S. Dinesh, M. Lavanya, M. Jeevan, V. Mythily 1, and M. Elanchezhiyan *

Department of Microbiology, Dr. ALM. PG Institute of Basic Medical Sciences,

University of Madras, Taramani, Chennai - 600 113, India.

1Blood Bank, Voluntary Health Services, Adyar, Chennai - 600 025, India.

e-mail: emanickan@yahoo.com*

Hepatitis B virus (HBV) causes significant morbidity and mortality worldwide. HBV infection can be acute which can be cured completely or chronic during which the virus persists in the liver for a long time and occasionally for their life. Despite the universal vaccination of neonates and infants during the last years and the subsequent reduction in the incidence of new infections with hepatitis B virus (HBV), chronic HBV infection remains a significant public health problem worldwide (Maddrey, 2000). It is estimated that there are approximately 40 billions people with chronic HBV infection and that more than 500 000 people die every year due to complications of HBV related chronic liver disease (Maddrey, 2000). Although considerable improvements in the evaluation and treatment of patients with chronic HBV infection have occurred during the last decade, several issues regarding the optimal management of such patients still challenging. All patients with chronic HBV infection are at increased risk for hepatocellular carcinoma (HCC) compared with the general population and the risk increases substantially in patients with prolonged high viremia and cirrhosis (Chen et al., 2006). Recent data suggest that patients with chronic HBV infection and HBV DNA above 10⁴ copies/mL (approximately 2000 IU/mL) are at increased risk for cirrhosis and HCC regardless of alanine aminotransferase (ALT) activity and are therefore possible candidates for treatment (Chen et al., 2006)

Lamivudine treatment is widely employed to curtail chronic HBV infection (Alberti and Caporaso, 2011). Treatment with Interferon α is also an approved method of treatment for chronic HBV infections (Dusheiko, 1995). However, interferon α therapy is associated with several side effects and often unsatisfactory response rates. Among the reasons that have been cited for explaining the poor response rate to interferon are: 1) the immune tolerance to HBV after infection at birth or during early childhood (Lai et al., 1998) and 2) persistence of the viral covalently closed circular (ccc) DNA in the liver (Lin et al., 1998). Thus the optimal first-line anti-HBV therapy with the best long-term cost/benefit ratio is yet to be developed.

Now there is a need for the clinical development of more efficacious yet safe and non-toxic cytoprotective agents for the adequate management of hepatitis. In this context antiviral and hepatoprotective drugs development from alternative natural sources play important role. Several medicinal herbs has been reported in Ayurvedha, Sidda and Unani for their medicinal properties. In recent times, many attempts to identify the active anti-HBV substances in Phyllanthus extracts is being investigated (Thyagarajan et al., 1988; Yoon et al., 2000). Though Phyllanthus amarus (P. amarus) extracts shown to possess bioactivities to clear the carrier status of HBV infections its efficacy during chronic HBV infections is reported to be equivocal (Venkateswaran et al., 1987). This necessitates the search for novel anti HBV drugs from marine sources.  Drug from the sea program is a major initiative by the Government of India to develop novel drugs against infectious diseases. Significant interest on marine organisms has developed recently due to their possession of pharmacologically bioactive substances that had been used against bacteria, viruses and tumors (Smit, 2004). Despite the increasing number of new findings about seaweed metabolites possessing biological activity on the last three decades few products having actual potential have been identified or developed (Smit, 2004). Antiviral activities of extracts derived from various marine algae have been documented (De Almeida et al., 2011). The present study documents the possible anti HBV activity of Padina tetrastromatica  by HBsAg binding inhibition assay.

The marine alga, Padina tetrastromatica (Fig. 1) was collected from Rameshwaram coast, India. The impurities were removed by rinsing in sterile distilled water and authenticated at the Department of Botany, University of Madras, Chennai. The algae was shade dried, powdered and stored at room temperature until use. Methanol extract of P. tetrastromatica was prepared by adding 50 gms of algal powder in 500 ml of methanol and then filtered using Whatman filter paper (No.1). The filtrate was allowed to evaporate for about 2-3 days. The dried filtrate was collected, weighed and stored at 4°C until use. 

Fig. 1. Padina tetrastromatica - simple, freely branched filaments to highly differentiated forms

and distinguished into blades, stipes and holdfast.

Equal volume of pre-titrated HBV and varying concentration of methanolic extract was mixed and incubated at 37°C for 5 days. The mixture was assayed on day 5 for the presence of bound/unbound HBsAg using ELISA kit (Hepanostika HBsAg kit). Controls included in the experiment were drug positive control (Elan-PA001) and drug negative control (Nonoxynol-9). Other controls included the kit positive and negative controls. ELISA was performed as per the manufacturer protocol. Briefly, to the anti HBsAg antibody precoated plates extract treated HBV virus was added and incubated for 1 hour at 37°C. Then the plates were washed and secondary antibody-HRP conjugate was added and further incubated for 1 hour at 37°C. Then the plates were washed and TMB substrate was added and incubated at room temperature for 30 minutes. To this stop solution was added and the plates were read at 450 nm in ELISA reader (BioTek). Experiments were conducted thrice and one representative experiment was described. Results are represented as ELISA optical density (OD) and percentage (%) inhibition.

Percentage inhibition = OD of Test-OD of the control/OD of the control × 100.

Anti HBV property of P. tetrastromatica was evaluated by studying the inhibition of HBsAg binding by the extracts. In this evaluatory study first varying concentration of P. tetrastromica (dose response) were tested for its anti HBV activity. Secondly two different doses of virus were tested for the drug efficacy. Table 1 represents the dose response profile of P. tetrastromatica,percent inhibition above 90% was considered significant. As shown in the table above 90% inhibition (virus concentration 1.5pg/ml) of HBV was noticed at a drug concentration of 5 mg/ml and above. Up to 10 mg/ml concentration of P. tetrastromatica, the algal extract did not show any drug toxicities as described by MTT assay (data not shown). An in-house preparation, Elan-PA001 served as drug positive control which completely inhibited HBV. A potent anti HIV drug Nonoxonol-9 was used as negative HBV drug which showed <1.0% activity. As anticipated the group received sterile distilled did not show any anti HBV activity.

Table: 1. Anti-HBV activity of methanolic extract of Padina tetrastromatica

Various concentrations of Padina tetrastromatica were tested for its anti HBsAg inhibition activity on day 5 by ELISA. Virus concentration = 1.5 pg/ml; Untreated = group that received sterile distilled water; OF = OD over 4.0.

In order to assess whether the anti HBV activity of P. tetrastromatica was virus dose dependent, varying concentrations of HBV such as 3 pg/ml, 1.5 pg/ml and 0.75pg/ml were tested against 5 mg/ml concentration of the P. tetrastromatica extract. The results revealed that P. tetrastromatica extracts completely inhibited the 1.5 pg/ml of HBV as shown in the above experiment (Table 2). A similar inhibitory activity was noticed with the lower virus dose of 0.75 pg/ml also. When the virus dose was increased to 3 pg/ml this inhibitory activity was abrogated suggesting the 1.5 pg/ml may be the optimum concentration that could be nullified by the P. tetrastromatica (data not shown).

Table 2. Anti HBV activity against two different doses of HBV

Stock virus was diluted to 1.5 pg/ml and 0.75 pg/ml and tested for the drug’s anti HBV activity by ELISA

Exploring novel drugs to combat HBV infections especially the chronic HBV hepatitis is needed very desperately. Marine sources serve as a potential treasure hunt platforms for unique drug development that could be used to treat viral hepatitis. In this evaluatory study varying concentration of P. tetrastromatica were tested for the anti HBV activity and it was found that 5 mg/ml concentrations of P. tetrastromatica completely neutralized the HBV suggesting its medicinal scope to treat viral hepatitis. P. tetrastromatica extracts inhibited 1.5 pg/ml of HBV suggesting the magnitude of inhibition. Bioactivities of several marine algae have been reported but medicinal value of P. tetrastromatica has not been studied so far suggesting the pioneering nature of our study.

Chronic HBV infections can lead to liver cirrhosis and hepatocellular carcinoma. Considering the severity of clinical outcome proper treatment modalities must be in place to fight against human HBV infection. One of the very important proteins of HBV is the surface antigen (HBsAg) which helps the virus in adherence to the target tissue (Neurath et al., 1990). Importance of HBsAg is multifold and it is highly immunogenic (Carman et al., 1993). HBsAg is also known as “australia antigen” and found in 4 phenotypes namely adw, ayw, adr and ayr and each phenotype is epidimiologically important (Tiollais et al., 1985). Presence of HBsAg in a patient is an indication that it is a recent infection and antibodies to HBs (anti HBs antibody) are efficient in clearing the HBV (Halliday et al., 1992). Besides that there are two other important antigens namely HBcAg and HBeAg are important for the complete clearance of the virus during chronic infections. (Liaw et al., 1984). In chronic HBV infection both HBsAg and antibodies to HBs (anti HBs) are found in the patients and presence of HBsAg helps in the new infection of hepatocytes.

HBV infects hepatocytes and causes viral hepatitis. Receptors for HBV is not fully known and it is speculated that preS domain of surface protein of the virus bind to carboxypeptidase D molecules found on hepatocytes. Thus the surface antigen (HBsAg) plays an important role in virus attachment to the hepatocytes and any methodology that would interfere with this initial binding can prevent the virus attachment to the host tissue. In this context the current investigation is very important as the study clearly showed that P. tetrastromatica extract inhibits HBsAg binding to its receptor, anti HBs antibody. This study also clearly demonstrated that 5 mg/ml of the extract inhibited the virus binding and this inhibition was noticed upto 1.5 pg/ml concentration of the virus. This report is of first of its kind in augmenting the efficacy of P. tetrastromatica extract in inhibiting HBV binding to its receptor. This study open up new vistas on the molecular mechanism of HBV viral entry inhibition. Therefore cataloging marine algae and their medicinal property have more scope in combating infectious diseases.

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