INTRODUCTION:

Hepatocellular carcinoma is a leading cancer globally whose incidence is increasing, and is closely related to advance liver disease. The diagnosis of hepatocellular carcinoma can uniquely be made on characteristic multiphase contrast based cross-sectional imaging rather than a strict need for tissue sampling. Despite advances in medical, locoregional and surgical therapies, hepatocellular carcinoma remains one of the most common causes of cancer-related death globally¹. Most of the available drugs, can act only on part of the pathogenic process and only to a partial extent. Demand for medicinal plants is increasing worldwide due to the growing recognition of natural products, being non-toxic, and more efficacies, easily available at affordable prices. In the series of various medicinal plants, Punica granatum (Pomegranate) is well known for its folkloric use.

Punica granatum (Pomegranate) belongs to family Punicaceae and is unique among plants. Various parts of pomegranate including roots, leaves, flowers, rind, seeds and the reddish brown bark are used medicinally. Pomegranate bark and root contains several alkaloids including isopelletierine that fights against tapeworms. Pomegranate bark, leaves, immature fruit and fruit rind extracts are given to combat diarrhea, dysentery and hemorrhages, whilst powdered flower buds act as a remedy for nose bleeding^2,3.

Pomegranate is used as a gargle for sore throat, and it is applied to the skin to treat hemorrhoids. It has immuno-stimulatory, anti-oxidant, anti-inflammatory anti-diabetic and anticancer. It is widely used in treating certain types of cancer including leukemia, breast, prostate and colon cancer, dysentery, diarrhea, excessive bleeding, intestinal worms and parasites⁴. Pomegranate fruit rind extract possessess a wide range of beneficial as well as pharmacological properties. Hence in the present study, an attempt has been made to evaluate the antioxidant properties of fruit rind extract against liver cancer using HepG2 cell line.

MATERIALS AND METHODS:

Preparation of Punica granatum (Pomegranate) fruit rind extract:

Matured Punica granatum (Pomegranate) fruit were collected and authenticated. Punica granatum (Pomegranate) fruit rind were washed and dried in a hot air oven at 40°C and subsequently ground into powder in an electrical grinder, which was stored in an airtight brown container at 5°C until further use. The powdered rind were delipidated with petroleum ether (60-80°C) for 24 hrs. It was then filtered and soxhalation was performed with 95% ethanol. Ethanol was evaporated in a rotary evaporator at 40-50°C under reduced pressure. The yield was around 14.5 % of dry weight.

IN VITRO STUDIES:

HepG2 cells were procured from National Center for Cell Science (NCCS), Pune and the cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 0.1% Antibiotic-antimycotic solution which contains 100 U/mL penicillin, 30 μg/mL streptomycin and 20 μg/mL gentamycin in a 5% CO₂ atmosphere (Thermo Scientific, USA). Cells at 85% confluence were used for all the assays.

Cell viability assay.

The proliferation of HepG2 cells was assessed by MTT assay, (Safadi et al., 2003).

Experimental protocol

Group 1: Control HepG2

Group 2: HepG2 cells treated with 30µM of extract for 24 hours

Group 3: WRL-6 Normal cells.

Isolation of Protein in HepG2 cells.

After treatment of HepG2 cells for 24h and 48h with extracts were prepared by sonication in 50 mMTris, 5 mM (EDTA), 10 μg/mL of phenyl methyl sulfonyl fluoride (PMSF), and pH 7.6. The cell debris was removed by centrifugation at 4000 rpm for 5 min at 4°C. The levels of Protein content of the supernatant was determined by the method Lowry et al (1951). The intracellular concentrations of SOD, GPx and Catalase were determined in the supernatant.

Antioxidant Assays:

An activity of Superoxide dismutase was determined by the method of Marklund and Marklund⁵, 1974. The activity of catalase was assayed by the method of Aebi⁶ (1984). The amount of reduced glutathione (GSH) in the samples were estimated by the method of Boyne and Ellman⁷(1972).

Malate Dehydrogenase Assay:

Malate dehydrogenase (MDH) catalyzes the interconversion of L-malate and oxaloacetate using nicotinamide adenine dinucleotide (NAD) as a coenzyme⁸.

Statistical analysis:

The statistical package SPSS (Statistical Package for Social Science), version 10.0 for windows are used for statistical analysis. Results are expressed as mean ± SD. Multiple comparisons of the significant ANOVA were performed by Duncan’s multiple comparison tests. A p- value of <0.05 was considered as statistically significant.

RESULTS AND DISCUSSION:

Figure 1 shows the MTT assay in Punica granatum fruit rind extract on HepG2 cells. Figure 1 Protective effects of pomegranate rind extract on HepG2 cells for 24 & 48 hrs. Results are expressed as % of cellular death in terms of MTT reduction. IC 50 value found to be 200µg. The different Concentration of pomegranate rind extract is expressed in µg/ml.

Figure 1 shows the MTT assay in Punica granatumfruit rind extract on HepG2 cells.

The Protective effects of pomegranate rind extract on HepG2 cells for 24 & 48 hrs.

IC 50 value found to be 200µg/ml (Figure 1). The morphological changes of extracts on HepG2 cell for 24 hrs should show a normal morphological structural similarly to extract of 200µg/ml.

Figure 2 Morphological changes in HepG2 liver cancer cells. Cells were treated with extracts after 24 hours as captured using microscope at 10×10 magnification. Control represents the cells incubated with medium only. (A) Control (B) 5µg/ml Crude Extract Treated (C) 10 µg/ml crude extract treated (D) 15µg/ml crude extract treated.

Figure 2 Morphological changes in HepG2 liver cancer cells.

Figure 3: Status of superoxide dismutase (SOD) activities in hepatoma cell line (HepG2) and in normal cell line (WRL-68) with and without extract.

Figure 3: Status of superoxide dismutase (SOD) activities in hepatoma cell line (HepG2) and in normal cell line (WRL-68) with and without extract. Data are presented as mean±standard deviation, SD from triplicate wells. *SOD activities were significantly lower (p<0.05) in HepG2 cells at 200 and 500µg/ml of extract compared to untreated hepatoma cell line, while the changes seen in normal culture were not significant (p >0.05) at all in the concentrations of extract when compared to the untreated normal cells. # SOD activities were significantly higher in HepG2 cells (p<0.001) when compared to normal cell line without extract treatment.

Figure 3: represents the status of SOD activities in normal and liver cancer cell lines (HepG2) with and without treatment of extract. In untreated normal and liver cancer (HepG2) cell lines, SOD activities were 77.81±3.8unit/mg protein and 157.37±22.8unit/mg protein respectively. SOD activity was significantly reduced (p<0.05) by 26.6% and 72.32% in HepG2 cell line when 200µg/ml and 500µg/ml extracts were introduced in the cultures respectively. Changes observed in normal cultures were not significant.

Figure 4: Status of glutathione peroxidase (GPx) activities in HepG2 and in normal cell lines (WRL-68) with and without treatment.

Figure 4: Status of glutathione peroxidase (GPx) activities in HepG2 and in normal cell lines (WRL-68) with and without treatment of Fruits rind extract. Data are presented as mean±SD from triplicate wells. GPx activities were significantly reduced (p<0.05) in HepG2 cells at all concentrations of Fruits rind extract when compared to the untreated culture. GPx activities were significantly reduced (p<0.05) in normal cell at 200 µg/ml extract compared to the untreated culture.

Figure 4 represents the status of GPx activity in normal (WRL-68) and liver cancer (HepG2) cell lines with and without treatment of extract. In untreated WRL-68 and HepG2 cell lines, GPx activities were 0.651±0.18 unit/ mg protein and 0.529±0.09 unit/mg protein respectively. GPx activities were significantly reduced (p<0.05) by 77.16%, 87.35% and 71.05% in HepG2 cell line at 100, 200 and 500 µg/ ml of extract respectively. In normal culture, GPx activity was significantly reduced (p<0.05) at 200µg/ml extract when compared to the untreated culture.

Figure 5: Status of catalase (CAT) activities in HepG2 and in normal cell lines WRL-68 with and without extract.

Figure 5: Status of catalase (CAT) activities in HepG2 and in normal cell lines WRL-68 with and without extract. Data are presented as mean±SD from triplicate wells. CAT activities were significantly reduced (p< 0.05) in HepG2 cell line at 200 and 500µg/ml extract when compared the control.

Figure 5 represents the status of CAT activities in normal (WRL-68) and liver cancer (HepG2) cell lines with and without treatment of extract. In untreated cells and HepG2 cell lines, CAT activities were 0.86+0.035 unit/mg protein and 1.00+0.19 unit/mg protein respectively. CAT activities were significantly reduced (p<0.05) at 200 and 500 µg/ml of extract in HepG2 cell lines when compared to the untreated culture. In normal cell line, CAT activities were not significantly reduced (p>0.05) at all concentration of extract compared to the untreated culture.

Figure 6: Status of GRD activity in HepG2 and in normal cell lines with and without extract.

Figure 6: Status of GRD activity in HepG2 and in normal cell lines with and without extract. Data are presented as mean±SD (triplicate wells). Changes in glutathione content was not significant (p>0.05) in both cell lines at all concentrations of extract when compared to untreated cultures.

Figure 6 depicts the GRD activity in normal (WRL68) and liver cancer (HepG2) cell lines with and without treatment of extract. GRD activity were 36.91±0.79µM and 36.95±0.82µM in normal and in liver cancer cell (HepG2) lines respectively. Addition of extract had no effect on both normal and liver cancer cell lines.

Figure 7: Status of malonaldehyde (MDA) in HepG2 and in normal cell lines with and without treatment of extract.

Figure 7: Status of malonaldehyde (MDA) in HepG2 and in normal cell lines with and without treatment of extract. Data are presented as mean±SD (triplicate wells). MDA levels were not significantly different in HepG2 and WRL-68 cell lines at all concentrations of extract when compared to control (untreated culture).

Figure 7 depicts the status of MDA in HepG2 and in normal cell lines with and without treatment of extract. In untreated WRL-68 and HepG2 cell lines, MDA levels were 73.4±0.75µM and 73.94±0.37µM respectively. Changes in MDA levels were not significant both in HepG2 and normal cell lines at all concentrations of extract when compared to the respective untreated culture.

DISCUSSION:

Chemotherapy, radiation therapy, and surgery have been used for cancer treatment as a standard. However, these therapies have not been fully effective and the side effects are of concern. Most antitumor agents currently used in chemotherapy are toxic to normal cells and cause weakening of the immune system. Therefore, there is an increased demand to seek out new anticancer agents with lower side effects than those of current agents. Medicinal plants are of immense value to the health and communities. The therapeutic value of the medicinal plants lies in some chemical substances that are produced as secondary metabolites with definite physiological action on the human body. Medicinal plants are the reservoirs of ecologically derived secondary metabolites such as phenolic acids, alkaloids, flavonoids, saponins, anthocyanins and hydroxycinnamic acid derivatives. Among the secondary metabolites, the phenolic classes have gained extensive attention in recent years due to their physiological functions including free radical scavenging, antidiabetic, anticarcinogenic and anti-inflammatory effects⁹_.

Oxidative stress (OS) produces toxic metabolites which can initiate and promote cancers^10,11. Consumption of polyphenols and flavonoids are beneficial for the prevention of cardiovascular, inflammatory, and other diseases by preventing OS that induces lipid peroxidation in arterial macrophages and in lipoproteins _12,13. The presence of antioxidants has been reported in Punica granatum. Punica granatum contains some species of flavonoids and anthocyanidins (delphinidin, cyaniding and pelargonidin) in its seed oil and juice and shows antioxidant activity three times greater than green tea extract^{14, 15}.

Earlier study^16,17 has shown the anti- tumour effect of extract by inhibiting proliferation and inducing apoptosis in human hepatoma cell line, HepG2. In an attempt to elucidate the baseline levels of antioxidant enzyme status in HepG2 cell line, we measured the activities of SOD, GPx, CAT, and GSH and MDA contents. We also explored the possible antitumor and antioxidant effects of rind extract, in HepG2 cell line by measuring the endogenous antioxidant levels in this cell line when treated with rind extract. It has been highly suggested that in the process of carcinogenesis excessive accumulation of reactive oxygen species may play an important role in causing oxidative damage. In an attempt to defend the situation, antioxidant enzymes (SOD, GPx and CAT) may be elevated or reduced in these cells¹⁸. This study showed that SOD activity was higher in hepatoma cell line (HepG-2) by 2fold compared to control, which may reflect the higher superoxide radicals in the former. This is comparable to the study of (Erel, 1997)¹⁹whereby SOD activity in HepG-2 cell line was 2.8fold higher than normal cell line. However, other antioxidant enzymes, CAT and GPx activities in HepG2 cell line were not significantly different when compared to normal cell line.

GSH and MDA contents were also not significantly different in hepatoma cell line when compared to the respective control. An increase of 4.3-fold, 2.9-fold and 1.4-fold of CAT, GPx activities and GSH content respectively in hepatoma cells was reported by Kono, 1982. However, low levels of CAT, and GSH have been observed in chronic liver disease such as alcoholic liver disease, viral hepatitis and liver cancer. Such observations could be due to modulations of the defence system in the tumour cells in relation with their growth and maintenance. Chidambaram et al 2002, had noted 90 - 70% decrease in the level of antioxidant enzymes, SOD and CAT respectively and an absence of GPx activity in human hepatoma cell line HepG2.

The findings with excessive accumulation of oxygen free radicals and DNA damage in hepatoma cells, are considered to be important in carcinogenic mechanism. This study also showed that the antitumour effects of Fruits rind extract may be exhibited by influencing the status of antioxidant enzymes in HepG2 cells. SOD, GPx and catalase activities which were higher in untreated hepatoma cells, were reduced significantly when fruits rind extract at concentrations of 200-500 ug/ml was introduced into the culture. Fruits rind extract may be taking over the role of antioxidants SOD, GPx and catalase in eliminating the superoxide radicals and hydrogen peroxide accumulation in HepG2 hepatoma cell line. However, Fruits rind extract had no effect on the levels of GSH and MDA contents of HepG2 cells at all concentrations.

In summary, this study suggests the possible chemoprotective role of extract by eliminating superoxide radicals and hydrogen peroxide in the liver cancer cell line HepG2 thus replacing the activities of SOD, GPx and CAT. Fruits rind extract had no effects on the activities of SOD, GPx and CAT in the normal cell line. Future studies should include the mechanism by which extract acts as exogenous antioxidants in scavenging free radicals.

CONCLUSION:

The results of the present study indicate the antioxidant activity of Punica granatum fruit rind extract which might be attributed to its phytochemical components most especially flavonoids and phenols. This property makes it a potential agent that might be used to prevent and manage the complications of free radical medicated diseases such as cancer.

REFERENCES:

1. Hartke J, Johnson M, Ghabril M. The diagnosis and treatment of hepatocellular carcinoma. Semin Diagn Pathol 2017; 34(2):153-159.

2. Murthy KNC, Jayaprakasha GK, Singh RP. Studies on antioxidant activity of pomegranate peel extract using in vivo models. Journal of Agricultural and Food Chemistry 2002); 50: 4791–4795.

3. Noda Y, Kaneyuki T, Mori A, Packer L. Antioxidant activities of pomegranate fruit extract and its anthocyanidins: delphinidin, cyanidin, and pelargonidin. Journal of Agricultural and Food Chemistry 2002; 50, 166–171.

4. Kaushik V, Kulkarni. A Complete Overview on Profile and Medicinal uses of Punica grantum (Pomegranate) and its Health Benefits, International Journal for Pharmaceutical Research Scholars 2018, 7(1).

5. Marklund S, Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 1974 16; 47(3):469-74.

6. Aebi H. Catalase in vitro. Methods Enzymo.1984; 105: 121-126.

7. Boyne AF Ellman GL. A methodology for analysis of tissue sulfhydryl components. Anal. Biochem 1972, 46 (2), 639-53.

8. Bleile DM, Foster M, Brady JW, Harrison JH. Identification of essential arginyl residues in cytoplasmic malate dehydrogenase with butanedione. J Biol Chem. 1975; 25; 250(16):6222-7.

9. Manthey JA, Biological properties of flavonoids pertaining to inflammation. Microcirculation 2000, 7 (1), S29–S34.

10. Karageuzyan KG. Oxidative stress in the molecular mechanism of pathogenesis at different diseased states of organism in clinics and experiment. Curr Drug Targets Inflamm Allergy. 2005; 4(1):85-98.

11. Ohshima H, Tazawa H, Sylla BS, Sawa T. Prevention of human cancer by modulation of chronic inflammatory processes. Mutat Res 2005; 591(1-2):110-22.

12. Noda Y, Kaneyuki T, Mori A, Packer L. Antioxidant activities of pomegranate fruit extract and its anthocyanidins: delphinidin, cyanidin, and pelargonidin. J Agric Food Chem. 2002; 50(1):166-71.

13. Miguel G, Dandlen S, Antunes D, Neves A, Martins D. The Effect of Two Methods of Pomegranate (Punica granatum L) Juice Extraction on Quality during Storage at $4^\circ$ C. J Biomed Biotechnol. 2004; 2004(5):332-337.

14. Mori-Okamoto J, Otawara-Hamamoto Y, Yamato H, Yoshimura H. Pomegranate extract improves a depressive state and bone properties in menopausal syndrome model ovariectomized mice. J Ethnopharmacol 2004; 92(1):93-101.

15. Seeram NP, Schulman RN, Heber D. Pomegranates: Ancient Roots to Modern Medicine. Boca Raton: Taylor and Francis Group 2006; 5–8.

16. Kang HS, Kim KR, Jun EM, Park SH, Lee TS, Suh JW, et al. Cyathuscavins A, B, and C, new free radical scavengers with DNA protection activity from the Basidiomycete Cyathusstercoreus, Bioorg Med Chem Lett 2008; 4047-50.

17. Lü JM, Lin PH, Yao Q, Chen C, Chemical and molecular mechanisms of antioxidants: Experimental approaches and model systems, J Cell Mol Med 2010, 840-60.

18. Yunfeng Li; Changjiang Guo; Jijun Yang; Jingyu Wei; Jing Xu. and Shuang Cheng. Evaluation of antioxidant properties of pomegranate peel extract in comparison with pomegranate pulp extract. Food Chemistry 2006,96; 254-260.

19. Erel O, Kocyigrit A, Aktepe S, Bulut V. Oxidative stress and antioxidant status of plasma and erythrocytes in patients with malaria. Clin Biochem1997; 30: 631-639.

Received on 05.04.2019 Modified on 23.05.2019

Research J. Pharm. and Tech. 2019; 12(10): 4719-4723.

DOI: 10.5958/0974-360X.2019.00813.8