332 Genes Nutr (2010) 5:331–341 on molecular analysis to support the beneﬁcial effects of the pulp extracts are, however, still lacking. Therefore, in this study, we decided to analyse the global gene expres- sion in response to low concentration of the fruit pulp extracts of T. indica in HepG2 cell line, a widely used in vitro model for human liver hepatocytes. chosen to study the modiﬁcations of gene expression patterns in HepG2 cells in response to treatment with methanol extracts of the T. indica fruit pulp. The concen- tration of the methanol extracts of the T. indica fruit pulp that reduced cell viability by 50% (IC50) was also calculated from the plot. Materials and methods Preparation of T. indica fruit pulp extracts Whole, ripe T. indica fruits were collected from Kedah in the northern region of Malaysia. The voucher specimen of the sample with an identiﬁcation number, KLU 45976, was deposited in the Rimba Ilmu Herbarium, the University of Malaya. The fruit pulp extracts were prepared as previously described by Razali et al. [37]. Brieﬂy, the fruit pulp was separated from the seeds, air-dried and then powdered. The powdered T. indica fruit pulp (2.5 g) was then placed in a conical ﬂask and soaked in 50 ml methanol at room tem- perature for 24 h. The resulting extracts were then ﬁltered, roto-evaporated and redissolved in 10% DMSO. The samples were kept at -20 C until further analysis. Cell culture The human hepatoma cell line HepG2 (ATCC, Manassas, VA, USA) was grown in Dulbecco’s modiﬁed Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (Flowlab, Australia), 1% penicillin (Flowlab, Aus- tralia) and 1% streptomycin (Flowlab, Australia). Cells were maintained in humidiﬁed air with 5% CO2 at 37 C. Cell viability assay A cell viability assay was carried out using 3-(4,5-dimethyl- thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as described by Mosmann [28], with minor modiﬁcations. Brieﬂy, HepG2 cells at a density of 5,000 cells per well were seeded in a 96-well ELISA microplate. The cells were incubated at 37 C in 5% CO2 for 24 h. After 24 h, the pulp extracts, at various concentrations (100–9,000 lg/ml), were added into the wells. The cells were left to grow in the incubator for 48 h. After 48 h, MTT reagent (Merck) was added, and the mixture was further incubated for 4 h. Next, the mixture in each well was removed, and formazan crystals formed were dissolved in 10 ll of 75% isopropa- nol. Spectrophotometric measurement of the mixture was performed in a microplate reader (Bio-Rad) at wavelengths of 570 and 620 nm. A log plot of cell viability (%) against the concentrations of plant extracts were constructed. From the plot, a near non-toxic concentration of the extracts was 123 Treatment of HepG2 cells with methanol extracts of T. indica fruit pulp Conﬂuent HepG2 cells maintained in DMEM were treated with methanol extracts at 300 lg/ml, a non-toxic concen- tration determined from the MTT assay. The cells were then incubated at 37 C for 24 h. As a control, cells were incubated in the absence of the methanol extracts. After 24 h, cells were trypsinized and then precipitated by cen- trifugation at 1,300 rpm for 5 min. Cells were washed with PBS twice before total cellular RNA (tcRNA) was extracted from the cells. Total cellular RNA preparation from the control and treated HepG2 cells tcRNA from both untreated (control) and T. indica-treated HepG2 cells was isolated and then puriﬁed using RNAEasy kit (Qiagen) according to the manufacturer’s instructions. Brieﬂy, the cells were lysed with guanidine thiocyanate- containing buffer (RLT buffer) followed by homogeniza- tion with a QIAshredder. Ethanol (75%) was then added to the mixture, and the mixture was then loaded into RNA spin columns. Following this, the columns were washed with RW1 and RPE buffers that were supplied by the manufac- turer. After washing with a reduced volume of buffer RW1, the RNA was treated with DNase I using RNase-free DNase Set (Qiagen) while bound to the RNAEasy membrane. The DNase I was removed by a second wash with buffer RW1. Then, washing with buffer RPE and elution of RNA were then performed. In the ﬁnal step, the tcRNA was eluted using RNase-free water into collection tubes. The quality of the tcRNA was estimated by measuring the absorbance ratio of 260–280 nm. Values above 1.8 indicate that the tcRNA is of good quality. The integrity of the tcRNA was also ana- lysed using denaturing gel electrophoresis. The integrity of the tcRNA is indicated by the presence of two distinct bands corresponding to the ribosomal 28S and 18S subunits, with the intensity of the larger 28S band approximately twice than that of the smaller 18S band. Preparation of the sense strand DNA for microarray gene expression analysis Gene expression analysis was performed on the Affymetrix Human Gene 1.0 S.T (sense target) arrays according to the