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.