Estimation of phyllanthin and hypophyllanthin by high performance liquid chromatography in Phyllanthus amarus


Sharma, A.; Singh, R.T.; Handa, S.S.

Phytochemical Analysis. 4(5): 226-229

1993


A reversed phase high performance liquid chromatographic procedure for standardizing a popular hepatoprotective Indian herb "Bhumyamlaki" (Phyllanthus amarus) on the basis of two of its bioactive lignans, phyllanthin and hypophyllanthin, has been developed. The method has been found to be sensitive, precise and to record more than 98% recovery of the two lignans. The leaves of P. amarus were found to contain the highest amounts of phyllanthin (0.7% w/w) and hypophyllanthin (0.3% w/w) as compared to the other parts of the plant.

PHYTOCHEMICAL
ANALYSIS,
VOL.
4,
226-229
(1993)
Estimation
of
Phyllanthin
and
Hypophyllanthin
by
High
Performance
Liquid
Chromatography
in
Phyllanthus
amarus
Anupam
Sharma,
Ravneet
T.
Singh
and
Sukhdev
S.
Handa
*
ICMR
Centre
for
Advanced
Research
on
Standardization,
Quality
Control
and
Formulation
of
Traditional
Remedies/Natural
Products,
Department
of
Pharmaceutical
Sciences,
Panjab
University,
Chandigarh
160014,
India
A
reversed
phase
high
performance
liquid
chromatographic
procedure
for
standardizing
a
popular
hepatopro-
tective
Indian
herb
"Bhumyamlaki"
(Phyllanthus
amarus)
on
the
basis
of
two
of
its
bioactive
lignans,
phyllanthin
and
hypophyllanthin,
has
been
developed.
The
method
has
been
found
to
be
sensitive,
precise
and
to
record
more
than
98%
recovery
of
the
two
lignans.
The
leaves
of
P.
amarus
were
found
to
contain
the
highest
amounts
of
phyllanthin
(0.7%
w
/w)
and
hypophyllanthin
(0.3%
w/w)
as
compared
to
the
other
parts
of
the
plant.
Keywords:
Phyllanthus
amarus;
phyllanthin;
hypophyllanthin;
lignans;
high
performance
liquid
chromatogra-
phy.
INTRODUCTION
Phyllanthus
amarus
Schum.
and
Thonn.
(Euphorbia-
ceae),
known
popularly
in
the
Indian
System
of
Medicine
as
"Bhumyamlaki",
has
been
traditionally
used
in
the
treatment
of
a
variety
of
ailments
including
hepatic
disorders
(Kirtikar
and
Basu,
1933;
Nadkarni,
1976).
The
plant
is
currently
being
investigated
by
the
Indian
Council
of
Medicinal
Research
(ICMR)
under
its
multicentric
double-blind
clinical
trial
programme
to
evaluate
its
efficacy
in
the
treatment
of
viral
hepatitis.
P.
amarus
has
been
reported
to
exhibit
marked
anti-
hepatitis
B
virus
surface
antigen
activity
both
in
in
vivo
(Thyagarajan
et
al.,
1988;
Blumberg
et
al.,
1989,
1990a,b;
Thumlikitkul
et
al.,
1991;
Shead
et
al.,
1992)
and
in
vitro
studies
(Mehrotra
et
al.,
1991;
Unander
and
Blumberg,
1991)).
Furthermore,
the
plant
has
been
reported
to
possess
antibacterial,
anticrustacean,
antifungal
and
antiviral
activities
(Verpoorte
and
Dihal,
1987;
Macrae
et
al.,
1988).
The
major
lignans,
phyllanthin
(1)
and
hypophyllanthin
(2),
reported
from
P.
niruri
(Row
et
al.,
1966)
and,
in
the
present
work,
from
P.
amarus
have
been
shown
to
exhibit
antihepato-
toxic
activity
against
carbon
tetrachloride
and
galacto-
samine
in
primary
cultured
rat
hepatocytes
(Syama-
*
Author
to
whom
correspondence
should
be
addressed.
Me0
CH
2
OMe
Me0
CH
2
OMe
OMe
OMe
1
sunder
et
al.,
1985).
No
method
of
standardizing
this
potentially
bioactive
plant
has
been
reported
to
date.
The
only
report
available
for
P.
amarus
is
on
its
macro-
and
micro-morphology
(De
and
Datta,
1990).
A
method
has
therefore
been
developed
to
standardize
1
and
2
using
high
performance
liquid
chromatography
(HPLC).
The
method
has
been
used
for
estimating
these
lignans
in
different
parts
of
the
plant
and
also
the
plant
material
packed
in
the
capsules
prepared
at
this
centre
for
the
clinical
trials
being
conducted
by
the
ICMR.
EXPERIMENTAL
Plant
material.
P.
amarus,
dried
whole
plant,
was
obtained
through
the
courtesy
of
the
Central
Drug
Research
Institute
(CDRI),
Lucknow,
and
the Central
Biostatistical
Monitoring
Unit,
Institute
for
Research
in
Medical
Statistics,
Madras.
The
identity
of
the
plant
material
received
from
these
two
sources
(hereinafter
referred
to
as
the
Lucknow
and
Madras
samples)
was
reconfirmed
by
comparison
with
reference
specimens
of
P.
amarus
preserved
at
the
Herbarium-cum-Museum
of
the
Department
of
Pharmaceutical
Sciences,
Panjab
University,
Chandigarh.
Estimation
of
lignans
was
performed
on
the
roots,
CH
2
OMe
Me0
CH
2
OMe
Me0
OMe
O
\-0
2
0958-0344/93/050226-04
$07.00
Received
6
October
1992
C)
1993
by
John
Wiley
&
Sons,
Ltd.
Accepted
(revised)
12
April
1993
LIGNANS
IN
PHYLLANTHUS
AMARUS
227
Table
1.
Mean
peak
areas
obtained
from
HPLC
analysis
of
phyllanthin
and
hypophyllanthin
Peak
area
(eV
x
sec)
Amount
injected
(Mean'
±
SE)
(4g)
Phyllanthin
Hypophyllanthin
0.05
323±6.5
245±
8.6
2
0.10
591
±
38.3
793
±
35.6
0.20
1069
±
177.3
1483
±
169.8
0.40
2069
±
128.2
2882
±
116.3
0.80
4108±97.1
5855
±
283.8
1
1.00
4987
±
181.6
7478
±
390.9
2.00
9539
±
173.5
13
906
±
125.4
3.00
13
956
±
186.6
15
667
±
225.2
4.00
18
775
±
176.3
20
924
±
212.1
n=3.
stem,
branches,
leaves
and
fruits,
collected
manually
from
the
whole
plant,
and
also
in
the
materialt
used
for
preparing
the
capsules
(hereinafter
referred
to
as
cap-
sule
material)
for
the
double-blind
clinical
studies
for
viral
hepatitis.
HPLC
analysis.
A
Waters
chromatographic
system
com-
prising
a
Model
510
delivery
pump,
a
Model
680
gradi-
ent
controller,
a
Model
U6K
injector,
a
Model
481
UV
detector
and
a
Model
730
integrator
was
employed.
A
jtBondapak
C18
column
(30
cm
x
3.9
mm)
was
employed
for
isocratic
resolution
of
phyllanthin
and
hypophyllanthin
using
methanol
:
water
(66
:
34)
as
the
mobile
phase
at
a
flow-rate
of
1.8
mL/min.
The
detec-
tor
was
operated
at
230
nm.
The
two
lignans
were
isolated
from
P.
amarus following
the
method
reported
by
Row
et
al.
(1966),
and
the
identity
of
the
lignans
was
confirmed
by
comparing
the
optical
rotation,
UV,
IR
and
1
H
NMR
spectra
of
the
isolates
with
those
reported
in
the
literature
(Anjaneyulu
et
al.,
1973).
These
com-
pounds
were
used
as
external
standards
for
their
esti-
mation
in
the
plant
samples.
All
analyses
were
run
in
triplicate.
Calibration
curves.
1
and
2
were
dissolved
separately
in
methanol
to
obtain
solutions
containing
2
mg/mL
of
each.
Three
separate
sets
of
such
stock
solutions
were
t
For
the
preparation
of
the
capsules
for
clinical
trials,
dried
plant
material
comprising
the
tender
aerial
parts
of
P.
amarus
(received
from
CDRI)
was
garbled
and
passed
through
a
coarse
sieve
(#18)
to
separate
out
the
finer
material.
The
composition
of
the
sieved
material,
as
determined
by
manual
separation
from
aliquot
samples
and
weighing,
was
observed
to
be
75-76%
w/w
leaves
and
24--
25%
w/w
rachis/fruits.
This
material
was
ground
to
a
fine
powder
(#100)
prior
to
filling
into
hard
gelatin
capsules.
0
5
10
15
20
min
0
5
10
15
20
min
(
a
)
(b)
Figure
1.
Chromatograms
of
(a)
hypophyllanthin
(peak
1)
and
phyllanthin
(peak
2)
and
(b)
leaves
of
P.
amarus.
prepared.
The
solutions
of
the
two
lignans
were
mixed
in
equal
proportions
to
obtain
a
working
stock
solution
of
standards
containing
1
mg/mL
of
each
lignan.
Dilutions
containing
0.05,
0.10,
0.20,
0.40,
0.80,
1.00,
2.00,
3.00
and
4.00
µg/mL
each
of
1
and
2
were
pre-
pared
from
the
working
stock
solution.
Aliquots
(10
!IL)
of
each
dilution
were
subjected
(in
triplicate)
to
HPLC,
and
the
area
under
the
peak
of
either
lignan
was
recorded
(Table
1).
Figure
1(a)
shows
a
typical
chromatogram
of
the
mixture
of
the
two
lignans.
Standard
plots
(Fig.
2)
of
phyllanthin
and
hypophyllan-
thin
were
drawn
by
plotting
mean
peak
area
against
the
injected
amount.
Optimization
of
the
extraction
procedure.
The
capsule
mat-
erial
(1
g)
was
mixed
with
lime
(300
mg)
and
water
(2.5
mL),
and
macerated
at
room
temperature
for
18
h.
The
macerated
material
was
refluxed
in
a
boiling
water
bath
with
methanol
(30
mL)
containing
3%
potassium
hydroxide.
Three
reflux
periods
were
investigated:
30
min,
60
min
and
90
min.
The
refluxed
material
was
filtered,
the
residue
washed
with
methanol
(3
x
5
mL),
Table
2.
Amount
of
lignans
extracted
by
repeated
extraction
of
P.
amarus-
containing
capsules
Reflux
time
(min)
Extraction
cycle
Mean'
±
SE
(8g)
Phyllanthin
Hypophyllanthin
Cumulative
%
extracted
b
Phyllanthin
Hypophyllanthin
30
1
0.958
±
0.003
0.265
±
0.006
60
1
1.460`
±
0.010
0.402
d
±
0.006
83.14
84.81
2
0.296
±
0.005
0.072
±
0.004
100.00
100.00
3
NDe
N
D
e
90
1
1.390`
±
0.011
0.368
d
±
0.004
n
=
3.
b
Calculated
on
the
basis
of
total
extractable
quantity.
"
Statistically
insignificant
difference
(p=
0.05;
t-test).
e
ND
=
not
detected.
228
A.
SHARMA
ET
AL.
20
16
(/)
Z
12
M"
8
=4
ro
a)
0
1.0
2.0
3.0
4.0
Amount
(lig)
Figure
2.
Standard
plots
of
phyllanthin
(-LI-)
and
hypophyllanthin
(-0--)
Table
3.
Recovery
of
phyllanthin
and
hypophyllanthin
from
P.
amarus-
containing
capsules
Sample
Phyllanthin
Content
(µg/mg)
Control
Spiked'
3.404
4.386
Recovery
1%)
98.2
Hypophyllanthin
Content
(ug/mg)
Recovery
Control
Spiked'
1%)
0.828
1.818
99.0
2
3.388
4.372
98.4
0.842
1.836
99.4
3
3.420
4.408
98.8
0.882
1.874
99.2
Mean
±
SD
98.5
±
0.31
99.2
±
0.200
a
1
mg
each
of
phyllanthin
and
hypophyllanthin
added
to 1
g
of
the
control
sample.
and
volume
of
the
combined
filtrate
and
washings
was
made
up
to
50
mL.
A
sample
(10
[AL)
of
this
solution
was
subjected
to
HPLC
and
the
lignans
were
estimated
(Table
2).
The
mart
from
the
60
min
reflux
period
was
again
extracted
twice
(1
h
each
time),
the
resulting
Table
4.
HPLC
estimation
of
phyllanthin
and
hypophyllan-
thin
in
different
plant
parts
of
P.
amarus
(Lucknow
and
Madras
samples)
and
in
the
capsule
material.
Plant
part
and
source
Roots
Mean'
-
±SE
(%
w/w,
dry
basis)
Phyllanthin
Hypophyllanthin
Lucknow
0.012
b
±
0.0006
0.003`
±
0.0003
Madras
0.003
b
±
0.000
0.010`
±
0.000
Stem
Lucknow
0.064
±
0.002
0.016
±
0.0006
Madras
0.058
±
0.0006
0.013
±
0.0003
Branches
Lucknow
0.011
d
±
0.0003
0.027
±
0.001
Madras
0.067
d
±
0.001
0.022
±
0.0003
Leaves
Lucknow
0.709
±
0.051
0.271
±
0.019
Madras
0.685
±
0.019
0.271
±
0.020
Fruits
Lucknow
0.017
±
0.0003
0.004
±
0.000
Madras
0.015
±
0.0007
0.004
±
0.000
Capsule
material
0.510
±
0.022
0.181
±
0.008
a
n=
3.
b
'
c
'
d
Statistically
significant
difference
(
p=0.05;
t-test).
solutions
concentrated
under
reduced
pressure,
their
volumes
made
up
to
5
mL
and
the
two
lignans
were
quantitated
using
10
[AL
of
each
solution.
Since
the
reflux
period
involving
two
cycles
each
of
60
min
exhibited
optimum
extraction
(Table
2),
this
procedure
was
consequently
used
for
all
successive
analyses.
Efficiency
of
HPLC
assay.
The
efficiency
of
the
extraction
procedure
was
determined
by
estimating
the
recovery
of
1.00
mg
each
of
1
and
2
added
to
1g
of
capsule
material
and
comparing
with
that
of
an
unspiked
control
sample.
The
samples
for
HPLC
analysis
were
prepared
as
described
above
using
two
cycles
each
of
the
60
min
reflux
period,
pooling
the
resulting
solu-
tions,
concentrating
under
reduced
pressure
and
finally
making
up
their
volume
to
50
mL.
A
10
[AL
sample
of
this
solution
was
employed
for
HPLC
analysis
of
the
control
as
well
as
of
the
spiked
samples.
Table
3 shows
the
recovery
percentage
of
the
two
lignans.
Estimation
of
phyllanthin
and
hypophyllanthin
in
test
sam-
ples.
Roots,
stem,
branches,
leaves
and
fruits
of
Lucknow
and
Madras
samples
of
P.
amarus
(each
ground
to
a
fine
powder)
and
the
capsule
material
were
extracted
following
the
aforementioned
procedure.
The
pooled
solutions
from
two
extraction
cycles
were
concentrated
under
reduced
pressure
and
their
volume
was
made
up
to
5
mL
in
all
cases
except
for
the
leaves
and
the
capsule
material
where
the
volume
was
made
up
to
50
mL.
Figure
1(b)
shows
as
typical
chromato-
LIGNANS
IN
PHYLLANTHUS
AMARUS
229
gram
of
leaves
of
P.
amarus.
Table
4
shows
the
mean
content
of
phyllanthin
and
hypophyllanthin
in
the
test
samples
estimated
by
HPLC
using
10µL
of
each
test
solution.
RESULTS
AND
DISCUSSION
An
HPLC
method
for
the
standardization
of
P.
amarus
has
been
attempted
using
two
of
its
bioactive
lignans,
phyllanthin
(1)
and
hypophyllanthin
(2),
as
markers.
Mean
retention
times
for
hypophyllanthin
and
phyllan-
thin
under
the
conditions
described
above
were
observed
to
be
8.6
min
(capacity
factor,
k'
=3.5)
and
9.6
min
(k'
=4.2),
respectively.
The
calibration
curve
for
phyllanthin
was
found
to
be
linear
over
the
range
0.05-4.00m/mL
while
that
of
hypophyllanthin
exhi-
bited
linearity
over
the
range
0.05-2.00n/mL
(Fig.
2).
The
respective
regression
equations
for
phyllanthin
and
hypophyllanthin
were:
y
=
207.71
+
4636.16x
(r
2
=
0.99984)
and
y
=
105.81
+
7011.28x
(r
2
=
0.99917).
The
mean
coefficient
of
variation
for
the
peak
areas
of
replicate
injections
did
not
exceed
3.06.
Extraction
of
the
capsule
material
using
three
reflux
periods
(30,
60
and
90
min)
showed
that
a
60
min
reflux
time
extracts
a
maximal
amount
of
the
two
lignans
(Table
2).
Increasing
the
reflux
time
to
90
min
had
no
added
effect
on
the
amount
of
phyllanthin
and
hypo-
phyllanthin
extracted,
as
is
obvious
from
the
statistical
insignificance
(
p
=
0.05;
t-test)
between
the
yields
during
the
60
min
and
90
min
reflux
periods.
Thus,
a
60
min
reflux
time
was
chosen
from
among
the
three
extraction
periods
investigated,
and
to
ensure
complete
extraction
of
phyllanthin
and
hypophyllanthin
the
resi-
dual
marc
from
the
first
extraction
cycle
(60
min)
was
subjected
to
two
successive
extractions
each
of
60
min.
Neither
phyllanthin
nor
hypophyllanthin
could
be
detected
in
the
solution
obtained
from
the
third
extrac-
tion.
However,
the
second
extraction
yielded
about
16%
of
the
lignans
that
were
not
extracted
during
the
first
cycle
(Table
2).
Thus,
the
optimal
conditions
for
isolation
of
extractable
amounts
of
the
two
lignans
were
observed
to
be
two
successive
cycles
of
extraction
each
of
60
min,
and
these
conditions
were
adhered
to
for
the
quantitation
of
phyllanthin
and
hypophyllanthin
in
all
the
test
samples.
Recoveries
calculated
from
the
concentration
differ-
ences
between
the
control
and
the
spiked
capsule
material
(Table
3)
showed
that
more
than
98%
of
phyllanthin
and
hypophyllanthin
could
be
extracted
using
the
extraction
procedure
described
above.
The
highest
amounts
of
phyllanthin
(-0.7%
w/w)
and
hypophyllanthin
(-0.3%
w/w)
were
found
in
the
leaves
of
P.
amarus
(Table
4).
The
phyllanthin
content
varied
significantly
in
the
roots
and
the
branches
of
the
Lucknow
and
Madras
samples,
whereas
hypophyllan-
thin
exhibited
significant
variations
in
the
roots
only
from
these
two
sources.
P.
amarus-containing
capsules
were
estimated
to
contain
0.510%
w/w
and
0.181
w/w
phyllanthin
and
hypophyllanthin,
respectively.
CONCLUSION
The
assay
method
described herein
is
simple,
precise
and
capable
of
detecting
and
reproducibly
quantifying
concentrations
as
low
as
0.05m/mL
of
phyllanthin
and
hypophyllanthin,
the
two
bioactive
lignans
in
P.
amar-
us.
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