memory booster, research backed multifunctional properties make it a promising potential agent to target multiple pathways in neurogenerative diseases such as Alzheimer’s. Being rich with diverse phytochemicals, the occurrence of chemotype variations of this plant are reported in the production of these metabolites due to origin and growth conditions. Current study intended to optimise extraction, chemical profiling, targeted quantification, unfolding possible patho-mechanisms and insight into bioactivities of potential neuroprotective phytochemicals in C. asiatica extracts. Among the tested extraction techniques, ultrasound assisted extraction and steam distillation were chosen to extract liquid and volatile fractions. A solid to liquid ration of 1:25, a binary solvent system with 9:1 methanol to water (v/v) and a 20 minutes extraction time offered the optimum conditions in extracting total triterpenes of the plant in terms of major triterpenes; madecassoside, asiaticoside, madicassic acid and asiatic acid. Study of different solvent systems based on water and methanol also revealed information on the extraction behaviour of Total Triterpene Content (TTC), Total Polyphenolic Content (TPC), Total Flavonoid Content (TFC) and the antioxidant capacity variations. Initial HPLC-DAD quantification was followed by a multiple reaction monitored (MRM), UHPLC-ESI-triple quadrupole tandem mass spectrometry to further confirm and to enable more specific and sensitive determination of major saponins and sapogenins together with chlorogenic acid simultaneously. C. asiatica leaf extracts of a winter harvested (in August) sample contained high amounts of triterpenic glycoside asiaticoside (52.30 ± 3.92 mg/g DW), followed by madecassoside (14.27 ± 1.19 mg/g) and chlorogenic acid (3.98 ± 0.25 mg/g). However, insignificant quantities of aglycone levels, compared to those reported elsewhere, indicated the occurrence of distinct chemotype of C. asiatica in New Zealand. This study characterised the phytochemical profile of C. asiatica leaf extract; liquid and volatile fractions identifying 69 compounds in the liquid fraction and 38 compounds in volatile fraction including triterpenes, flavonoids, caffeoylquinic acids, sesquiterpenes and phytosterols. Q-extractive orbitrap high- resolution mass spectrometry and gas chromatography-mass spectrometry enabled profiling of phytochemicals in liquid and volatile fractions respectively. The liquid fraction comprised of pentacyclic triterpenoid compounds; mainly the ursane and oleanane subtypes, and flavonoids as major compounds. Sesquiterpenoids and monoterpenoids included as the significant compounds in essential oil fraction. Among 69 compounds positively identified in liquid fraction 33 had not been previously reported elsewhere; however, further investigation is needed to confirm them unambiguously. In silico, in vitro and in vivo bioactivity testing; acetylcholine esterase inhibition, anti-amyloid aggregation and anti-oxidant potential, evaluated the possible target pathways of bio-activities. Different extracts were explored for bioactivity since these compositional changes seem to give differences in biological activity. While the methanolic extract comprised mainly of triterpenes, and showed the highest potency in AChE enzyme inhibition, single triterpenes did not show strong inhibitory activity. Among the triperpenes, asiaticoside showed the highest activity which was also supported by molecular docking studies. At the same time the flavonoids showed strong inhibitory potential as single compounds. On the other hand, flavonoids, triterpenes as well as caffeoylquinic acids seemed to possess good anti-amyloid aggregation activity based on initial screening and qualitative results; however this activity should be further explored quantitatively. According to the present study, synergistic effect of the bioactive compounds were more prominent since when taken alone, the individual compounds did not show strong activities. Thus, it can be recommended to consider the possible use of the whole plant as a medicinal or food ingredient in the diet. However, even though these compounds are present in this plant in abundance, the amount of intake with whole food consumption and the bioavailability in the body should be taken in to consideration. Bioactivities indicated the potential of Centella asiatica as a functional brain food in promoting general neuroprotection as well as arresting multiple disease pathways in neurodegenerative disorders.