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.