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Isolation, characterization and

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Pranav Shrivastav

Chemico-biological Interactions

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 Chemico-Biological Interactions 181 (2009) 351–358

Contents lists available at ScienceDirect

Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint

Isolation, characterization and antioxidative effect of phyllanthin against

CCl4 -induced toxicity in HepG2 cell line
Rajesh Krithika a , Ramasamy Mohankumar b , Ramtej J. Verma a,∗ , Pranav S. Shrivastav c ,
Illiyas L. Mohamad d,1 , Palani Gunasekaran d , Srinivasan Narasimhan b
a
Zoology Department, School of Sciences, Gujarat University, Ahmedabad 380009, India
b
Asthagiri Herbal Research Foundation, East Tambaram, Chennai 600059, India
c
Chemistry Department, School of Sciences, Gujarat University, Ahmedabad 380009, India
d
Virology Department, King Institute of Preventive Medicine, Guindy, Chennai 600032, India

a r t i c l e i n f o a b s t r a c t

Article history: The present study was an attempt to investigate the hepatoprotective and antioxidative property of Phyl-
Received 21 April 2009 lanthus amarus (P. amarus) extract and phyllanthin. Phyllanthin, one of the active lignin present in this
Received in revised form 22 June 2009 plant species was isolated from the aerial parts, by silica gel column chromatography employing gradient
Accepted 23 June 2009
elution with hexane–ethyl acetate solvent mixture. It was obtained in high yields (1.23%), compared to
Available online 1 July 2009
reported procedures and the purity was ascertained by HPTLC and reversed-phase HPLC analysis. Char-
acterization of phyllanthin was done by mp, UV–Visible spectrophotometry, elemental analysis, FT-IR,
Keywords: 1
H NMR, 13 C NMR and mass spectral analysis. Free radical scavenging activity of P. amarus extract and
Phyllanthus amarus
Phyllanthin
phyllanthin was also examined using DPPH assay. The protective effect of P. amarus extract and phyl-
Isolation lanthin was studied on CCl4 -induced toxicity in human hepatoma HepG2 cell line. The results indicated
Characterization that CCl4 treatment caused a significant decrease in cell viability. In addition, the toxin treatment ini-
Antioxidative tiated lipid peroxidation (LPO), caused leakage of enzymes like alanine transaminase (ALT) and lactate
HepG2 cell line dehydrogenase (LDH) with a significant decrease in glutathione (GSH) levels. It was observed that phyl-
lanthin effectively alleviated the changes induced by CCl4 in a concentration-dependent manner, with
much smaller strengths as compared to P. amarus extract.
© 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction ventive action of P. amarus extract on the corrosion of mild steel in

Phyllanthus amarus (family: Euphorbiaceae) is an annual herb
which is distributed throughout India. The plant is incorporated as
one of the important ingredient in various ayurvedic preparations
available to treat liver ailments [1]. Previous reports have confirmed
the ability of this plant in inhibiting Hepatitis-B virus thus proving
its potential as an antiviral agent [2]. The plant is also given credit
for its capacity to destroy human immunodeficiency virus (HIV)
[3]. The hepatoprotective property of crude extract of P. amarus has
been studied against paracetamol [4], carbon tetrachloride (CCl4 )
[5,6], ethanol [7], aflatoxin B1 [8] and galactosamine [9] induced
liver toxicity in various animal models. The obstruction of ethanol-
induced gastric lesion and carrageenan-induced mice paw edema
by P. amarus has also been reported in the past [10]. Apart from
its hepatoprotective behavior and other medicinal values, the pre-
∗ Corresponding author. Tel.: +91 79 2630 2362; fax: +91 79 2630 8545.
E-mail address: ramtejverma@gmail.com (R.J. Verma).
1
Present address: Neurovirology Department, National Institute of Mental Health
and Neuro Sciences, Bangalore 560029, India.
acidic media has also been described [11].
Phyllanthin and hypophyllanthin [6] are now well-characterized
bioactive compounds isolated from the organic extracts of this herb.
These two lignins are reported to inactivate Hepatitis-B, both in vitro
and in vivo [12]. Recently, Yadav et al. [13] have studied synergistic
effect of silymarin and standardized extract of P. amarus against
CCl4 -induced hepatotoxicity in rats. The pronounced effect was
attributed to the higher concentration of phyllanthin in the extract
as determined by HPLC analysis. Syamasundar et al. [14] have iso-
lated phyllanthin from hexane extract of P. niruri and studied its
antihepatotoxic property against CCl4 and galactosamine-induced
toxicity by measuring the ALT activity.
The current research on plant based medications focuses on iso-
lation of biologically active substances from potent plants, their
characterization and commercialization. Research in this direc-
tion has been greatly facilitated by modern physico-chemical
techniques of isolation and structural elucidation. Notable work
in the process of extraction, separation and characterization of
different constituents of P. amarus has been well documented
in previous reports [15–18]. Subsequently, Somanabandhu et al.
[19] have unambiguously elucidated the structure of phyllan-

0009-2797/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.cbi.2009.06.014
352 R. Krithika et al. / Chemico-Biological Interactions 181 (2009) 351–358
thin and hypophyllanthin by extensive 1 H NMR and 13 C NMR
including homonuclear two-dimensional NMR correlation spec-
troscopy (COSY), homonuclear decoupling, Attached-Proton-Test
(APT), heteronuclear chemical shift correlation (HETCOR), Nuclear
Overhauser Effect (NOE) difference, selective Insensitive Nuclei
Enhanced by Polarization Transfer (INEPT), and Correlation by
means of Long range Coupling (COLOC) experiments. Since then
several analytical methods based on HPLC with fluorescence detec-
tion have been developed for the determination of phyllanthin and
other lignins from P. amarus [20–22].
CCl4 is a hepatotoxic haloalkane whose mechanism has been
studied intensively over the past years. It is one of the best stud-
ied solvent in regards to liver toxicity. It is well known that CCl4
undergoes metabolic activation by a cytochrome P-450 dependent
step to free radical products which can initiate lipid peroxidation
[23]. The toxicity induced by CCl4 in vivo and in cultured hepato-
cytes, involves the stimulation of lipid peroxidation, detected as an
increase in malondialdehyde (MDA) formation [6,24].
Naturally derived antioxidants counteract the oxidative stress
induced by many hepatotoxins [25]. The quest to discover such
naturally occurring antioxidants has become a major scientific chal-
lenge over the past few decades. CCl4 is conventionally used to
induce liver toxicity in vitro/in vivo models [26], followed by test-
ing of plant based drugs for their liver protecting property. Many
previous reports confirm the decrease of both toxicity and lipid
peroxidation induced by CCl4 by many antioxidants [27,28].
The present study was designed to ascertain the antihepato-
toxic potential of P. amarus extract and phyllanthin against this
well characterized hepatotoxin in human hepatoma HepG2 cell
line. Immortalized hepatoma cell lines from man are widely used
to investigate the hepatoprotective property of crude plant extracts
and their isolated compounds. HepG2 cells are preferred to primary
cultures from animals, as these are cells from humans which can
minimize any species related differences that can occur during the
extrapolation of the results [29].
Thus, in the present study phyllanthin was isolated from P.
amarus and its structural elucidation was carried out by different
spectroscopic techniques. The free radical scavenging activity of
phyllanthin and P. amarus extract was ascertained by DPPH (1,1-
diphenyl-2-picrylhydrazyl) assay. The hepatoprotective nature of
phyllanthin in HepG2 cell line was demonstrated by inducing CCl4
toxicity.
2. Materials and methods
2.1. Plant material and chemicals
The aerial parts of P. amarus were collected in the month
of August and September 2007 from the herbal garden of
Gujarat University, Ahmedabad, Gujarat, India. Herbarium spec-
imen was prepared and authenticated by National Institute
of Science Communication and Information Resources (Ref:
NISCAIR/RHMD/consult/-2007-08/938/122), New Delhi, India. All
chemicals used were procured from Sigma–Aldrich (St. Louis, MO,
USA), unless specified. HPLC grade ethyl acetate, hexane, acetone
and CCl4 were obtained from Merck Specialties Pvt. Ltd. (Mumbai,
India). Water used in the entire analysis was prepared from Milli-
Q water purification system procured from Millipore (Bangalore,
India).
2.2. Instrumentation
Melting points were taken in a single capillary tube on a Toshni-
wal melting point apparatus (Mumbai, India) and are uncorrected.
Elemental analysis was carried out on Heraeus Carlo Erba 1108
elemental analyzer (Milan, Italy). UV absorption studies were car-
ried out on a Varian Cary 400 UV–Visible spectrophotometer (Palo
Alto, CA, USA) with 10 mm matched quartz cells. FT-IR spectra were
recorded on Bruker Tensor 27 Infrared Spectrophotometer (Bil-
lerica, MA, USA) as KBr pellets. Thin layer chromatography was
performed on pre-coated silica gel 60 F254 plates from E. Merck
(Darmstadt, Germany) with a semi-automatic Linomat IV band
spotter from CAMAG (Switzerland). The plate was developed to a
height of 10 cm with 20 mL of mobile phase of hexane:acetone:ethyl
acetate (7:2:1, v/v/v) using CAMAG twin trough glass tank. Devel-
oped plates were dried in a stream of air and then immersed in
a freshly prepared solution of vanillin in concentrated sulphuric
acid: ethanol (5:95, v/v) mixture [30]. After drying, the plates
were heated at 110 ◦ C to develop the colour of the spots. Band
scanning was done with CAMAG TLC scanner 3 using win-CATS soft-
ware at 540 nm. 1 H NMR (500.13 MHz) and 13 C NMR (125.75 MHz)
spectra were recorded on Bruker DPX-500 AVANCE in CDCl3 with
tetramethylsilane as internal standard. A Shimadzu LC-VP HPLC-
UV system (Kyoto, Japan) consisting of LC-10AD prominence pump,
SIL-HTc autosampler, CTO 10 ASvp column oven and a DGU-14A
degasser was used for setting the reverse-phase liquid chromato-
graphic conditions. Chromatographic separation was achieved on a
Phenomenex C18 (250 mm × 4.6 mm, 5 ␮m particle size) analytical
column, maintained at 40 ◦ C in a column oven under isocratic con-
ditions. The mobile phase consisting of acetonitrile:water (60:40,
v/v) was filtered through 0.2 ␮m membrane filter and pumped at a
flow rate of 0.5 mL/min. The injection volume was kept at 20 ␮L and
the wavelength of detector was set at 230 nm. Mass measurements
were done on a JEOL GCmate II GC–MS (Peabody, MA, USA) bench
top double-focusing magnetic sector mass spectrometer operating
in electron ionization (EI) mode with TSS-20001 software.
2.3. Extraction, isolation and purification of phyllanthin from P.
amarus
Freshly cut aerial parts of P. amarus were shade dried, pow-
dered (100 g) and extracted with ethanol:water (3 × 1000 mL,
50:50, v/v) at room temperature. The solvent was evaporated
under reduced pressure in a rotary evaporator at 40 ◦ C. The
residue (8.5 g) obtained was mixed with silica gel (16 g) to
form an admixture and chromatographed over silica gel col-
umn (500 mm length × 20 mm diameter, 63–200 ␮m particle size,
70–230 mesh size). Gradient elution was done in the following
sequence, hexane (100 mL) → hexane–ethyl acetate (200 mL; 98:2,
v/v) → hexane–ethyl acetate (200 mL; 95:5, v/v) → hexane–ethyl
acetate (500 mL; 90:10, v/v) → hexane–ethyl acetate (300 mL;
80:20, v/v). TLC was performed using hexane–acetone–ethyl
acetate (70:20:10, v/v/v) as the mobile phase. The fractions
(51–100) collected were combined, re-chromatographed and the
solvent evaporated to give 2.0 g of crude phyllanthin. The product
was purified by crystallization (3 times) in petroleum ether to afford
pure phyllanthin (yield, 1.23%).
2.4. Characterization data for phyllanthin
Colour: white crystals, mp: 96 ◦ C (uncorrected). UV max /nm
(methanol): 230 (molar absorptivity, log ε 4.31) and 280 (2.2); IR
/cm−1 (KBr): 3100 (C–H aromatic stretch), 2999, 2917, 2869 (C–H
aliphatic stretch), 1516, 1464 (C C ring stretch), 1182, 1140 (C–O–C
stretch), 964. 1 H NMR (500 MHz, CDCl3 , TMS): ı 6.75 [2H-5(5′ ),
d, J = 8.0], 6.65 [2H-6(6′ ), dd, J = 8.0, 1.9], 6.60 [2H-2(2′ ), d, J = 1.9],
3.83 [6H-4(4′ )-OCH3 , s], 3.78 [6H-3(3′ )-OCH3 , s], 3.30 [6H-9(9′ )-
OCH3 , s], 3.25 [4H-9(9′ ), m], 2.65 [4H-7(7′ ), m], 2.05 [2H-8(8′ ), m].
Anal. calcd. for C24 H34 O6 : C, 68.87%; H, 8.91%, Found: C, 68.78%;
H, 8.86%.
 R. Krithika et al. / Chemico-Biological Interactions 181 (2009) 351–358
2.5. DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging
assay
The antioxidative property of the plant extract and phyl-
lanthin was measured in vitro using DPPH, a stable free
radical [31]. The reaction mixture contained 0.1 mL of 1 mM
DPPH, 0.8 mL of 99% ethanol and 0.1 mL of P. amarus extract
(50–500 ␮g/mL) or phyllanthin (2.5–25 ␮mol/mL). The solution
was mixed rapidly and allowed to stand for 30 min in dark.
The scavenging activity was measured by noting the decrease in
absorbance at 517 nm as compared to DPPH control. The anal-
ysis was done in triplicates. The antioxidative property of the
tested plant and isolate were expressed as IC50 , which is defined
as the concentration required for inhibition of DPPH radical
by 50%.
2.6. Cell culture
HepG2 were obtained from ATCC, USA and grown on Eagle’s
minimum essential medium (EMEM, GIBCO, USA) supplemented
with 10% (v/v) heat inactivated fetal calf serum, 2 mM l-glutamine,
MEM non-essential amino acids, gentamycin (50 ␮g/mL) and peni-
cillin (100 ␮g/mL) [32]. Cells were passaged every 48–72 h in 1:3
ratio.
2.7. CCl4 treatment
HepG2 cells were seeded in 24-well plates at a concentration of
3 × 104 cells/mL EMEM/well and incubated for 48 h at 37 ◦ C under
5% CO2 to attain confluency. The cells were treated with 0.4% (v/v)
CCl4 in 0.25% DMSO prepared in serum free culture medium for
various time intervals of 0.16, 0.33, 0.5, 3, 6, 12 and 24 h and the
following assays were carried out.
2.7.1. Cell viability
Trypan blue exclusion test was carried out according to the
method of Wu and Cederbaum [33] with slight modification. Cells
were trypsinized from culture plates, pooled with floating cells
from medium and centrifuged at 1000 × g for 10 min at 4 ◦ C. They
were resuspended in 1 mL PBS and treated with 0.1 mL of 0.2%
trypan blue. Living cells were counted in haemocytometer under
a light microscope and results were calculated and expressed as
percentage of live cells compared to control using the following
formula
(Total number of cells − Trypan blue stained cells)
%Viability =
Total number of cells
× 100%
CCl4 treated group − group treated with P.amarus extract/phyllanthin
Percent recovery =
2.7.2. ALT and LDH leakage
The culture medium and cells were collected separately. The
medium (0.2 mL) was used for measuring activities of ALT and LDH.
The transaminase activity was measured by IFCC method as kinetic
reaction at 340 nm using kit procured from Agappe Diagnostics,
Ernakulam, Kerala, India. The collected cells were resuspended in
0.5 mL PBS and sonicated for 5 s in an ice bath. The LDH activity in
the medium and cell lysate was determined based on SCE kinetic
353
method using the kit procured from the same company. The cyto-
toxicity index based on the LDH assay was expressed as the ratio of
LDHmedium
× 100%
LDHlysate+medium
2.7.3. Lipid peroxidation
HepG2 cells were seeded in 24-well plates at a concentration of
3 × 104 /mL EMEM per well and treated with 0.4% CCl4 for various
time intervals. After trypsinization the cells were suspended in
0.5 mL of PBS and sonicated for 10 s. To this 0.5 mL of TCA–TBA
reagent was added and heated at 100 ◦ C for 1 h and centrifuged. The
extent of lipid peroxidation was quantified by estimating the levels
of malondialdehyde. The absorbance was measured at 535 nm and
the formation of thiobarbituric acid reactive substances was calcu-
lated using a molar extinction coefficient of 1.56 × 105 M−1 cm−1
[33].
2.7.4. Glutathione levels
The HepG2 cells were seeded and treated with CCl4 . The cells
were scraped and harvested by trypsinization and resuspended
in 0.5 mL PBS to which 0.5 mL of 10% TCA was added and cen-
trifuged. The supernatant was mixed with buffer A (0.1 M sodium
phosphate, 5 ␮M EDTA, 0.6 mM 5,5′ -dithio-bis 2-nitrobenzoic acid,
0.2 mM NADPH) and buffer B (10 U/mL glutathione reductase in
0.1 M sodium phosphate buffer) and its absorbance was read at
412 nm. The reduced glutathione levels in the HepG2 cells were
then determined by constructing a curve using reduced glutathione
as standard [33]. Protein content of the cells was determined using
standard bovine serum albumin [34].
The 24 h incubation period with 0.4% (v/v) concentration of CCl4
was found to be most suitable for our further experiments along
with P. amarus extract/phyllanthin.
2.8. Treatment with P. amarus/phyllanthin
HepG2 cells were plated in 24-well plate (3 × 104 cells/well in
1 mL of medium) and incubated at 37 ◦ C, under 5% CO2 . On attain-
ing confluency after 48 h the cells were treated with 0.5 mL each
of 0.4% (v/v) CCl4 dissolved in 0.25% DMSO in serum free culture
medium; 0.4% (v/v) CCl4 + P. amarus extract (200, 400, 600 ␮g/mL);
0.4% (v/v) CCl4 + phyllanthin [10 (4.18 ␮g/mL), 20 (8.36 ␮g/mL), 30
(12.54 ␮g/mL) ␮mol] for 24 h.
Trypan blue exclusion test, ALT and LDH leakage, lipid per-
oxidation and glutathione assays were performed using standard
methods as described in Section 2.7.
2.8.1. Percent recovery
The recovery observed after P. amarus extract/phyllanthin treat-
ment along with the toxin in three different concentrations was
calculated from the mean value of the respective groups in the form
of percent recovery [13] using the formula
× 100
CCl4 treated group − control group
2.9. Statistical analysis
The result of the DPPH assay was expressed as mean ± SEM. The
IC50 values of P. amarus extract and phyllanthin were calculated by
regression analysis. The HepG2 in vitro data were statistically ana-
lyzed using SPSS software, version 10. The results were expressed as
mean ± SEM. Hypothesis testing method included one-way Anal-
ysis of Variance (ANOVA) followed by least significant difference
(LSD) multiple comparison tests. The level of significance was
accepted with p < 0.05.
354 R. Krithika et al. / Chemico-Biological Interactions 181 (2009) 351–358
3. Results and discussion
3.1. Extraction, isolation, purification and structural confirmation
of phyllanthin
The plant extract in ethanol:water (50:50, v/v) showed
seven prominent spots on TLC plates with retention factors of
0.15, 0.32 (phyllanthin), 0.38, 0.51, 0.57, 0.63 and 0.72 using
hexane–acetone–ethyl acetate (70:20:10, v/v/v) as the mobile
phase. After extensive column chromatography, complete separa-
tion of phyllanthin was achieved with hexane–ethyl acetate (90:10,
v/v) solvent mixture. The retention time for phyllanthin on the
reversed-phase column was 22.3 min in a total run time of 40 min.
The capacity factor which describes the rate at which the analyte
migrates through the column was 3.95 based on the dead time of
4.5 min and the number of theoretical plates was 1989. The results
of HPTLC and reversed-phase HPLC analysis of plant extract and the
extracted phyllanthin were compared with standard phyllanthin
sample.
To ascertain the identity of the extracted compound, character-
ization was done with FT-IR, 1 H NMR (Fig. 1A), 13 C NMR (Fig. 1B),
mass and elemental analysis. In one of the earlier reports, Soman-
abandhu et al. [19] have unambiguously characterized phyllanthin
and hypophyllanthin by IR, 1 H NMR, 13 C NMR and mass spec-
troscopy. The results obtained in the present study were in good
Fig. 1. (A) 1 H NMR (500 MHz in CDCl3 with TMS as internal standard) and (B)
NMR (125 MHz in CDCl3 ) spectra of extracted phyllanthin.
agreement with their structural elucidation of phyllanthin. 1 H NMR
and 13 C NMR gave complete assignment for all the protons and car-
bon atoms. The aromatic protons at 6(6′ ) appear as double doublet
at ı 6.65 with J value of 8.1 and 1.8 due to long range coupling. A
multiplet at ı 2.65 and 3.25 were assigned to four benzylic protons
at 7(7′ ) and 9(9′ ) respectively. Mass spectrum showed a molecu-
lar ion peak at m/z 418 and a base peak at m/z 151 corresponding
to dimethoxy benzylic fragment (Fig. 2). The probable fragmenta-
tion pathway reveals ions at m/z 386 and 354 due to successive
elimination of methanol.
3.2. Antioxidative effect of P. amarus extract and phyllanthin
The DPPH radical scavenging activity of the extract and phyl-
lanthin is shown in Fig. 3A and B respectively. It was observed
that the DPPH free radical scavenging activity was concentration-
dependent in both cases and reaches a maximum at a concentration
of 300 ␮g/mL for P. amarus extract and 20 ␮mol/mL for phyllan-
thin. No difference in inhibition was noted with further increase
in concentration of either of the compounds. Our results indicated
that phyllanthin exhibited very high antioxidative property as com-
pared to P. amarus extract which is clearly evident by its low IC50
value of 7.4 ␮mol/mL.
The DPPH assay is based on the reduction of stable radical
DPPH to yellow coloured diphenyl picryl hydrazine. Thus, the abil-
ity of the tested products to quench this radical is a measure of its
antioxidative ability. Many previous studies have reported strong
to moderate DPPH free radical activity of crude extracts of plants
belonging to Phyllanthus species [6] and phyllanthin [35].
3.3. Carbon tetrachloride-induced lipid peroxidation in HepG2
cells
HepG2 cell line has been proposed as an alternative model to
hepatocytes. These are used in various metabolic and drug tox-
icity studies [36]. They have added advantage of being available
in plenty, easy maintenance, rapid cryopreservation and ability to
retain drug metabolic and enzyme activities [37]. HepG2 possess
many of the morphological and biochemical features of normal hep-
atocytes [38]. Since it retains many of the phenotypic and genotypic
characteristics of liver cells, this cell line has been used in vari-
ous studies related to medicinal plants for their liver protecting
property [29,39].
The CCl4 mediated liver injury in vitro is reported in the literature
to be caused by two components, a direct solvent effect or by gener-
ation of free radicals and subsequent lipid peroxidation depending
on its concentration, the duration of exposure and the sensitivity of
the in vitro system to which it is added [40]. Many previous reports
suggest involvement of lipid peroxidation as a major mechanism of
cellular damage in HepG2 cells by a variety of toxicants [39,41].
Berger et al. [42] have reported the induction of membrane dam-
age in rat hepatocyes by virtue of direct solvent effect of CCl4 at
concentrations commonly used in in vitro studies. According to
them exposure to a maximum concentration of 4 ␮L CCl4 as a 20%
solution in ethanol resulted in immediate and almost complete
destruction of hepatocytes as assessed by various markers of liver
injury. They have observed a rapid change in all parameters stud-
ied in the first 10–30 min after exposure to this toxicant, which
they postulate to have been caused by the direct solvent action
affecting the membranes, which was not protected by antioxidants.
Similarly they have also quantified MDA levels as a marker of lipid
peroxidation in those cells. A modest rise in the MDA content after
a minimum exposure period of 3 h and a highly significant eleva-
13
C tion after 3 h of incubation with CCl4 was observed by them. This
second phase of damage was marked by significant reduction in
 R. Krithika et al. / Chemico-Biological Interactions 181 (2009) 351–358
Fig. 2. Electron impact mass spectra of phyllanthin with probable fragments at m/z 418 (molecular ion peak), 386, 354, 203, 177, 151 (base peak) and 107.
Fig. 3. DPPH scavenging activity of (A) P. amarus extract and (B) phyllanthin showing
IC50 values of 162.3 ␮g/mL and 7.4 ␮mol/mL respectively.
355
the intracellular glutathione levels and was effectively mitigated
by promethazine an exogenous antioxidant.
To ascertain the extent of lipid peroxidation (Fig. 4A) caused by
0.4% of CCl4 in HepG2 cells, we quantified the MDA formed and
intracellular GSH content at various time intervals of 0.16, 0.33,
0.5, 3, 6, 12 and 24 h. Trypan blue exclusion (Fig. 4B), ALT (Fig. 4C)
and LDH (Fig. 4D) levels were also determined. As compared to
control cells, no significant change in lipid peroxidation as well
as glutathione depletion, trypan blue exclusion, ALT and LDH lev-
els were observed until an exposure period of 3 h, thus overruling
any direct solvent mediated damage by CCl4 in HepG2 cells. There
after a time-dependent increased leakage of ALT and LDH and loss
of cell viability was observed. Similarly, increased lipid peroxida-
tion with concurrent decrease in glutathione was noted when cells
were incubated with CCl4 for 24 h. Our experiment thus proved
the involvement of lipid peroxidation on exposure to CCl4 in a
time-dependent manner as evident by increase in MDA levels of
these cells which paralleled well with other parameters indicative
of hepatocellular damage. Duthie et al. [37] have observed a sim-
ilar 50% increase in MDA levels in HepG2 cells after exposure to
a 10 mM concentration of CCl4 for a period of over 3 h. Holden et
al. [43] have reported that CCl4 caused a time-dependent produc-
tion of ROS and subsequent lipid peroxidation mediated through
Interleukin 8 in HepG2 cells which was found to be maximum
after an incubation period of 24 h. Harries et al. [32] have stan-
dardized the concentration and exposure time for CCl4 -induced
cytotoxicity on HepG2 cells by studying the gene expression pro-
files specific to CCl4 exposure. They have reported the involvement
of lipid peroxidation via free radical formation in these cells after
challenge with this hepatotoxin. There results indicated that when
HepG2 cells were exposed to CCl4 for a time period of only 2 h,
the number of genes which were up-regulated was negligible
and only 7 genes were down-regulated. But prolonged exposure
356 R. Krithika et al. / Chemico-Biological Interactions 181 (2009) 351–358

Fig. 4. Time-dependent changes observed in (A) lipid peroxidation and GSH levels (B) trypan blue exclusion test (C) ALT and (D) LDH leakage after exposure to 0.4% of CCl4 in
HepG2 cells. Group 1 - Control; Group 2 - DMSO control (0.25% (v/v); Group 3 - 0.4% (v/v) CCl4 in 0.25% DMSO. Results are expressed as mean ± SEM (n = 5). Level of significance
p < 0.05. a Compared to vehicle control.

Table 1
Protective effect of P. amarus extract and phyllanthin on CCl4 -induced toxicity in HepG2 cell line.

S. No. Experimental groups Trypan blue exclusion ALT (U/L) LDH (%) MDA (nmol/mg Glutathione (nmol/mg
(%) protein) protein)

(I) Control
1. Control 99.95 ± 0.24 12.43 ± 0.22 8.36 ± 0.16 1.65 ± 0.09 31.69 ± 0.37
2. DMSO control (0.25%, v/v) 99.93 ± 0.45 12.77 ± 0.51 8.56 ± 0.52 1.65 ± 0.04 32.40 ± 0.57
3. P. amarus control extract 99.95 ± 0.67 12.54 ± 0.30 8.35 ± 0.41 1.61 ± 0.06 32.42 ± 1.01
(600 ␮g/mL)
4. Phyllanthin control (30 ␮mol) 99.95 ± 0.32 12.51 ± 0.63 8.41 ± 0.27 1.67 ± 0.04 32.71 ± 1.60

(II) Toxin treatment

5. Carbon tetrachloride (0.4%, v/v) 42.84 ± 0.46a 40.73 ± 0.32a 70.13 ± 2.12a 7.70 ± 0.36a 14.50 ± 0.45a

(III) Phyllanthus amarus treatment

6. CCl4 (0.4%, v/v) + P. amarus extract 55.15 ± 0.47b (21.6) 33.10 ± 0.86b (27.28) 60.69 ± 0.58b (15.33) 5.05 ± 0.12b (43.80) 19.84 ± 0.63b (29.83)
(200 ␮g/mL)
7. CCl4 (0.4%, v/v) + P. amarus extract 62.93 ± 0.65b (35.0) 27.32 ± 0.26b (47.96) 40.02 ± 0.41b (48.90) 3.93 ± 0.09b (62.31) 23.03 ± 0.34b (47.63)
(400 ␮g/mL)
8. CCl4 (0.4%, v/v) + P. amarus extract 72.99 ± 0.35b (52.8) 21.51 ± 0.32b (68.74) 19.80 ± 0.69b (81.74) 2.75 ± 0.91b (81.81) 27.38 ± 0.63b (71.95)
(600 ␮g/mL)
(IV) Phyllanthin treatment
9. CCl4 (0.4%, v/v) + Phyllanthin 68.02 ± 0.34b (44.1) 29.17 ± 1.42b (41.34) 48.11 ± 1.41b (35.76) 3.56 ± 0.12b (68.42) 22.02 ± 0.54b (43.01)
(10 ␮mol)
10. CCl4 (0.4%, v/v) + Phyllanthin 87.20 ± 0.41b (77.7) 20.58 ± 0.40b (72.06) 27.31 ± 0.56b (69.54) 2.40 ± 0.13b (87.60) 27.47 ± 0.42b (72.45)
(20 ␮mol)
11. CCl4 (0.4%, v/v) + Phyllanthin 99.00 ± 0.43bc (98.4) 12.81 ± 0.55bc (99.85) 8.59 ± 0.27bc (99.95) 1.72 ± 0.05bc (98.84) 32.38 ± 0.59bc (99.88)
(30 ␮mol)

Results are expressed as mean ± SEM (n = 5). Values shown in parenthesis indicate percent recovery. No significant difference was noted between groups 1, 2, 3 and 4.
a
Compared to vehicle control (group 2) and CCl4 treated (group 5); b compared to CCl4 treated (group 5) and CCl4 + antidote treated (groups 6, 7, 8, 9, 10 and 11); c compared
to CCl4 + highest dose of phyllanthin (group 11) and CCl4 + highest dose of P. amarus (group 8). Level of significance p < 0.05.
 R. Krithika et al. / Chemico-Biological Interactions 181 (2009) 351–358
until a time period of 24 h gave rise to several changes in gene
expression.
Marinovich et al. [44] have observed that HepG2 cells were
some what less sensitive to toxic effects of CCl4 compared to
isolated rat hepatocytes requiring higher dosage (33 and 23 ␮L
respectively) and longer incubation time (8 and 2 h respectively)
to obtain a comparable degree of damage between the two. They
concluded that HepG2 cells could effectively act as substitute for
hepatocytes in toxicity studies as they form a homogenous cell
population, possessing longer life span with no involvement of
animals. Based on our results and other reports [32,43] a 24 h incu-
bation period was chosen to study the protective effects of P. amarus
extract and phyllanthin against CCl4 mediated toxicity in HepG2
cells.
3.4. Cytoprotective effect of P. amarus extract and phyllanthin in
HepG2 cells
Table 1 depicts the results of trypan blue exclusion test, leakage
parameters—ALT and LDH, LPO and GSH levels in all experimen-
tal groups. Trypan blue exclusion test was used as a standard
indicator of cellular viability. It was observed that surviving cells
did not take up the trypan blue stain and were counted. Treat-
ment with CCl4 caused significant loss of viability of cells as
measured by this assay. Both P. amarus extract and phyllanthin
treatments along with CCl4 significantly increased cell viability.
The highest concentration of phyllanthin (30 ␮mol/mL) was most
effective as compared to all concentrations of P. amarus extract.
The extent of cellular damage was measured in terms of release of
leakage enzymes—ALT and LDH. Increased release of these intra-
cellular enzymes was observed at 24 h exposure to CCl4 . This
indicated membrane damage and instability owing to oxidative
injury created by the hepatotoxin. Likewise, toxin treatment caused
significant increase in MDA levels, with a concurrent decrease in
glutathione content in HepG2 cells. However, no significant dif-
ference is noted in all parameters among control groups (Group
1, 2, 3 and 4). Glutathione plays a unique role in cellular defense
against active oxygen species and reactive intermediates. It is estab-
lished that cellular oxidative stress is often preceded by depletion
of intracellular GSH [45]. Glutathione has the capacity to quench
reactive metabolites and free radical intermediates during con-
ditions of oxidative stress. Thus, glutathione exhaustion results
in lipid peroxidation, which is believed to cause hepatocellular
damage [46]. The CCl4 -induced changes in the HepG2 cells were
significantly ameliorated by co-treatment of the toxin along with
the P. amarus extract/phyllanthin. It was observed that the high-
est concentration of phyllanthin used (30 ␮mol/mL) exhibited the
maximum protective effect towards various parameters studied as
compared to all other concentrations of phyllanthin and P. amarus
extract.
The overall experimental strategy followed evaluated the plau-
sible protective nature of P. amarus extract and phyllanthin against
CCl4 -induced toxicity in HepG2 cell line. The loss of cell viability
was measured as the end product of toxicity by means of trypan
blue assay. The mechanism involved was ascertained by select-
ing relevant parameters like leakage of enzymes, quantification of
lipid peroxidation and measurement of intracellular glutathione
levels. The possible underlying mechanism for the hepatoprotec-
tive effect of phyllanthin is because of its ability to inhibit lipid
peroxidation and maintenance of glutathione in reduced state by
virtue of its antioxidative powers. These findings suggests that
phyllanthin can be used for treatment of liver diseases, however,
further studies are required to have a clear insight to understand the
molecular mechanism responsible for its hepatoprotective prop-
erty.
357
4. Conclusion
The present study reported the isolation and characterization
of phyllanthin from P. amarus. Oxidative stress was introduced in
HepG2 cell line by means of free radical inducer CCl4 . Treatment
with P. amarus extract/phyllanthin along with the toxin effectively
improved cell viability, prevented leakage of enzymes, reduced lipid
peroxidation and improved glutathione status in a concentration-
dependent manner. To conclude, data suggested that phyllanthin
treatment exhibited much higher antioxidative and hepatoprotec-
tive property in smaller concentrations as compared to the extract
of P. amarus.
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgements
The financial assistance to Krithika, R. from Birla Smarak Kosh
in the form of Junior Research Fellowship is acknowledged with
thanks. The authors thank the Director, King Institute of Preventive
Medicine, Chennai, for granting permission and providing neces-
sary infrastructural facilities to carry out HepG2 cell line work. The
authors also gratefully acknowledge the instrumentation facility
provided by IIT, Madras and Zydus Cadila, Ahmedabad. The authors
also extend their sincere thanks to Ms. Aishwariya Bhaskaran, for
her valuable support in literature collection.
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