PHYLLANTHIN AS A POTENT INHIBITOR OF HEPATITISC

VIRUS REPLICATION

FIELD OF THE INVENTION

The present invention generally relates to inhibitors of Hepatitis C virus replication. Specifically, embodiments of the invention relates to a naturally occurring compound phyllanthin, obtained from Phyllanthus amarus extract.

BACKGROUND

Hepatitis C infection is a major health problem worldwide. It is an infectious disease primarily affecting the liver caused by the Hepatitis C virus (HCV). HCV belongs to a member of family Flaviviridae and is an infectious pathogen causing chronic hepatitis that can further progress to hepatocellular carcinoma. Most common causes for the spread of the disease are blood transfusion. Other sources of HCV infection include use of contaminated needles sexual transmission and transmission of HCV from infected mothers to the babies.

HCV is a positive sense single stranded RNA virus. The genome of HCV comprises 9600 nucleotides and has a single open reading frame (ORF) that encodes structural proteins and non structural proteins. Both the structural and non structural proteins have been the target for development of HCV therapies. Of these the proteases namely NS2 cysteine auto-protease and NS3-4A serine protease encoded by the viral genome have been targeted for curbing the HCV infection. HCV non-structural protein 3 (NS3) plays a critical role in polyprotein processing and viral RNA replication. This multifunctional enzyme has two major domains: the serine protease domain and helicase domain. Saumitra Das group have demonstrated that the NS3 protease domain alone can specifically bind to HCV-IRES (Internal Ribosomal Entry Site) near initiator AUG and dislodge the binding of a host factor the human La protein. The interplay leads to inhibition of translation in favor of replication (Ray and Das 2011).

One of the methods of curbing HCV infection has been through inhibition of activity of NS3-4A. Various inhibitors have been designed and targeted at the NS3-4A. The inhibitors include but are not limited to small molecules derived synthetically and obtained from natural sources oliognucleotides and peptides.

There are many synthetically derived compounds that have been targeted for HCV inhibition. One of the disadvantages of employing a synthetically derived compound is cytotoxicity. Another disadvantage is presence of undesirous side effects. Hence compounds extracted from natural sources such as plants have shown to be hepato protective. Plants are regarded as a promising source of novel therapeutic agents due to their higher structural diversity as compared to standard synthetic chemistry and may provide a suitable high throughput platform for HCV inhibition.

Phyllanthus Amarus belonging to species is indicated as a potential hepatoprotective agent (Thyagarajan et al. 1988) For example: P. amarus has been used in a multi herbal extract for inhibition of HBV. Individually P.amarus extract has shown significant inhibition of Hepatitis B Virus (HBV) in both chronic and acute conditions of infection. Recent scientific literatures have shown profound anti-viral activity of this plant against Hepatitis B virus (Thyagarajan et al. 1988) increase in the life span of rats with HCC (Rajeshkumar and Kuttan 2000) and in inhibition of HIV (Notka et al 2004).

P.amarus is used as one of the important constituent in many ayurvedic formulations which include but are not limited to Diabecon (Glucocare) Liv.52 Protec and Chyavanaprasha.

phyllanthin is one of the major constituents of P.amarus extract. There have been studies which have shown phyllanthin as a potential hepatoprotective compound along with other identified compounds in the multi herbal extract. Hence there is a need to isolate phyllanthin and evaluate the hepato protective characteristic of phyllanthin against HCV infection.

OBJECT OF THE INVENTION

One object of the invention is to provide a naturally occurring lignan derivative for example phyllanthin as a hepatoprotective compound for targeting HCV replication.

Another object is to provide a phyllanthin based formulation for the inhibition of HCV infection.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the manner in which the above recited features of the present invention can be understood in detail a more particular description of the invention briefly summarized above may be had by reference to various embodiments some of which are illustrated in the appended drawings. It is to be noted however that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope for the invention may admit to other equally effective embodiments.

FIG.1 shows the Schematic representation of extraction and purification of phyllanthin from Phyllanthus amarus.

FIG. 2 illustrates thin layer chromatography of pure phyllanthin.

FIG.3a shows the 1H NMR spectra of extracted phyllanthin from P.amarus.

FIG.3b shows the 13C NMR spectra of extracted phyllanthin from P.amarus .

FIG.4 shows a graph indicating the effect of phyllanthin when compared with the other compounds of P.amarus on HCV-NS3 protease activity.

FIG.5 shows the effect of phyllanthin on HCV Replication at different concentration.

FIG.6 shows a graph of combined effect of phyllanthin and pegylated interferon alfa on HCV replication.

FIG. 7 shows a graph of the cytotoxic effect of phyllanthin at various therapeutic doses during MIT assay.

BRIEF DESCRIPTION OF THE INVENTION

Various embodiments of the invention provide a method for extracting phyllanthin from the extract of P.amarus. The invention also provides method for targeting phyllanthin for inhibition of HCV replication.

FIG.1 shows the Schematic representation of extraction and purification of phyllanthin from P. amarus. Fresh plants of P.amarus are collected during the flowering season from their natural habitats. In an embodiment of the invention the plants of P.amarus were collected during the flowering season from habitats in and around Bangalore. The species of the plant collected is authenticated and certified by an authorized body for authentication and certification. The collected plants of P.amarus are deposited with a voucher specimen number 74099 at the Bio-Cultural Repository of Indian Medicinal Plants Resources -. The collected plants of P.amarus are also deposited with Institute of Ayurveda and Integrated Medicine and Foundation for Revitalization of Local Health Traditions (FRLHT) Bangalore.

In an embodiment of the invention the process of isolation of phyllanthin from P.amarus includes
Digesting the fresh leaves of P.amarus with an organic solvent obtain a filtrate; Concentrating the filtrate to obtain a dried powder; and Isolating phyllanthin from the dried powder through solvent fractionation process.

In an embodiment of the invention the fresh leaves of P. amarus are digested with 100% methanol. The digestion referred to herein means partial digestion of the plant materials. During digestion the temperature is maintained in a range of 65oC to 70oC. The digestion of the P.amarus leaves is carried out for 18hrs. At the end of 18 hrs the digested P. amarus leaves are filtered through a filter paper. In an example of the invention Whatman filter paper is used. The filtrate obtained through digestion is subsequently concentrated by draining the remaining solvent to form a paste.

The paste obtained is then vacuum dried in the presence of magnesium carbonate to obtain a dried powder extract of P.amarus. In an embodiment of the invention magnesium carbonate is added to the paste obtained in the ratio of 1:1 (W/W) to obtain the dried powder extract of P.amarus. In an example of the invention 100gram of magnesium chloride is added to 100 gram of paste obtained and vacuum dried to form a dried powder extract of P.amarus.

The powder extract of P.amarus is then dissolved in a methanol n-hexane mixture taken in the ratio 1:20 (V/V). The methanol dissolved extract is fractionated to obtain a top layer and a bottom layer. The top layer is a hexane layer comprising of non polar compounds present in the dissolved powder extract of P.amarus. The bottom layer is a methanolic extract of the dissolved powder extract of P.amarus comprising of polar compounds. The top layer is discarded and the methanolic extract is fractionated with chloroform taken in the ratio 1:20 (V/V). The fractionation of the methanolic extract with the chloroform yields a colored chloroform layer. The fractionation of the methanolic extract with chloroform is repeated a plurality of times until a colorless chloroform layer is obtained.

The colorless chloroform layer is separated and subsequently concentrated at 30oC to form a paste. The concentrated paste is given at least one mild wash with distilled water followed by at least one wash with petroleum ether (PE). The washed paste extract is treated with refrigerated absolute alcohol repeatedly at least ten to 15minutes until it turns colorless and left overnight for re-crystallisation of phyllanthin.

The extracted and purified phyllanthin is characterized by running the purified phyllanthin through Thin Layer Chromatography (TLC). In an embodiment 10% sulphuric acid and 90% of methanol was used for detection of phyllanthin on the TLC (FIG. 2).

Further the extracted and purified phyllanthin is characterized through determination of various physico – chemical properties. The physico-chemical properties include but are not limited to solubility stability and structure of phyllanthin. In an embodiment of the invention the structure of phyllanthin is determined through Nuclear Magnetic Resonance (NMR) Spectroscopy. In an embodiment of the invention 1H NMR spectra is determined. In an example of the invention the determination of 1H NMR spectra is performed on a Bruker AR X 400 spectrometer at 400 MHZ. The obtained spectra is calibrated with water as internal standards. FIG.3a shows the 1H NMR spectra of extracted phyllanthin from P.amarus. In another embodiment of the invention 13C NMR spectra is determined on a Bruker AR X 400 spectrometer at 100 MHZ. The obtained spectra is calibrated with methanol as internal standard (FIG..3b shows the 13C NMR spectra of extracted phyllanthin from P.amarus.

The salient features of phyllanthin as determined through 1H NMR spectra and 13C NMR spectra show that the purified form of phyllanthin has needle like structure. phyllanthin is soluble in organic solvents which include but are not limited to chloroform absolute alcohol and methanol. The solubility of phyllanthin in organic solvents is a characteristic typical of lignan group.

The inhibitory action of phyllanthin is determined through in-vitro NS3 protease inhibition assay. Along with phyllanthin chemical compounds known to be present in the P.amarus extract including hypophyllanthin oleonolic acid and triacontanol are also tested for NS3 protease inhibition. Each of the chemical compounds is individually screened in a concentration range varying between 100ng to 500ng. FIG 4a shows the inhibitory activity of phyllanthin alone and FIG 4b shows concentration dependent inhibition of NS3 protease activity. The results indicate that at lower concentration ranges (100ng) all chemical compounds present in P.amarus extract including phyllanthin show identical inhibition of NS3 protease activity. However at higher concentrations of chemical compounds used only phyllanthin shows significant increase in the inhibition of NS3 protease activity up to a maximum of 70-80 % inhibition.

It is also observed that there is a gradual decrease in the NS3 protease activity with simultaneous increase in the concentrations of phyllanthin in a dose dependent manner (IC50= 250ng) indicating that phyllanthin exhibits the NS3 serine protease activity. phyllanthin and hypophyllanthin are selected to test their effect on HCV replication in Huh7 cells harboring HCV monocistronic replicon RNA. Interestingly out of 2 compounds selected in this assay only phyllanthin showed significant decrease in HCV RNA levels in a dose dependent manner compared to hypophyllanthin (FIG.-5). Results of these studies clearly demonstrate that phyllanthin is one of the most important compounds of P. amarus.

In one embodiment of the invention phyllanthin was added with Interferon alpha (IFNa). Interferon is commonly used in anti HCV therapies to enhance the inhibitory effect of the anti-HCV drug. In an example of the invention phyllanthin is treated to Huh7 cells harboring HCV monocistronic replicon RNA with pegylated-IFNa. this combination showed further decrease in HCV RNA levels with increase in the dose levels of phyllanthin (FIG.-6 ) Synergetic effect of phyllanthin and pegylated intereferon alfa on HCV replication is achieved significantly when compared with the phyllanthin and pegylated-IFN alone (FIG.-6).

In an embodiment of the invention the cytotoxic effect of phyllanthin is studied on a mammalian cell. The mammalian cell is a human hepatocyte line Huh 7 cell line. Cytotoxic effect of phyllanthin (upto 500ng) on Huh-7 cell proliferation is measured by MTT [3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazoliumbromide; Sigma] assay by harvesting after 72h [Gianni Ciofani et al. 2010].

In an embodiment of the invention Huh7 cells are plated on a 96 well plate at a density of 5 x 103 cells per well. During plating the confluence of the cells are maintained at 50% confluence. A humified atmosphere with 5% carbon dioxide (CO2) is maintained during plating. Dulbeco’s Modified Eagle Medium (DMEM) having 10% serum is chosen as the medium for growth of cells. After allowing growth of cells for 24hr at 37oC a predetermined inhibitor is added. In order to study the effect of various inhibitors including phyllanthin isolated from the method described herein before and pegylated interferon alpha (IFNa) are added individually. In an example of the invention the concentration of phyllanthin is varied from 100 nanograms (ng) to 500ng The concentration of IFNa is varied from 100 picograms (pg) to 1000pg. Also combination of phyllanthin and IFNa are added as inhibitors. In one combination the concentration of phyllanthin added is a maintained at a preset value and the concentration of IFNa is varied. In another combination concentration of phyllanthin added is varied and the concentration of IFNa is maintained at a preset value. In an example of the invention the concentration of phyllanthin is preset at 150ng and the concentration of IFNa is varied from 100pg to 500pg. Further the concentration of IFNa is preset at 250pg and the concentration of phyllanthin is varied from 25ng to 500ng.

Subsequent to addition of inhibitors the plates containing the Huh7cells are incubated for a further period of 72hrs. At the end of 72hrs of incubation 20 micro liter of MTT solution having a MTT concentration of 5mg/ml in a phosphate buffer saline solution is added to each well and incubated at 37oC for duration of 4hrs. At the end of 4hrs the MTT solution is decanted and formazan is extracted from the cells with 100micro liter of DMSO in each well. Fluorescence at absorbance of 550nm is measured with an ELISA (Enzyme Linked ImmunoSorbent Assay) plate reader. Cell viability is measured as the concentration of drug required to kill 50% of the Huh7 cells and denoted as CC50. FIG.7 shows the results of MTT assay performed according to an embodiment of the invention. Results show that cell viability is not affected upon treatment with phyllanthin especially after 72h. These results further suggest that phyllanthin does not induce any cytotoxicity at the therapeutic dose levels. This encourages using phyllanthin as a safe drug at the therapeutic dose levels.

Further embodiments of the invention along with detailed explanation of various embodiments and further illustrations shall be explained in detail in the complete specification to follow. The aforesaid description is enabled to capture the nature of the invention. It is to be noted however that the aforesaid description and appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope for the invention may admit to other equally effective embodiments.

ADVANTAGES:

• A method for rapid isolation of phyllanthin.

• Reduction of cytotoxicity in hepatocytes treated with phyllanthin alone compared to the treatment with P.amarus extract.

• Inhibition of HCV replication.

• Inhibition of NS3 protease activity

• Potential candidate for combination therapy.

• Potential candidate for anti hepatitis B activity

We Claim:

1. A process for isolation of phyllanthin from P.amarus comprising:

digesting fresh leaves of P.amarus with an organic solvent obtain a filtrate;

concentrating the filtrate to obtain a dried powder; and

isolating phyllanthin from the dried powder through solvent fractionation process.

2. The process as claimed in Claim 1, wherein the digestion is a a partial digestion of the plant material in 100% methanol with temperature maintained in the range of 65°C to 70°C for 18 hours to obtain a filtrate.

3. The process as claimed in Claim 1, wherein the dried powder is obtained through vacuum drying of the filtrate in presence of light magnesium carbonate.

4. The process as claimed in Claim 1, wherein the solvent fractionation comprises of
dissolving the powder extract of P.amarus in a methanol,n-hexane mixture in the ratio 1:20 to obtain a methanol extract;

fractionating the methanol extract with the chloroform to obtain a colorless chloroform layer;

concentrating of the chloroform layer at 30°C to form a paste;

washing the paste with water, petroleum ether to obtain a dried chloroform extract; and

repeatedly washing the dried chloroform extract with absolute alcohol for at least 10-15 minutes to obtain a colorless paste.

5. The process according to Claim 1, wherein the phyllanthin is obtained through crystallization of the colorless paste.

6. A formulation comprising phyllanthin isolated from P.amarus, wherein the formulation is targeted for inhibition of HCV replication.

7. The formulation according to claim 6, wherein optionally phyllanthin is targeted in combination with pegylated interferon for enhancing the inhibition of HCV replication.

8. The formulation according to claim 6, wherein the inhibition of HCV replication is mediated through irreversible inhibition of NS3 protease activity.

9. A method for isolating phyllanthin from P.amarus as described in the specification and as illustrated in the accompanying drawings.

10. A formulation comprising phyllanthin as described in the specification and as illustrated in the accompanying drawings.