Genes Nutr (2010) 5:331–341 Fig. 7 Validation of the microarray data using real-time RT–PCR. The bar chart shows the gene expression patterns (presented as fold change) of selected signiﬁcantly regulated genes calculated using semi-quantitative RT–PCR, real-time RT–PCR and microarray anal- ysis. The down-regulated genes selected were MTTP, PDE3A and CYP1A1, while the up-regulated genes are GSTA1 and IFIT1. All PCR data were normalized to the intensity of GAPDH as a housekeeping gene. *NQ IFIT1 gene expression was not included in the semi-quantitative RT-PCR from blood. Mutations in the APOA5 gene have been shown to cause hypertriglyceridaemia [35]. In our study, we found that in the methanol extract-treated HepG2 cells, APOA5 gene was up-regulated by 1.9-fold. This could be contributed by procyanidin, a phenolic compound found in grape seed that was able to lower TG levels in rats, up- regulate apoA5 gene expression in mouse liver [12] and was shown to also be present in the T. indica fruit pulp [44]. APOA4 gene codes for the apolipoprotein A-IV (ApoA- IV) which is like cholecystokinin (CCK), a known gas- trointestinal satiety signal proteins whose synthesis and secretion from the gut are stimulated by fat absorption [24]. Apart from the intestine, apoA-IV is also synthesized by the hypothalamus, and the gene has also been shown to be expressed in the liver [23]. In human hepatoma HepG2 cells, APOA4 mRNA was up-regulated when cells were treated with liver X receptors (LXR) agonists. LXRs are oxysterol receptors that can mediate cholesterol homeo- stasis. Treatment of C57B6 mice with a synthetic LXR agonist, T0901317, resulted in signiﬁcant increases in plasma apoA-IV that was associated with an increase in high-density lipoprotein. Examination of both intestinal 339 and liver apo A-IV mRNA revealed speciﬁc increases in liver mRNA only [23]. ApoA-IV had also been shown to be a potent endogenous antioxidant that inhibits lipid per- oxidation [14, 36]. In this study, the gene encoding an ATP-binding cassette sterol transporter, ABCG5, was also up-regulated by the methanol extracts of the T. indica fruit pulp. ABCG5 has been shown to be regulated in the liver and intestine by the LXRs [38, 48]. An overexpression of ABCG5 was found to promote cholesterol excretion through bile and reduce absorption of dietary cholesterol [48]. In addition, muta- tions in the ABCG5 gene were implicated in sitosterola- emia [17, 19, 25], characterized by elevated plasma levels of plant sterols and cholesterol due to increased absorption and decreased excretion of the neutral sterols into biles [3, 4]. The increased circulating levels of these sterols are associated with the deposition of cholesterol in the skin and tendons (xanthomas), and in the walls of the coronary arteries, resulting in premature coronary atherosclerosis. Peroxisome proliferator-activated receptor alpha (PPARa) is a key regulator in hepatic lipid metabolism, and apoA-IV had been identiﬁed as a potential therapeutic target for dyslipidaemia [30]. Oral administration of PPARa agonist KRP-101 in dogs caused a decrease in serum TG accompanied by an increase in serum apoA-IV levels [30]. In addition, administration of a potent and selective PPARa agonist, LY570977 L-lysine decreased TG and increased circulating apoA-V levels in cynomolgus monkeys [41]. Functional PPAR-binding element in the promoter of the human APOA5 gene was found in human, which is somewhat degenerate and non-functional in the corresponding mouse Apoa5 [34]. APOA5 resides on the APOAI/CIII/AIV gene cluster on human 11q23 [33]. Gene coding for the apolipoprotein C-III which is a marker of TG-rich lipoproteins was up-regulated in this study but at a lower fold change compared to that of the APOA5 with APOA5/APOC3 ratio of 1.3. In our study, we found that the crude extracts from the T. indica fruit pulp up-regulated the expression of genes that code for apoA-IV, apo A-V and ABCG5. Since the methanol extracts up-regulated three genes, we postulated that there are components in the crude extracts that may act as PPARalpha agonist which can regulate TG and HDL levels and may eventually be able to be used as a lipid- lowering agent. It is postulated that there may be compo- nents in the extracts that could also regulate cholesterol homeostasis by increasing the expression of the ABCG5 gene that could promote biliary cholesterol secretion and reduce dietary cholesterol absorption which could poten- tially be used to reduce TG levels in blood and help to prevent coronary heart disease. We also found that the crude extract down-regulated the expression of the MTTP gene that codes for microsomal 123