Psidium guajava Leaf Extracts and Their Quercetin Protect HepG2 Cell Lines Against CCL₄ Induced Cytotoxicity

Abstract

The present study was carried out to evaluate the in vitro cytoprotective effects of Psidium guajava and their isolated quercetin fraction to reduce the CCl₄ (carbon tetrachloride) induced toxicity in HepG2 cell lines (Hepatocellular carcinoma G2). Silymarin was used as a standard drug to compare the protective effects of plant extracts in infected cell lines. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay, cell viability assay, leakage parameters [Aspartate aminotransferase (AST), Alanine aminotransferase (ALT) and Lactate dehydrogenase (LDH)], lipid peroxidation and reduced glutathione (GSH) levels were used to find out the protection of human derived HepG2 cells against CCl₄-induced damage. The levels of cytotoxicity, viability and GSH were reduced. While the activities of AST, ALT, LDH and lipid peroxidation was increased in CCl₄-treated groups. The treatment of P. guajava and their isolated quercetin fractions (100, 200, 300 µg/mL) decreased the elevated levels of all these parameters. The results of the present study suggest that the ethanolic extract of P. guajava leaf and their isolated quercetin fractions can able to reduce the CCl₄-induced cytotoxicity in HepG2 cell lines.

Introduction

Although a lot of carcinogens entail metabolic activation to counter with DNA, the utilization of cells that have endogenous bio-transforming action can diminish few of the problems linked with the usage of exogenous activation mixtures such S9 (Supernatant fraction obtained from liver homogenate by centrifuging at 9000 g for 20 mts) mix [1]. Human hepatoma cell lines appear to be a practical substitute for reviewing genotoxicity or antigentoxicity [2]. Hepatocellular carcinoma G2 (HepG2) cells are deemed as a sensible model for analyzing in vitro xenobiotic metabolism and toxicity to the liver, since they continue majority of focused actions like normal human hepatocytes [3]. Recently, hepato protective activity of plant extracts has been reported by in vitro cytotoxicity assay.

P. guajava leaves belong to the family Myrtaceae. It is used as an important food as well as a medicinal plant in tropical and subtropical countries, therefore its nickname as the poor man’s apple. Many people habitually take P. guajava leaf decoction for its antispasmodic and antimicrobial properties for the treatment of dysentery and diarrhea [4]. P. guajava leaf extracts cure the diseases caused by pathogenic protozoa, fungi, bacteria and viruses [5]. The isolated bioactive compounds like quercetin may contribute significantly to the antioxidant activity of P. guajava leaves [6]. The leaf extract of P. guajava plants can inhibit quorum-sensing and pathogen induced lysis of hepatoma cells [7]. In our previous study, we found that the ethanolic extract of P. guajava leaves and their isolated quercetin fractions have a protective effect against CCl₄-induced oxidative damage [8]. The present investigation was planned to study the in vitro cytoprotective effects of ethanolic extract of P. guajava and their isolated quercetin fractions against CCl₄-induced toxicity in HepG2 cell lines.

Materials and Methods

The fresh leaves of P. guajava were collected from Tiruchirappalli and authentication (PV 001) was obtained from Department of Botany, St. Joseph College, Tiruchirappalli, Tamilnadu, India. The plant leaves were dried under shade at room temperature, and pulverized by a mechanical grinder. The coarse powder was extracted successively with petroleum ether, water and ethanol by a percolation method in a Soxhlet apparatus for 24 h. All the extracts were filtered through Whatman No.41 filter paper and dried-powder samples (9.1, 7.42 and 10.25%, respectively) were obtained by using rotary evaporator and stored in a refrigerator.

The air-dried, powdered samples (50 g) were extracted exhaustively with petroleum ether (60–80 °C). The marc (35 g) was extracted to exhaustion with 95% ethanol to give a greenish mass 3.75 g. The crude ethanolic extract was suspended in water and successively extracted with ethyl acetate (2 × 100 mL) and evaporation of the solvents at reduced pressure gave 1.25 g of ethyl acetate (Greenish brown) was used to isolated and the isolated compound subjected to TLC (Thin layer chromatography) for conformation [9].

HepG2 cells were obtained from National Centre for Cell Science (NCCS), Pune, India and grown on Eagle’s minimum essential medium (EMEM, GIBCO, USA) supplemented with 10% (v/v) heat inactivated fetal calf serum, 2 mM l-glutamine, MEM (Minimum essential medium), non-essential amino acids, gentamycin (50 µg/mL) and penicillin (100 µg/mL) by according to the method of Harries et al. [10]. Cells were passaged every 48–72 h in 1:3 ratio.

HepG2 cells were seeded in 24-well plates at a concentration of 3 × 10⁴ cells/mL and incubated for 48 h at 37 °C under 5% CO₂ to attain confluence. The cells were treated with 0.4% (v/v) CCl₄ for 24 h in 0.25% DMSO (Dimethyl sulfoxide) prepared in serum free culture medium for the following study.

Group I

HepG2 cell line (Control)

Group II

HepG2 cell line + CCl₄

Group III

HepG2 cell line + CCl₄ + ethanolic leaf extract of P. guajava (100 µg/mL)

Group IV

HepG2 cell line + CCl₄ + ethanolic leaf extract of P. guajava (200 µg/mL)

Group V

HepG2 cell line + CCl₄ + ethanolic leaf extract of P. guajava (300 µg/mL)

Group VI

HepG2 cell line + CCl₄ + isolated quercetin fraction of ethanolic extract (100 µg/mL)

Group VII

HepG2 cell line + CCl₄ + isolated quercetin fraction of ethanolic extract (200 µg/mL)

Group VIII

HepG2 cell line + CCl₄ + isolated quercetin fraction of ethanolic extract (300 µg/mL)

Group IX

HepG2 cell line + CCl₄ + silymarin (250 µg/mL)

MTT Assay

The cytotoxic effect of CCl₄-treated HepG2 cells was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay based on the detection of mitochondrial dehydrogenase activity in living cells as per the method of Mosmann [11]. HepG2 cells (1 × 10⁶ cells/mL) were taken into a 96 well plate. Then the cells were pretreated with different concentration of ethanolic leaf extract of P. guajava and their isolated quercetin fraction (100, 200, and 300 µg/mL), and incubated for 1 h. The treated cells were exposed to CCl₄ and incubated at 5% CO₂ and 95% O₂ environment at 37 °C for 24 h. MTT (0.5 mg/mL) was added to the treated cells and then further incubated at same growth conditions for 4 h. The cells were centrifuged for 10 min and the supernatant was removed, 200 μL of DMSO were added into each tube. Absorbance was measured in a microplate reader at 540 nm. Percentage of cytotoxicity was calculated as follows:

$$\%\text{Cytotoxicity}=\frac{\text{Test optical density}}{\text{Control optical density}}\times 100$$

Tryphan Blue Dye Exclusion Viability Test

The standard trypan blue dye exclusion viability test was carried out to understand the effect of ethanolic leaf extract of P. guajava and isolated quercetin fraction on CCl₄-induced cell death in HepG2 cells, according to the method of Darzynkiewiez et al. [12]. The CCl₄-treated cells were kept at room temperature for 1 h and washed twice with PBS (phosphate-buffered saline) and then transferred to sterile tube. Few drops of trypan blue dye (0.4%) were added to the HepG2 cells (total 200 cells) in the eppendrof centrifuge tube and viability was recorded using haemocytometer under a microscope. The percentage of viable cells was calculated as follows:

$$\%\text{Viability}=\frac{\text{Total number of viable cells}}{\text{Total number of viable and nonviable cells}}\times 100$$

AST, ALT and LDH Leakage Assay

The culture medium and cells were collected separately. The medium (0.2 mL) was used for measuring activities of Aspartate aminotransferase (AST), Alanine aminotransferase (ALT) and Lactate dehydrogenase (LDH). The transaminase activities were measured by IFCC (International federation of clinical chemistry) method as a kinetic reaction at 340 nm using kit procured from Agappe Diagnostics, Ernakulam, Kerala, India. The cell leakage of enzyme activities in culture medium was expressed as Unit (U)/L.

Lipid Peroxidative Marker Assay

HepG2 cells were cultured in 24-well plates at a concentration of 3 × 10⁴/mL and treated with 0.4% CCl₄ at various time intervals. After trypsinization, the cells were suspended in 0.5 mL of PBS and sonicated for 10 s. The supernatant was mixed with 0.5 mL of TCA–TBA (trichloroacetic acid–thiobarbituric acid) reagent and heated at 100 °C for 1 h. The extent of lipid peroxidation was quantified by estimating the levels of malondialdehyde (MDA). The absorbance was measured at 535 nm and the formation of TBA reactive substances was calculated using a molar extinction coefficient of 1.56 × 10⁵ M⁻¹ cm⁻¹ [13].

Estimation of Glutathione Levels

The HepG2 cells were treated with CCl₄ and harvested by trypsinization, and resuspended in 0.5 mL PBS to which 0.5 mL of 10% TCA was added. The supernatant was mixed with buffer A (0.1 M sodium phosphate, 5M EDTA, 0.6 mM 5,5′-dithio-bis 2-nitrobenzoic acid, 0.2 mM NADPH) and buffer B (10 U/mL glutathione reductase in 0.1 M sodium phosphate buffer) and its absorbance was read at 412 nm. The GSH levels in the HepG2 cells were determined by using GSH standard [13]. The protein content of the cells was determined using standard bovine serum albumin [14]. The quantification of protein was used to calculate the values of MDA and GSH.

Results were presented as means ± SD. Data were analyzed by one way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT) using a commercially available statistics software package (SPSS^® for Windows, V. 13.0, Chicago, USA). p values < 0.05 were regarded as statistically significant.

Results and Discussion

From our previous study, quercetin was isolated and identified from P. guajava by column and thin layer chromatography [15]. MTT test was used as an indicator of cytotoxicity induced by ethanolic leaf extract of P. guajava and their isolated quercetin fractions in infected HepG2 cells (Fig. 1). The cells were exposed to increasing concentrations of P. guajava extract and their isolated quercetin fractions (100, 200 and 300 µg/mL) showed that the cytotoxicity increased in lower concentration of leaf extract. The 300 µg/mL concentrations induced 32.4% cell death, whereas in lower concentration (100 µg/mL), 78.29% cytotoxicity was observed. The isolated quercetin fractions were found to be cytotoxic activity against HepG2 cells. Isolated quercetin fractions at higher concentration (300 µg/mL) showed 18% cytotoxicity, whereas at lower concentration (100 µg/mL) expressed 71.37% cytotoxicity.

Fig. 1.

Effect of P. guajava leaf extractand their isolated fractions on cell growth inhibition of HepG2 cell line. Means followed by the same letter (a, b, c, d, e, f) are not significantly different (p ≤ 0.05) as determined by DMRT. LE leaf extract; IF isolated fraction

The results of cell viability, leakage parameters such as AST, ALT, LDH, MDA and GSH levels in control and experimental groups were given in Fig. 2 and Table 1. A significant decrease in viability of cells and significant increases in the levels of AST, ALT and LDH was examined in the HepG2 cells exposed to CCl₄ when compared with control. Similarly, toxin treatment caused a significant raise in MDA levels, with a concomitant diminishes in GSH content in HepG2 cells. These cells, when treated with different concentrations (100, 200 and 300 µg/mL) of ethanolic leaf extract of P. guajava and their isolated quercetin fractions showed a significant restoration of the altered biochemical parameters towards the normal compared to CCl₄-treated group and is the effect was dose dependent manner.

Fig. 2.

Effect of P. guajava leaf extract and their isolated fractions on cell viability of HepG2 cell line. Means followed by the same letter (a, b, c, d, e, f) are not significantly different (p ≤ 0.05) as determined by DMRT. LE leaf extract; IF isolated fraction

Table 1.

Effect of P. guajava leaf extract and their isolated quercetin fraction on biochemical contents in CCL₄ infected HepG2 cell line

Experiment	AST (U/L)	ALT (U/L)	LDH (U/L)	MDA (nmol/mg protein)	GSH (nmol/mg protein)
Control	16.82 ± 1.40f	10.32 ± 1.30d	105.45 ± 6.25e	3.12 ± 0.53d	27.24 ± 1.37a
CCl4	50.35 ± 3.81a	24.96 ± 1.97a	210.34 ± 7.47a	7.35 ± 0.64a	10.45 ± 0.99e
CCl4 + P. guajava (100 µg/mL)	44.52 ± 1.49b	21.68 ± 1.93a	184.66 ± 3.58b	5.99 ± 0.53b	14.79 ± 0.80d
CCl4 + P. guajava (200 µg/mL)	36.34 ± 2.98c	18.28 ± 1.23b	145.41 ± 3.11c	4.45 ± 0.43c	19.55 ± 0.61c
CCl4 + P. guajava (300 µg/mL)	29.48 ± 1.96d	13.51 ± 1.27c	118.46 ± 4.34d	3.73 ± 0.27d	23.85 ± 1.80b
CCl4 + isolated quercetin fraction (100 µg/mL)	40.23 ± 3.43b	21.20 ± 1.87a	181.59 ± 6.20b	5.63 ± 0.49b	13.35 ± 0.73d
CCl4 + isolated quercetin fraction (200 µg/mL)	28.54 ± 3.76d	16.13 ± 1.19b	145.12 ± 5.11c	4.72 ± 0.63c	20.69 ± 1.69b
CCl4 + isolated quercetin fraction (300 µg/mL)	17.45 ± 0.93f	11.11 ± 1.06c	111.23 ± 6.01d	3.35 ± 0.24d	26.59 ± 1.72a
Silymarin (250 µg/mL)	19.03 ± 0.99e	11.89 ± 0.93c	113.32 ± 10.21d	3.98 ± 0.19d	25.48 ± 1.76a

Means followed by the same letter (a, b, c, d, e, f) are not significantly different (p ≤ 0.05) as determined by DMRT

AST Aspartate aminotransferase, ALT Alanine aminotransferase and LDH Lactate dehydrogenase, GSH Glutathione, MDA Malondialdehyde

The long-term use of the commercial drug, including silymarin causes cytotoxicity and alters the blood biochemical parameters [16]. Alternative to chemical drugs, plant extracts have several pharmaceutical properties including antioxidants. The chemical composition of P. guajava leaf and their anti-inflammatory activity, free radical scavenging activity and insulin-resistant activity was reported by few studies [17–19]. Recently, Ibrahim et al. [20] noted that the seeds of Convolvulus arvensis showed their antioxidant potential due to the presence of quercetin and other phenolics and flavonoids. Till date, no studies revealed that hepato protective effect of quercetin fraction collected from P. guajava leaf extracts against hepatotoxicity induced by CCl₄ in HepG2 cell line. In current study revealed that P. guajava leaf extract and their quercetin fractions reduced the toxicity in hepatic cells and an effort was undertaken to discover the link between endogenous antioxidant and hepato protective activity.

The mitigation effect of plant extract and their isolated quercetin against the cytotoxic activity of HepG2 cells was evaluated by MTT assay. Plant extracts showed the growth inhibition of HepG2 cells after the incubation for 24 h. The results of the present study were in concurrent with the earlier report, which proved that CCl₄ produced a time-dependent formation of ROS and succeeding lipid peroxidation in HepG2 cells, which was effective after the incubation period of 24 h. Therefore, HepG2 cells were incubated with CCl₄ for 24 h to examine the defensive activity of ethanolic leaf extract of P. guajava and their isolated quercetin fractions against CCl₄-induced toxicity. However, the dosage of treatments influences the cell growth. The cytotoxicity of CCl₄ was increased by lower concentration of extract and isolated quercetin fractions whereas; higher concentration of the extract and isolated quercetin fractions reduced the cytotoxicity in HepG2 cell line. Roy et al. [21] revealed that higher dose of P. guajava leaf extract was more effective than lower doses in CCl₄-induced chronic liver damage. The high mortality rate of cells was caused by the extract and isolated quercetin fractions due to the loss of mitochondria, which is one of the key factors to apoptosis pathway. It is clearly evident that the optimal concentration of a leaf extract activates the apoptotic pathways and results in death of HepG2 injured cell lines.

Apoptosis causes loss of membrane integrity, which enhances the permeability of trypan blue into the CCl₄ affected cells, indicating the cellular damage. The promising defensive nature of ethanolic leaf extract of P. guajava and their isolated quercetin fractions against CCl₄-induced toxicity in HepG2 cell line was evaluated by the protective mechanisms such as leakage of enzymes (AST, ALT and LDH), lipid peroxidation and measurement of intracellular GSH levels. The higher section of intracellular enzymes such as AST, ALT and LDH was observed in CCl₄ injured cells than their controls. Similarly, CCl₄-induced toxin caused lipid peroxidation, and declined GSH content in HepG2 cells. These results conformed the CCl₄ damages the structure and function of cellular membranes and creates the oxidative stress in hepatic cells. These infected cells, when treated with different concentrations of ethanolic leaf extract of P. guajava and their isolated quercetin fractions significantly restored biochemical parameters and protect the cells against CCl₄ injury. CCl₄ generates free radicals, in hepatotoxic cells [22], while P. guajava leaf extract declined the formation of free radicals, which might be due to the regulation of triglyceride synthesis in hepatic cells [21, 23]. However, the CCl₄-induced reduction of GSH results the quinone to bind with cellular macromolecules causing cell death [24]. The current study showed that P. guajava leaf extract and their quercetin fraction enhanced the GSH content in hepatotoxic cell lines.

The possible chief mechanism for the hepato protective effect of ethanolic leaf extract of P. guajava and their isolated quercetin fractions is owing to its capacity to hinder the lipid peroxidation and continuance of glutathione in reduced state by the asset of its antioxidative powers primarily owing to the presence of flavonoids. The isolated quercetin fraction of P. guajava, has the capacity to interfere with the different targets identified as the hallmarks of hepatotoxicity, which makes this molecule, together with several other phytochemicals, a multi-target inhibitor with pleiotropic and synergistic effects in HepG2 cells [25]. The flavonoid, quercetin inhibits the growth and proliferation of cell lines [26]. The previous study of Anju [27], proves the mode of action of Caesalpinia pulcherrima extract and its isolated fraction in same concentration. This may proved in the present study also.

The present results revealed that the ethanolic extract of P. guajava and their isolated quercetin fractions protect the HepG2 cell lines from the oxidative stress induced by the CCl₄. Therefore, the in vitro cytoprotective effects of ethanolic extract of P. guajava and their isolated quercetin fraction diminish CCl₄-induced damage. HepG2 cell is one of the important sensible model to study human hepatocytes in vitro. This study gives a new dimension in hepatoprotective effect of ethanolic extract of P. guajava and their isolated quercetin fraction in CCl₄-induced damage by in vivo.

Compliance with Ethical Standards

Conflict of interest

The authors declare no conflict of interest.