IN VIVO CARBON TETRACHLORIDE-INDUCED HEPATOPROTECTIVE AND IN VITRO CYTOTOXIC ACTIVITIES OF GARCINIA HOMBRONIANA (SEASHORE MANGOSTEEN)

Plant materials

The analyzed plant material (G. hombroniana) was collected from Penang Botanical Garden, Malaysia. A voucher specimen; PBGK12 has been deposited at the herbarium of this garden for future reference.

Chemicals and instruments

The solvents; methanol and ethyl acetate of analytical reagent grade used for extraction were purchased from QRёC, Malaysia. Carbon tetrachloride; CCl₄ (toxicant) and silymarin (reference hepatoprotective agent), and solvent; DMSO-d₆ for the experiment of ¹H and ¹³C NMR spectral analysis were purchased from Sigma-Aldrich Co., USA. Silymarin (200 mg/kg) was dissolved in distilled water. The serum activities of alanine aminotranferase (ALT), aspartate aminotransferase (AST) and alkaline phosphate (ALP) were measured by using Hitachi 902 Automatic Chemical Analyser (Japan). The chemicals of cytotoxic activity; DBTRG (glioblastoma), MCF-7 (human breast cancer), U2OS (osteosarcoma) and PC-3 (prostate) cell lines were purchased from Sigma-Aldrich (USA). IR spectra were recorded by KBr using Perkin Elmer (USA) 2000 FT-IR spectrophotometer. The ¹H and ¹³C NMR experiments were performed at room temperature using a Bruker Ascend 500 MHz (¹H) and 125 MHz (¹³C) spectrometer (Bruker Biospin, Switzerland).

Extraction and isolation

The air dried ground defatted bark of G. hombroniana was extracted by Soxhlet extractor with a mixture of polar solvents of 20% methanol in ethyl acetate (1:4). The filtered extract was evaporated and concentrated using a rotary evaporator at 40°C and the obtained concentrated extract then dried by passing oxygen free nitrogen gas. A shiny brown dried 20% methanolic ethyl acetate (MEA) extract was obtained and screened for hepatoprotective and cytotoxic activities.

Hepatoprotective activity

CCl₄-induced toxicity

A slightly modified method of Zakaria et al., (2011) for CCl₄ toxicity assay was used. The rats were randomly divided into six groups each consisting of six rats (n = 6). Group I which served as normal control only received 10% DMSO. Group II served as negative control received 10% DMSO and was intoxicated by induction of 1.0 mL CCl₄/kg body weight diluted in olive oil with a ratio 1:1 intraperitoneally (i.p). Group III served as positive control group was treated with 200 mg/kg silymarin, followed by induction of toxicity with CCl₄. Other three groups; IV, V and VI were administered orally with MEA extract of 50, 250 and 500 mg/kg, respectively and toxicity in them was induced by CCl₄. All the samples and drugs to all the groups were administered orally. Prior to treatment, all the animals were fasted for 48 h under standard laboratory conditions. Each group received respective oral dose of test solution for seven consecutive days. After 3 hours of the solution administration on seventh day, CCl₄ was induced i.p. At the end of induction for 48 h, the rats were fasted again and anesthetized with the help of diethyl ether. For hepatoprotectivity test of the MEA extract, the blood was collected for biochemical assay, and then the animals were sacrificed by cervical dislocation. The livers were removed and preceded for histopathological study.

Biochemical evaluation

Liver damage was assessed by evaluation of serum activities of ALT, AST ALP and measured by Hitachi 902 Automatic Chemical Analyser using standard methods. A 3 mL of the blood was collected by cardiac puncture with disposable sterile syringe and kept at room temperature for 45 min to clot. To separate the serum, it was centrifuged at 2500 rpm at 30 °C for 15 min, and used for the evaluation of ALP, ALT and ASP. Then, the rats were sacrificed, their livers were removed and separated from the body immediately, its weight per 100 g body weight was measured, and fixed in 10% formalin for histopathology studies.

Histopathological studies

The liver was fixed in 10% formalin after dissected out, dehydrated in gradual ethanol (50–100%), cleared in xylene, and then embedded in paraffin wax. The sections were trimmed to approximately 4-5 μm thickness and prepared for staining with hematoxylin and eosin dye for microscopic evaluation. Pathological changes (normal hepatocyte, steatosis, coagulative necrosis, haemorrhage and inflammation) of the liver structure were scored according to the severity of the hepatic injury using modified El-Beshbishy et al., (2010) method.

Cytotoxic Activity

Cytotoxicity assay was conducted according to the modified method of Mosmann (1983). Briefly, a stock solution containing 500 μg/mL of MEA extract was prepared by dissolving 0.5 mg of extract in 1 mL of dimethyl sulfoxide (DMSO). Working solutions of 10, 25, 50, 75 and 100 μg/mL in triplicates were prepared from the stock solution. MCF-7 and DBTRG cell lines were grown in the Roselle’s Park Memorial Institute (RPMI) 1640 medium, U2OS cells in the Dulbecco’s Modified Eagle’s Medium (DMEM), whereas PC-3 were grown in the Ham F12 K medium. These all cells were then supplemented with 10% of fetal bovine serum and 100 units/mL penicillin. They were kept at 37°C in a humidified condition, 5% of C0₂ in air and passage 2 to 3 times per week by light trypsinization. Control cells received the vehicle alone (<0.1%). A cytotoxicity detection kit was used to determine the cytotoxic effect of MEA extract on the aforementioned cell lines. The amount of lactate dehydrogenase (LDH) was measured by using a test kit. Cells were seeded in 24-well plates at 50,000 cells per mL, incubated for 24 h and allowed to attach overnight. Prior to expose cells to MEA extract (10, 25, 50, 75 and 100 μg/mL), the medium was replaced with fresh one containing 2% of fetal bovine serum for up to 72 h. The change in colour generated and its absorbance was determined spectrophotometrically at 490 and 620 nm referenced wavelengths using a microplate reader. The amount of LDH released was calculated according to the formula given in the manufacturer’s protocol. As a control for maximum releasable LDH activity, cells were treated with a lysis reagent (1% of triton X-100) for 10 min, whereas spontaneous LDH released in the supernatant of untreated cultures was measured. Additional cell-free wells containing assay medium alone were prepared which were used as a blank. The results were expressed as mean ± standard deviation.

Statistical analyses

All the numerical data presented as mean ± SEM were analyzed using one way analysis of variance (ANOVA) followed by Dunnett’s post hoc test (p < 0.05) with SPSS software, version 19.0 (SPSS Inc., Chicago, USA).

Hepatoprotective activity

Histopathological study

From Figure 1a, it can be seen that the histology of liver of normal control animals exhibited normal architecture of hepatic cells having well-defined cytoplasm and sinusoid, prominent nucleus, and central vein. However, section of liver tissue of a group of rats treated with 1.0 g/kg CCl₄-treated group (i.p) given in Figure 1b, showed massive coagulative necrosis, haemorrhage and infiltration of inflammatory cells. Section of liver tissue of another group serving as positive control, where the liver was treated with 200 mg/kg of silymarin followed by CCl₄ induction showed preservation of normal hepatocytes (Figure 1c). Rats treated with 50 mg/kg body weight of MEA extract along with CCl₄ intoxication exhibited both necrosis and inflammation (Figure 1d), whereas those treated with moderate dose of 250 mg/kg body weight of MEA extract showed less marked damage and vacuole formation, and the coagulative necrosis was less (Figure 1e). Furthermore, in rats which were treated with a dose of 500 mg/kg body weight, the liver appeared was normal but with mild inflammation, of similar texture and cell arrangement observed for the liver of rats which were treated with 200 mg/kg of silymarin (Figure 1f). So, MEA extract inhibited the CCl₄ induced hepatotoxicity in rats at a dose equal to the half of the standard drug; silymarin. The results of histopathological scoring of the liver tissues pre-treated with the respective test solutions are given in Table 2. These results provided evidence to support biochemical analysis which further suggested that MEA extract has the ability to regenerate the parenchyma cells, to protect the membrane fragility and to decrease the enzymes release into circulation. In histopathological study, the presence of marked necrosis, inflammation, and haemorrhage in CCl₄ intoxicated rats (shown by negative control group) was reduced remarkably when pre-treated either with MEA extract or silymarin. These changes in the liver histology were consistent with the corresponding changes in the enzyme levels, confirming the anti-hepatotoxic effects and inhibition of hepatocellular necrosis by the tested extract.

Figure 1a-f

Hepatoprotective activity of the MEA (20% methanolic ethyl acetate) bark extract of Garcinia hombroniana NH: normal hepatocyte, CN: coagulative necrosis, I: inflammation

Cytotoxic Activity

The results of cytotoxicity test of the MEA extract at a concentrations range of 10–100 μg/mL carried out against MCF-7, DBTRG, PC-3 and U2OS cells, showed that the tested extract is more toxic towards MCF-7 and DBTRG cells causing maximum cells death with 78.7% and 64.3% after 24 h, respectively. The MEA extract showed a significant dose-dependent activity against MCF-7 and DBTRG cancer cells which is also depicted from Figure 2.

Figure 2

Cytotoxic activity of MEA (20% methanolic ethyl acetate) bark extract of Garcinia hombroniana against PC3 and DBTRG cancer cell lines

However, the inhibition did not increase anymore with more time and no further increase up to 72 h was noted. The cell concentration decreased with increasing concentration of the extract. The inhibition of the cancer cell lines might be due to the presence of phenolic compounds such as anthocyanins, benzophenones, flavonoids and xanthones which have been previously reported to be the main constituents of Garcinia species (Gustafson et al., 1992; Kapadia et al., 1994; Masullo et al., 2008; Mackeen et al., 2000; Rukachaisirikul et al, 2006; Zhang et al, 2010; Jamila et al., 2015a; Jamila et al., 2015b).

¹H, ¹³C-NMR and IR spectral analysis

The tested extract was also analyzed for ¹H and ¹³C NMR, and IR experiments. The ¹H and ¹³C NMR spectra (Figures 3 and 4) displayed signals of the regions representing the glycosylated aromatic/phenolic compounds, most probably xanthones, benzophenones, flavonoids, biflavonoids and their glycosides. IR spectrum (Figure 5) exhibited absorption bands at 3255 cm^-1 for O-H, 1646 cm^-1 for C=0 and 1519 cm^-1 for C=C stretching, confirming the presence of phenolic compounds with carbonyl group. Plants which are used for the protection of liver contain an array of bioactive compounds particularly, phenolics such as flavonoids, benzophenones, coumarins, organic acids and xanthones (Kumar, 2012). Xanthones, benzophenones and biflavonoids are the important classes of compounds of Garcinia species which are reported to possess antioxidant activities due to the reducing abilities of their hydroxyl groups by donating hydrogen to free radicals, and thus, prevent the free radical chain reactions and consequently the complications arose from these radicals (Riaz et al., 2011). Adaramoye and Adeyemi (2006), and Adaramoye et al., (2008) in their study on G. kola found that “kolaviron”, a mixture of three biflavonoids (GB1, GB2 and kolaflavanone) has potent hepatoprotecive effects. From the NMR and IR spectral analysis results of MEA extract of G. hombroniana, it could be deduced that its hepatoprotective and cytotoxic activities might be due to the phenolic compounds present in it.

Figure 3

1H NMR spectrum (DMSO-d₆, 500 MHz) of MEA (20% methanolic ethyl acetate) extract of G. hombroniana

Figure 4

13C NMR spectrum of (DMSO-d₆, 125 MHz) MEA (20% methanolic ethyl acetate) extract f G. hombroniana

Figure 5

IR spectrum of MEA (20% methanolic ethyl acetate) extract of G. hombroniana