I. taste is bitter and acts as a

      I.           
Introduction

Medicinal plants are very important for
prevention of human diseases; there are wide varieties of medicinal herbs grown
in India. One such widely used herb is           Phyllanthus niruri 1.It belongs to the family
Euphorbiaceae 2. The herb has been the material of choice in the Indian
Ayurvedic and Unani system for Jaundice, ulcers, skin diseases,
diabetes, chest pain and urinary complications. Its taste is bitter and acts as
a stringent and show laxative effect 3. Studies on P.niruri have revealed
that its anti-viral activity can be extended to HIV and Ogata
et al 1992 and  Naik & Juvekar 2003
have shown that aqueous extract of the plant interfere with the replication of
the virus by inhibited HIV-1 reverse transcriptase. In human body researchers
have documented the diuretic, hypotensive and hypoglycaemic effects of P.niruri
15. Similar investigations by Ramakrishnan et al 1982 and Hukeri et al 1988
have shown significant reductions in the systolic blood pressure in
non-diabetic patients and blood glucose levels in diabetic patients by P.niruri. Beside this what is fascinating about P.niruri is the extract has been used to treat kidney and
gallbladder stones, for cold, flu, tuberculosis, and other viral infections;
liver diseases and disorders including hepatitis B, jaundice and liver cancer.
Studies have also shown that the plant has novel anti-viral properties against
Hepatitis –B virus (HBV) 2. Epidemiologically infection with
Hepatitis B virus has been associated with liver cirrhosis particularly
Hepatocellular carcinoma (HCC) 4.The mechanism by which HBV causes cancer is
still unclear but there is a good evidence that the virus itself exerts a direct
or indirect hepatocarcinogenic effect thus having implications for prevention
4. Although many strategies have been adopted for reducing the incidence of
HCC like the population based vaccination programmes, anti-viral therapy but
these remain uncertain and inefficient. Here we propose a novel way to treat
HCC with the help of nano-extract of P.
niruri which has shown to be effective in most of the clinical trials.

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Research on P. niruri
revealed that its antiviral activity

extends to human immunodeficiency virus (HIV) and a

simple aqueous extract of the plant inhibited HIV-1
reverse

transcriptase (Ogata et al 1992; Naik & Juvekar
2003)

Research on P. niruri
revealed that its antiviral activity

extends to human immunodeficiency virus (HIV) and a

simple aqueous extract of the plant inhibited HIV-1
reverse

transcriptase (Ogata et al 1992; Naik & Juvekar
2003)

  
II.           
Novelty

The
liver plays a crucial role in metabolism of drugs and xenobiotics, and in
maintaining biological equilibrium. Despite some of the noteworthy inventions
in medical sciences, there is hardly any drug that safe guard liver functions
or offers protection to the hepatic cells from damage or helps regeneration of
hepatic cells 5. Herbal drugs because of its low side effects and long
lasting nature are emerging as potent treatment weapons for hepatoprotection
today 5. Based on natural, nontoxic and bio available sources of
hepatoprotectants, we undertook to investigate the hepatoprotective activity of
Phyllanthus niruri (P.niruri). In
folkmedicine the plant has been in use for treating host of diseases
particularly hepatitis B. It contains many types of phytochemicals having
pharmacological properties which can be extensively used 6. The active
phytochemicals, flavonoids, alkaloids, terpenoids, lignans, polyphenols,
tannins, coumarins and saponins, have been identified from various parts of P.
niruri 6. However majority of these compounds are water insoluble so
there absorption on oral administration could be limited 5. Also most of the
oral formulations prepared to date with P.niruri
extract have direct or indirect involvement of metallic (gold or silver) nano
particles used in conjugation with the plant extract. Though this approach is
successful in achieving its target function but the metallic constituents can
pose to be detrimental in terms of their non-biodegradable nature, possibility
of accumulation and other side-effects. Therefore an attempt is made to enhance
the dissolution rates and hence bioavailability of the P.niruri extract by preparing its
nanoparticles (biodegradable) which can be used as a novel way for preparing
oral formulation of nanoparticles without any worry and concern 5.

III.           
Applicability

One of the most
serious liver infection in the world is Hepatitis B. Nearly two billion people
(or 1 in 3) have been infected and more than 240 million people knowingly or
unknowingly are living with a chronic hepatitis B infection. Each year up to 1
million people die from hepatitis B despite the fact that it is preventable and
treatable. Traditional treatments using interferon- ?, anti-viral
treatments or liver transplant has many side effects and is difficult to
afford. Alternative herbal medicine using extracts of Phyllanthus
niruri and Phyllanthus urinaria have been reported to be effective
against Hepatitis B and other viral infections (liver cirrhosis) 6. The
extract from P.niruri has been shown to exhibit marked antihepatitis B virus
antigen activity both in in-vivo and in-vitro studies. Thyagarajan et al conducted a study with 37 patients infected
with chronic viral hepatitis B who were treated with a daily dose of 600mg of Phyllanthus
niruri for 30 days. 59% of the patients lost the HBsAg two weeks after the
end of the treatment. Furthermore, none of the cases followed for up to 9
months had any symptoms of HBsAg 7. The researchers concluded that probably
the extract interfere with replication machinery of the virus thereby
inhibiting the proliferation of the virus. As per the research conducted in
India and Japan, 1980 it has shown the liver healing characteristics of -P.niruri. The primary compounds
responsible are phyllanthin, hypophyllanthin and triacontanal 6. Therefore if
the research is carried out we can develop a natural method to cure the
hepatitis infection without post treatment stress.

IV.           
Description

Nanotechnology
is principally an attractive area of research which has opened possibility for manipulating
and controlling structures at the molecular level. Due to the ease in
production of nanoparticles of variable size and shape the area has lead to the
generation of many applications in clinical medicine 8-10. Conventionally
nanotechnology has proved its worth in many bio-medical applications including biosensors,
tissue engineering and nanocomposites for implant design and controlled drug
release 11. Today nano –oriented methodologies are being extensively used to
optimize the technological aspects of drugs. The major impact of
nano-approaches has lead to increase in dissolution rates in vitro and increased bioavailabilities in vivo of many drugs 12.

In
the present study, our aim is to evaluate the hepatoprotective effects of the
extract of P. niruri and its nanoparticles for developing oral formulation
of the plant extract.

   V.           
Methodology

       I.           
Preparation of extract of P.niruri

The
freshly collected leaves of P.niruri will be dried in air followed by
tray drier under control conditions and powdered. About 1000g of powdered
leaves will be macerated with petroleum ether to remove fatty substances ;the
marc will further be exhaustively extracted with 95% ethanol for 3 d (3×3 l) by
cold percolation method and centrifugation at 10 000 rev min?1. The extract
will be separated by filtration and then dried in lyophilizer under reduced
pressure 5.

   
II.           
Preparation
of nano-suspension of P.niruri extract

Using Nano precipitation technique nanoparticles of the herb are
prepared. Briefly, 5 g of P.niruri will
be dissolved in 30 ml of acetone and ethanol (3:1) by sonication at 20W for 30
s. The resulting solution will then be slowly injected (1 ml min?1) with a
syringe connected to a thin Teflon tube, into 50 ml water containing polyvinyl
alcohol (PVA) 1.5% w/v with continuous magnetic stirring at 1000 rpm. The
resulting emulsion obtained will be diluted in 100 ml PVA solution (0.2% w/v in
water) in order to minimize coalescence and stirring will be accompanied
continuously (500 rpm) for 6 h at room temperature to allow solvent evaporation
and nanoparticle formation. The resulting nanosuspension will subsequently be
cooled down to ?18 C and freeze dried 5.

 III.           
Particle size analysis and morphological
characterization

 

Mean
particle size and size distribution ( i.e. polydispersity index) will be
determined using photon correlation spectroscopy .The size distribution
analysis will be performed at a scattering angle of 90 and at a temperature of
25 C using samples appropriately diluted with filtered water. In addition, the
morphological characterization of particles will be performed using transmission
electron microscope (TEM) 5.

 IV.           
Use
of biodegradable polymer

Poly
lactic-co-glycolic acid (PLGA) or Polylactic acid
(PLA) polymers will be used for drug loading. These drugs will specifically
target the hepatocellular protectivity. To avoid inconvenient surgical
insertion of large implants, inject able biocompatible PLGA particles will be
synthesized using Solvent Evaporation Method,
Phase Separation (Coacervation) or spray drying 13. Pre-synthesized lactic
acid (LA) monomers will be used which will subsequently be converted into PLA
polymers using a range of polymerization
processes, including polycondensation, ring opening polymerization or azeotopic
dehydration condensation reaction 14. The reason for choice of these polymers
can be elucidated from the fact that they are degraded in vivo either
enzymatically or non-enzymatically or both to produce toxicologically safe
by-products which are finally eliminated from the body using normal metabolic
pathways 13.

   
V.           
Cell
(in vitro) efficacy test on viral induced cytotoxicity

After treatment with the plant
extract the cell toxicity levels will be compared of the control group and the
experimental group (infected with viral HCC) using any one of efficacy tests: ADCC, ADCP, CDC, Lymphocyte subset analysis, Cell
proliferation analysis using BrdU, MTT. Agar Overlay or MEM Elution,
Keratinocyte/Fibroblast Viability Assay.

VI.           
Outcome: Alternative medicine

By conducting the proposed study we can
formulate an oral medication for treating virus inflicted hepatitis in remote
parts of the country where the herb P.niruri
is grown but is not used to its best potential. Thus, we suggest that the
nanoparticles system can be applied to overcome other water poorly soluble
herbal medicines and furthermore to decrease the treatment dosage. This study
could serve as a useful reference to allow the future exploitation of nanoparticulate
system as a novel preventive and therapeutic measure for the treatment of
hepatic and other various physiological disorders