## 3 Results  
### Lipid Accumulation by Differentiated hBM-MSCs  
Prior to assessing its anti-adipogenic properties, the cytotoxicity of loliolide was tested on hBM-MSCs via MTT assay. the results show that, with up to 10 uM loliolide treatment, there was no loss of viability (Figure 1B). The first significant decrease in viable cell amount was at 25 uM. Therefore, further loliolide treatment was carried out using 10 uM as the highest concentration. Adipogenesis of hBM-MSCs in response to loliolide treatment following adipogenic differentiation medium administration was evaluated at Day 10 of differentiation. Maturation of adipo-induced cells into adipocytes was confirmed by the lipid accumulation as intracellular lipid droplets. Most of the adipo-induced hBM-MSCs had lipid droplets displayed by positive Oil Red O stain compared to non-differentiated group (Figure 1C). Loliolide decreased the lipid accumulation dose-dependently starting at 5 \(\upmu\)M concentration. Inhibitory effect of loliolide in intracellular triglyceride droplet levels was further confirmed by the quantification of Oil Red O stain. Loliolide-treated adipocytes displayed 8.86% and 29.88% less lipid accumulation, at 5 and 10 \(\upmu\)M treatment, respectively, relative to the untreated control. Adipogenesis-induced accumulation of intracellular fat was also examined by fluorescence staining of the perilipin in adipocytes. Perilipin is a coating protein that bounds to the intracellular lipid droplets and has critical roles during adipogenesis. Perilipin expression in MSCs was reported to be upregulated during adipogenesis [23].  
Figure 1: The effect of loliolide on intracellular triglyceride accumulation in adipo-induced hBM-MSCs. (**A**) Chemical structure of loliolide. (**B**) The effect of loliolide treatment on the hBM-MSC viability after 24 h assessed by MTT assay. Viability of the hBM-MSCs were given as a relative percentage of loliolide-untreated group. Different letters (a–e) above the sample groups indicate that the difference between each group is statistically significant (\(p<0.05\)) compared to the untreated differentiated control. (**C**) The hBM-MSCs were differentiated into adipocytes in six-well plates with or without loliolide treatment (1, 5, and 10 \(\upmu\)M). One group was left untreated and non-differentiated as the negative control. At Day 10 differentiation, intracellular triglyceride stores of mature adipocytes and non-differentiated hBM-MSCs were stained with Oil Red O. The triglyceride levels stored inside adipocytes were quantified as the amount of retained Oil Red O which was calculated via colorimetric measurement. Numerical data are \(\pm\)SD (\(n=3\)). Different letters (a–d) above the sample groups indicate that the difference between each group is statistically significant (\(p<0.05\)) compared to the untreated differentiated control. (**D**) Images of fluorescence stained hBM-MSCs at incubation Day 10 after adipogenesis inducement (except undifferentiated untreated group which was fed growth medium only), stained with FITC conjugated anti-perilipin-1 antibody (red) and DAPI (blue) to highlight the nuclei. Scale bar, 100 \(\upmu\)m.  
As shown in Figure 1D, hBM-MSC adipocytes displayed over-expression of perilipin at Day 10 of differentiation. However, loliolide treatment (10 \(\upmu\)M) significantly inhibited perilipin expression, hence resulting in suppressed lipid accumulation.