Characterization secondary metabolites of butanol fraction ofTragia hispida (THB)
Although there chemical composition ofT. hispida , is not known
it has been shown that related species such as T. involucrata is
rich in flavonoids, particularly in several glycosides and derivatives
of quercetin 15,21. Flavonoids have shown to inhibit
sPLA2 activity, and has shown to inhibit sPLA2 in synovial fluid but not
from pancreatic fluid, showing that T.hispidia is most likely to
have compounds that inhibit sPLA2 II group of enzymes22. The chemical composition of T. hispida (THB
fraction) was characterised using thin layer chromotagraphy (TLC), which
showed several poorly resolved yellow, orange and blue bands, indicating
that it was likely to contact a mixture of flavonoid and phenolic
compounds (Figure 2).
Since it was difficult to identify the possible compounds based on the
TLC profile alone, HPLC was carried out to confirm the presence of those
secondary metabolites such as flavinoids and phenolic compounds. The
diode array UV showed the spectra of the four major peaks in HPLC
profile. The peaks at 3.207 min ( λmax 222 nm, 272 nm) and 7.972 min
(λmax 224 nm, 272 nm) were most likely to represent phenolics and that
the peaks at 11.883 min (λmax 276 nm, 366 nm) and 16.898 min ( λmax 254
nm, 370 nm) most likely to represent flavonoids (Figure 3).
SRB
assay for cytotoxicity of the aqueous extract of Tragia hispida(THA)
Although the THB fraction gave the highest sPLA2 inhibitory activity, we
sought to test the cytotoxicity of the aqueous extract of T.
hispida (THA), as this plant is prepared as an aqueous extract in
traditional medicine in Sri Lanka and THA is most likely to represent
what is consumed in practice. The cytotoxicity was assessed using the
SRB assay, comparing the cytotoxicity with that of Paclitaxel. THA was
used at concentrations of 25, 50, 100, 200 and 400 μg/mL while
Paclitaxel was used as a concentration of 1.25, 2.5, 5, 10, 20 μg /mL.
The viability of MRC-5 cells at different concentrations at different
time points with THA and Paclitaxel is shown in figure 4. The
differences in the viability of the MRC-5 with the different
concentrations of the drugs at different time points was assessed using
the Holm-Sidak method which corrects for multiple comparisons which
showed that the cell viability was significantly higher at 72 hours in
those treated with THA compared to those treated with Paclitaxel
(p=0.0005). The IC50 values for inhibition of cell
growth, at 24, 48 and 72 hours were assessed and are shown in table 2.