Clone 9 (ATCC number: CRL-1439) was obtained from Food Industry Research and Development Institute, fetal bovine serum (FBS) from Biological Industries, Israel, F-12K Nutrient mixture from GIBCO, Invitrogen, USA, Quick Cell Proliferation Assay Kit (wst-1) for from BioVision, USA, ALT-UV-IFCC kit (BC-0007 V3.0) from Formosa Biomedical Technology Corp., Taiwan, guava leaves were collected from Taiwan Guava Plantation and other chemicals used were analytical grade.

Extraction was done by using three different kinds of solvents; acetone, ethanol and water. Water extraction was done by two different temperatures; room temperature and 60˚C. Washed guava leaves were freeze dried and ground to powder. One gram guava leaf powder was mixed with 10 mL solvent and left to sit for 24 hours. The supernatant obtained was filtered (Whatman no.1), then evaporated by rotary evaporator at 40˚C and dried by freeze dryer [10].

The experiments were performed in clone 9 cells, a hepatocyte cell line derived from normal mice. The cells were grown in F12K medium supplemented with 10% heatinactivated fetal bovine serum (FBS) and 1% penicillinstreptomycin. Culture incubation was done at 37˚C with 5% CO₂. The cells were grown as monolayer and subcultures were performed with trypsinization using 0.05% trypsin in EDTA when cell growth reached confluence.

Cell viability was measured using a colorimetric assay for 96-well plates with 2-(4-iodophenyl)-3-(4-nitrophenyl)- 5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) reagent. Cells were added to 96-well plates in a concentration of 1 × 10⁵ cells/mL and cultivated for 24 hour. For alcoholinduced injury model, clone 9 cells were treated with 1%, 5%, 10% and 15% ethanol for 30 minutes and 60 minutes. Cell viability was measured with wst-1 assay. For cytoxicity and hepatoprotective assay, clone 9 cells were treated with 20 µL guava leaf extract (50, 100, 200, 400, 500, and 600 µg/mL) and 180 µL medium for 24 hours or guava leaf extract for 24 hours and alcohol for 30 minutes. To measured cell viability, the guava leaf extract or ethanol (liquid phase in 96 well plate) were removed for ALT assay and 10 µL of wst-1 and 100 µL of medium were added into cells and cells were incubated for an additional 2.5 hours. Absorbance was measured on ELISA plate reader (Epoch, BioTek Instruments, USA) with a test wavelength at 450 nm. Cell viability (%) was calculated by (cell number of sample group/cell number of control group) × 100%.

ALT (EC 2.6.1.2) was used as cell damage indicator. ALT was measured using commercially available reagent kit (BC-0007 V3.0, Formosa biomedical technology corp., Taiwan). For cytotoxic and hepatoprotective assay, after the cells were treated with guava leaf extract for 24 hours or ethanol for 30 minutes, 50 µL of the solution was removed into new 96-well plate and 800 µL R1 reagent was added into these solutions. After incubation in 37˚C for 5 minutes, 200 µL R2 reagent was added. Absorbance was measured in 340 and 405 nm (A = Abs 405-Abs 340) as A1 and after one minute, the absorbance was measured again in the same wavelength as A2. ALT value was determined by

Every analysis was done in three replications and statistical analyses were done by using SPSS 13.0. Statistical analyses that conducted were analysis of variance (ANOVA) using Duncan’s multiple range comparison post hoc with p < 0.05 considered significant.

After alcohol consumption, about 70% of alcohol will enter liver metabolism [10], and different cell lines will have different tolerance on alcohol concentration. This preliminary experiment was done to know optimum concentration and exposure time for ethanol-induced heaptotoxicity model on Clone 9 liver cells.

Results that are shown in Figure 1 suggest that cell survival rate declines as the alcohol concentration increases. Cell viabilities of Clone 9 cells treated for 30 minutes exposure time and 1%, 5%, 10%, and 15% of the ethanol concentrations were 107%, 79%, 37%, and 11%, and for 60 minutes were approximately 109%, 78%, 30%, and 12%, respectively. Hepatotoxicity caused by ethanol was dose-dependent, but different exposure times, 30 and 60 minutes, showed no apparent negative influence on cell viability. Therefore, for the purpose of the experiment 5% of the ethanol concentration with 30 minutes exposure time was used as alcohol induced liver cell injury model to analyze the protective effects of guava leaf extracts on alcohol induced liver injury on clone 9.

The effects of ethanol have been suggested to be a result of the enhanced generation of oxy-free radicals during its oxidation in liver. The peroxidation of membrane lipids results in loss of membrane structure and integrity. The decrease in activity of antioxidant enzymes superoxide dismutase, glutathione peroxidase are speculated to be due to the damaging effects of free radicals produced following ethanol exposure or alternatively could be due to a direct effect of acetaldehyde, formed by oxidation of ethanol [11].

Cytotoxicity assay was done to observe different guava leaf extract effect on healthy Clone 9 cell, to determine whether or not it can cause significant damage in healthy cells. After being cultured for 24 hours, ethanol and acetone extracts in high concentrations showed cytotoxic effects on clone 9 liver cells which is shown as reduced

cell viability by wst-1 assay (Figure 2). Water extract of guava leaves in 500 μg/mL concentration resulted in cell survival rate about 94%, and no toxicity occured at concentration of 500 μg/mL or lower, significantly. However, the cell that was treated with 400 μg/mL and 200 μg/mL hot water extracts has cell viability about 92% and 96%, but no significant toxicity occur on clone 9 liver cell in concentration of 400 μg/mL or lower.

Aside from the cell viability assay, this experiment also measured ALT value. When certain types of cells are damaged, they may effuse enzyme into the blood and alanin aminotransferase (ALT) is one of these enzymes; this indicates cell damage [12]. The upper range of normal in the present study was taken as less than 40 U, because this value is widely used by many clinicians as the upper normal [13]. ALT values from four different solvent extractions of the guava leaf extract were shown in Figure 3. Results shows that ALT increases extract concentrations increase, this can also be referred to as dosedependent cell damage caused by guava extracts on clone 9 liver cells. Cells that were treated by acetone extract at 200 μg/mL or below and ethanol extract at 500 μg/mL or below shows ALT value within normal range, while in the water and hot water extracts of guava leaves, transglutaminase enzyme activity (ALT) did not increase or exceed normal level.

This experiment aims to observe pre-protective effect of the different extraction of guava leaf extract for the alcohol-induced liver injury. The experiment was done by treated clone 9 liver cell with guava leaf extract for 24 hours, then 5% ethanol was added for 30 minutes to induce injury thereafter cell viability was measured. The survival rate of 5% alcohol induced injury on clone 9 liver cells was about 79% with ALT value and this result was used as the control group.

In this study, it was seen that administration of guava leaf extracts on ethanol-induced hepato-toxicity has potential as hepatoprotective agent in lower concentrations. Higher hepatoprotective effects were revealed by ethanol and hot water extracts. However, in high concentrations, cell viability of ethanol extract tends to decrease. These findings may be due to the cytotoxic ability of these guava leaf extracts, specifically acetone and ethanol extracts (Figure 4). The maximum protection against ethanol-induced hepatic aberrations was achieved by hot water extract.

Hot water extract of guava leaf can be considered to be an effective hepatoprotective herbal as it ameliorated the

damage cause by ethanol and lower cytotoxicity effect. The probable mechanism of guava leaf protective action against ethanol-induced hepatocellular metabolic alterations could be by its antioxidant properties, which may neutralize the effect of ethanol. Boiled water extract of guava leaf possess strong antioxidant properties especially in free radical-scavenging due to its high content of feluric and gallic acid [14]. In addition, concentration when administering of guava leaf extract must also considered since cytotoxic effect can be deleterious to cells in high concentrations.