Anti-hepatitis B viral activity of Phyllanthus niruri L ( Phyllanthaceae ) in HepG 2 / C 3 A and SK-HEP-1 cells

Purpose: To investigate the effectiveness of an ethanol extract of Phyllanthus niruri against hepatitis B viral (HBV) infection in human HepG2/C3A cells. Methods: An ellagic acid-rich ethanol fraction was obtained from P. niruri (Euphorbiaceae) by extraction and thin-layer chromatography. The anti-HBV activity of the fraction was evaluated in vitro against HepG2/C3A cells. The physicochemical characteristics of the fraction were assessed by nuclear magnetic resonance (H and C-NMR). Results: The isolated active compound showed a half-maximal inhibitory concentration (IC50) of 120 μg/mL. Ellagic acid had no effect on HBV DNA replication at the concentrations evaluated, and did not inhibit the reproduction of HBV. However, the ethanol fraction inhibited the growth of HBV-infected HepG2/C3A cells. Conclusion: The findings suggest that the ethanol fraction of P. niruri inhibits HBV, and that the active component is not ellagic acid.


INTRODUCTION
Hepatitis B virus (HBV) is a hepatotropic virus and the causative agent of hepatitis B. HBV infection may lead to hepatocellular carcinoma (HCC) and cirrhosis [1].HBV infection may be symptomless in many people during the initial stages.Globally, HBV is responsible for 0.5-1 million deaths annually.Although a considerable portion of carriers have varied level of hepatocyte obliteration, the infection can lead to HCC or cirrhosis [2].More than 300 million individuals are infected with HBV, which is thus considered a pandemic [3].Treatments for HBV infection include standard and pegylated interferon-alpha and nucleoside analogues such as entecavir, lamivudine and adefovir.However, current therapies are unable to eradicate HBV.Development of drug resistance and reactivation of viral replication can occur even when treatment is successful [4].The aim of therapies for HBeAg-positive and -negative individuals should be long-term eradication of HBV DNA, with an endpoint of hepatitis B surface antigen (HBsAg) seroconversion [5].
Phyllanthus niruri L. (Euphorbiaceae) is a medicinal herb distributed in South Asia.In traditional medicine, the plant has long been used as a hepatoprotectant and for treatment of hepatitis B [6].Current HBV therapies can result in moderate-to-severe side effects, development of resistance to HBV expansion, or limited efficacy in only a subset of HBV-infected patients [7].Despite the availability of numerous antiviral drugs, a number of issues remain, such as limited efficacy, dose-dependent side-effects, and drug resistance.Therefore, more-effective anti-HBV drugs are urgently needed.Despite their potential, few natural herbs have been evaluated in a systematic manner.The goals of HBV treatment are to prevent development of HCC and cirrhosis, regulate alkaline transaminase (ALT) levels, suppress viral replication, and reduce the liver damage in HBeAg-negative as well as with HBeAg-positive individuals [8].
It has been reported that water extract of P. niruri inhibits cellular DNA polymerase activity during HBV replication [9].A considerable proportion of untreated newborns of mothers who are longterm HBeAg carriers become infected, and 90 % become long-term carriers [10].The secretory non-particulate form of HBeAg is not necessary for HBV replication or infection.The immune system responds to the presence of HBeAg and suppresses infection [11].
The plant Phyllanthus niruri (also known as Phyllanthus amarus) exhibits anti-HBV activity and has long been used to treat jaundice in southern India [12].P. niruri belongs to the family Euphorbiaceae and can be used to treat kidney stones [13] and disorders of the gastrointestinal and genitourinary tracts [14].
In this study, we isolated and characterized ellagic acid from P. niruri and assessed its effect on HBV-infected HepG2/C3A cells as a potential agent to prevent the development and progression of hepatitis virus and HCC in chronic HBV carriers [15,16].

EXPERIMENTAL Evaluation of physicochemical properties
Cary 60 UV-Vis and 660 FTIR (together with potassium bromide) spectrophotometers (both from Agilent Technologies (Santa Clara, California, United States) were used to monitor UV-visible and infrared spectra, respectively.The acquired spectra were subjected to highthroughput mass spectrometry (electrospray ionization MS).
Bruker nuclear magnetic resonance (NMR) spectroscopy at 400 MHz, utilized to obtain the Electrothermal's melting point apparatus (Bibby Scientific Limited, Staffordshire, UK) was utilized to assess the melting point.Thin-layer chromatography was conducted on silica gel 60 F254 plates (250 μm).

Isolation and purification of an anti-HBV compound from P. niruri
Five hundred grams of dried P. niruri plant mass were subjected to aqueous extraction at 37 °C for 3 h.The extract was filtrated twice under low pressure at 40 °C (Figure 1).The supernatant was resolved on a gel column using a CHCl 3 /MeOH gradient twice.Fraction #12 was then separated on a silica gel column (70/10/1 CHCl 3 /MEOH/H 2 O) at low pressure.The resulting brown-colored powder was subjected to preparative HPLC using a mobile phase of acetonitrile/1 % formic acid (10/90 to 45/55 % gradients).Anti-HBV activity was evaluated as an increase in antigen secretion by HepG2/C3A cells (Figure 2).Extracellular HBeAg was detected using an ELISA kit.

Anti-HBV activity assay
HepG2/C3A cells were plated on 24-well flatbottom culture plates at 3  10 5 /mL.Following incubation for 24 h, test compounds were applied to the plates.
The compounds were dissolved in dimethyl sulfoxide (DMSO) to 1, 2, 4, 8, 16, 32, 64 and 128 µg.The highest concentration was 2.5 µg/mL, which did not affect HepG2/C3A cells.HBsAg levels were evaluated by enzyme immunoassay (Sigma-Aldrich Shanghai Trading Co Ltd, Shanghai, China).The reactions were assayed at a wavelength of 492 nm using the CLARIOstar® High-Performance Multimode Microplate Reader.Inhibition (%) was evaluated in comparison with DMSO as in Eq 1: where As and Ab are the absorbance of test sample and blank, respectively.Data are presented as the means of three replicates.Activity was classified as follows: 20 -35 % as weak, 30 -50 % as medium, 50 -65 % as strong, and > 65 % as very strong.Cell damage was assessed by aspartate transaminase (AST) assay (Abnova Corporation, Taipei, Taiwan).AST values of > 25 IU/L were considered indicative of cell damage.

HBeAg assay
For the initial assessment of HBV reproduction, secreted levels of HBeAg from HepG2/C3A cells following treatment of P. niruri extracts or ellagic acid were evaluated by ELISA (Hepatitis B Antigen ELISA kit; DRG International Inc., Springfield, NJ, USA).

Computation of half-maximal inhibitory concentrations (IC 50 )
Intracellular encapsidated HBV DNA was extracted and subjected to gel blot analysis.The IC 50 of each drug was determined as the concentration resulting in a 50 % decrease in the viral DNA level compared with that of untreated cells.

Statistical analysis
Data are shown as means ± standard deviation (SD, n = 3).IC 50 values were computed using Origin Lab version 18.0 software (Origin Lab Inc., Guangzhou, China).Students' non-paired t-test was used to compare factors between the sample and solvent control.A value of P < 0.005 was considered to indicate significance.

Anti-HBV activity of extract fractions
Figure 1 shows the antiviral activity of the various fractions of the plant extract.

Attributes of hepatitis B-reducing elements
The physicochemical properties of components with anti-HBV activity are shown in Table 1.Crystals of the active component were yellow in color.The component had a molecular weight of 302 Da, and infrared spectroscopy showed absorption at 3380, 1720, 1690, and 1610 cm −1 , indicating the presence of phenolic hydroxyl, αbyron C=O, and benzenoid C=C groups.These results together with other findings (melting point, UV spectra, R f , and solubility) indicated that the active component is ellagic acid, C 14 H 6 O 8 .The 1 H NMR spectrum of ellagic acid has a singlet at δ 7.47, which was associated with two protons at C-5 and C-5 (Table 2).The 13 C NMR spectrum of ellagic acid exhibited quinone carbonyl carbons at 168.60 and 145.86, showing C-2+C-2, C-4+C-4, and C-3+C-3, which were associated with δ 157.58 and δ 149.01.In addition, δ 119.73 and 121.79 were assigned to four carbons at C-5, C-5 and C-1, C-1, and δ 117.3 was assigned to C-6, C-6.The

Effect of ellagic acid on HBeAg secretion
Fraction #12 exhibited dose-dependent toxicity towards HepG2/C3A cells (Figures 3 and 4).Ellagic acid was incapable of increasing HBV polymerase activity.PEG 8000 precipitated virion particles with four deoxyribonucleic was used to observed anti-polymerase functions of ellagic acid.Lamivudine 3TC triphosphate was used as the positive control (Figure 5).

DISCUSSION
Chronic HBV infection can lead to development of cirrhosis and HCC, which increases the risk of death.The FDA has approved two drugs to treat HBV infection; however, both show poor efficacy and various side effects [20,21].
Medicinal plants have long been used to treat various illnesses worldwide.HBV infects millions of people worldwide and causes a severe, transmittable disease of the liver.The pharmaceutical industry is endeavoring to discover a natural anti-HBV agent.Therefore, it is important to explore phytochemicals as a new source of drugs.However, data on the antiviral activity of medicinal plants are limited.Phyllanthus species have long been used as herbal remedies for HBV infection.Ellagic acid, found in berry fruits, has components with anti-inflammatory, anti-fibrotic, and antioxidant activities.Ellagic acid was shown to reduce HBeAg production by HepG2 2.2.15 cells [22].
Ellagic acid has potent antioxidant activity.Moreover, ellagic acid has growth-inhibiting and apoptosis-inducing cytotoxic effects on cancer cells, including leukemic, pancreatic, breast, neuroblastoma, colon, prostate, tongue, and osteogenic sarcoma cells.Ellagic acid-treated cells show apoptosis, increased caspase 3/7 activities, activation of caspase 3, and cleavage of poly-ADP ribose polymerase [23].Although ellagic acid possesses potent activity against human DNA polymerases β, η, ι, and κ in vitro, our data did not indicate significant inhibition [24].This indicates that viral reproduction is not suppressed by ellagic acid.As HBV is a DNA virus, reverse transcriptase inhibitors may be a promising therapeutic to inhibit HBV DNA replication [25].

CONCLUSION
A potential anti-HBV agent, ellagic acid, was isolated from the medicinal plant P. niruri, and its physicochemical properties were characterized.The isolated compound exerted a cytotoxic effect against HepG2/C3A cells.Ellagic acid did not affect HBV replication.

1 H and 12 C
NMR spectra in CDCl 3 .

13 C
NMR data thus supported identification of the active component as ellagic acid.

Table 1 :
Physicochemical properties of ellagic acid