In this study, proteomic techniques were used to analyze the expression of proteins that were released by HepG2 cells in response to treatment with the methanol extract of T. indica fruit pulp. Serum-free DMEM culture media from HepG2 cells grown for 24 hours in the absence and presence of T. indica fruit pulp extract were initially subjected to 2D-GE. The use of serum-free medium was necessary for the proteomic analysis to avoid masking of the proteins released by the cells by those present in FBS. The results of our MTT assays showed that viability of the HepG2 cells was not affected by use of serum-free medium and neither was it significantly different when the cells were exposed to the T. indica fruit pulp extract.
Among the thousands of protein spots that were detected in the 2D-GE profiles of culture media isolated from HepG2 cells grown in the absence or presence of the methanol extract of T. indica
fruit pulp, only seven were found to be altered in expression. Five of the protein spots were found to be those of TTR, ENO1, GDI-2 and ApoA-I (2 isoforms), whilst two spots were not successfully identified Exposure of the HepG2 cells to the T. indica
fruit pulp extract appeared to have caused the increased release of ENO1 and GDI-2 but decreased secretion of TTR and ApoA-I. While the two latter proteins are known to be secretory proteins, ENO1 and GDI-2 are apparently cytosolic proteins 
. However, several earlier studies had also detected the presence of ENO1 and GDI-2 in the culture media of HepG2 cells 
When the differentially expressed proteins were subjected to analysis using IPA, all but ENO1 were found to be interconnected with interactomes in lipid metabolism. ENO1, although more popularly known as a glycolytic enzyme, is apparently a multifunctional protein that also acts as a receptor, activator and regulator molecule 
. Hence, the ENO1 that was detected in the culture media in this study may not be involved in glycolysis. Due to the multiple roles played by ENO1, it is difficult to speculate the reason why the release of the protein was increased when HepG2 cells were exposed to the T. indica
fruit pulp extract.
Interestingly, the three differentially expressed proteins that are involved in lipid metabolism appeared to be commonly associated with the same hormonal regulation, i.e., estradiol, and the homeostasis of cholesterol. GDI-2 functions to translocate prenylated Rab proteins from the cytosol to the membrane to form nascent transport vesicles. The protein also assists the subsequent retrieval of Rab proteins 
, which are key regulators for the transport of lipids and proteins between cell organelles from target membranes 
. To date, approximately 70 Rab proteins had been identified but their specific functions are still largely unknown 
. These include Rab11, whose overexpression has been shown to block the recycling of cholesterol from the endosome recycling compartment to the plasma membrane 
. On the other hand, Rab8 has been shown to assist the redistribution of cholesterol from late endosomes to the cell periphery and stimulate cholesterol efflux through the ABCA1/ApoA-I pathway 
. The increased release of GDI-2 by HepG2 cells when they were exposed to the methanol extract of T. indica
fruit pulp was probably to enable the recycling of Rab proteins that are involved in the cholesterol homeostasis in the cell.
TTR is a protein that is mainly synthesized in the liver and the choroid plexus of the brain 
. Its two main functions are to transport thyroxine and retinol through binding to the hormone and retinol-binding protein, respectively 
. However, a small fraction of plasma TTR (1–3%) is apparently associated with ApoA-I, the major apoprotein found in the anti-atherogenic lipoprotein HDL. ApoA-I is synthesized in the liver (as well as the intestine) and its secretion is believed to be either in a lipid-free/poor or pre-lipidated forms (intracellularly assembled nascent HDL) 
. Ohnsorg 
, Liz 
and their coworkers showed that TTR cleaves the C-terminus of ApoA-I, which is necessary for the transport of lipid-free ApoA-I through the aortic endothelial cells. When HepG2 cells were exposed to the tamarind extract in this study, secretion of both TTR and ApoA-I was reduced by more than 2-fold. The two proteins were also shown to be interconnected via our IPA analysis. This alteration reflects the indirect effects of T. indica
fruit pulp extract in regulating the function of HDL in the reverse transport of cholesterol and also in line with the earlier findings on the cholesterol and triacylglycerol lowering effects of the fruit extract in hypercholesterolaemic hamsters 
and in humans 
In our recent report, we have also described the up-regulated expression of the APOA1
gene by 1.2 fold when HepG2 cells were exposed to the same concentration of T. indica
fruit pulp extract 
. The high ApoA-I mRNA levels that were detected in HepG2 cells upon exposure to the extract as opposed to the low levels of ApoA-I that appeared in the media could be either due to the decrease in the rate of export of the apoprotein or that the mRNA may not be translated into ApoA-I. Our previous study also showed that the T. indica
fruit pulp extract induced the expression of the ABCG5
gene. ABCG5 apparently forms a dimer with ABCG8 to assist the secretion of cholesterol into the bile and its subsequent excretion via the faeces. Therefore, the lower ApoA-I secretion that was detected in the present study suggests that the T. indica
fruit pulp extract may be promoting the excretion of cholesterol via the ABCG5 instead of the ApoA-I transport system. Interestingly, the ABCG5/8-mediated cholesterol excretion and absorption and ABCA-1-mediated cholesterol efflux are apparently controlled by the liver X receptors, LXRs. Earlier studies have apparently shown that proanthocyanidins, which constitutes more than 73% of the total phenolic content of T. indica
, were able to modulate the activation of LXR/RXR 
. Hence, the data of this study, when taken together with that of our earlier report, suggest that the T. indica
fruit pulp extract exerts its lipid lowering effects through the modulation of the LXRs, a conception that was similarly derived from the canonical pathway analysis.
The methanol extract of T. indica fruit pulp altered the release of ENO1, ApoA-I, TTR and GDI-2 from HepG2 cells. Our results provide support on the predicted effect of T. indica extract on cellular lipid metabolism, particularly that of cholesterol.