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Characterizing the antioxidant activity of amla (Phyllanthus emblica) extract

Author(s): S. M. Khopde, K. Indira Priyadarsini, H. Mohan, V. B. Gawandi, J. G. Satav, J. V.
Yakhmi, M. M. Banavaliker, M. K. Biyani and J. P. Mittal
Source: Current Science, Vol. 81, No. 2 (25 July 2001), pp. 185-190
Published by: Current Science Association
Stable URL: https://www.jstor.org/stable/24106256
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 RESEARCH ARTICLE

Characterizing the antioxidant activity of

(Phyllanthus emblica) extract

S. Μ. Khopdef, Κ. Indira Priyadarsini1, Η. Mohanf, V. Β. Gawand

J. G. Satav*, J. V. Yakhmi1, Μ. M. Banavaliker**, Μ. K. Biyani*
J. P. Mittag*'*
'Chemistry Group, and 'Radiation Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, I
**Ajanta Pharma Ltd, Kandivali, Mumbai 400 067, India
"Also with the Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560 064, India

Amla is well-known for its rich vitamin C (ascorbic believed that the major constituent responsible for these
acid) and polyphenol contents. To assess its antioxi activities is vitamin C (ascorbic acid). Ascorbic acid
dant activity, we examined aqueous amla extract for shows antioxidant, anti-inflammatory and antimutagenic
its ability to inhibit y-radiation-induced lipid peroxi properties5-7. It is a very effective free-radical scaven
dation (LPO) in rat liver microsomes and superoxide ger. However, there are some in vivo studies indicating
dismutase (SOD) damage in rat liver mitochondria. that antioxidant activities of amla cannot be attributed
For the LPO experiment, amla extract was added as to ascorbic acid alone and that the overall effect is due
its aqueous solution; and irradiation was carried out
to other polyphenols such as ellagic acid, gallic acid,
at different time intervals. The extent of LPO was
measured in terms of thiobarbituric acid reactive
tannins, etc.8-10. It is in fact reported that autoxidation
substances. It was observed that the amla extract of ascorbic acid can actually increase free-radical pro
acts as a very good antioxidant against y-radiation duction". In the present paper, we studied the effect of
induced LPO. Similarly, it was found to inhibit the aqueous amla extract on the y-radiation-induced lipid
damage to antioxidant enzyme SOD. The antioxidant peroxidation (LPO) in rat liver microsomes and inhibi
activity of the amla extract was found to be both tion of superoxide dismutase (SOD) enzyme. Attempts
dose- and concentration-dependent. The amount of have also been made to understand the role of ascorbic
ascorbic acid in amla was standardized by HPLC acid and the antioxidant equivalents in its activity.
and titrimetric methods and was found to be 3.25 to
4.5% w/w. However in microsomes containing this
Materials and methods
composition of pure ascorbic acid alone, no inhibi
tion in LPO was observed. Cyclic voltammetry
of the amla extract was carried out to estimate the Thiobarbituric acid (TBA), butylated hydroxytolue
ascorbic acid equivalents, which was found to be (BHT), ascorbic acid and epinephrine were obtained
9.4% w/w of amla. This value was found to be in
from Sigma Chemicals. All the other reagents were
agreement when compared with the reactivity of reagent grade. Nitrous oxide (N20) gas, o
analytical
both amla and ascorbic acid towards ARTS'" radical,
tained from Indian Oxygen Ltd, Mumbai was of IOLA
a stable free-radical. Based on these results it is con
grade purity.
cluded that amla is a more potent antioxidant than
vitamin C.

Preparation of amla extract

Plant and plant products are being used as a source of
medicine since long. Among the most important Fresh
con fruits were freed from foreign matter like dust
stituents of edible plant products, low molecular weight
other organic matter. The cleaned raw material was the
antioxidants are the most important species. It is known
commuted to reduce its size. The commuted raw mate
that consumption of fruits and vegetables is essential
rial was extracted with the extraction medium and con
for normal health of human beings. Vegetarian diet can into powder form. Since the extract is
verted
reduce the risk of cancer, atherosclerosis, etc. Phyllan
hygroscopic, enough care was taken while handling the
thus emblica, also known as amla, has been used insample.
Ay
urveda, the ancient Indian system of medicine. It has
been used for treatment of several disorders such as
Method of estimation of ascorbic acid
common cold, scurvy, cancer and heart diseases1-4. It is
Sample solution equivalent to 0.2 mg ascorbic acid/ml
*For correspondence, (e-mail: mittaljp@magnum.bare.ernet.in)
was prepared in water containing 3% w/v metaphos

CURRENT SCIENCE, VOL. 81, NO. 2, 25 JULY 2001 185

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RESEARCH ARTICLE

phoric acid, added to increase the stability of ascorbic verted to hydroxyl radical (ΌΗ) as
acid. It was titrated against standard 2,6-dichlorophenol which can induce LPO in microsom
indophenol (2,6-DCPIP) solution of concentration
0.5 mg/ml, until the pink colour developed completely. H20 e-aq. ΌΗ, Η' (1)
The operation was repeated with a blank solution omit
ting the sample being examined. From the difference, + e- ^ + .qjj
the ascorbic acid in each mg of sample was calculated ®s -
from the ascorbic acid equivalent of the standard DCPIP Upjd peroxidation in the presence and
solution . The ascorb.c acid content in the amla extract am,a extrac{ 0f ascorbic acid was studjed
was determined to be 4.465% or 44.65 mg/g of amla. Two sets of sealed vials, one containing no
Ascorbic acid content of amla extract was also esti- somes dilute(J tQ 2 mi at a protein concen
mated by HPLC (Spectra Series, Ρ 100) with UV detec- 0 6 mg per ml and the other containing
tor at 265 nm, C-18 column and 5% v/v methanol in wjth am,a extract/ascorbic acid with the s
0.01 Μ KH2P04 as the mobile phase, at a flow rate of an(j protein concentration were prepared
1 ml/mm. Sample solution (0.2%) was prepared in the Dj,ution was done wUh pH ? 4 phosphate
mobile phase. Chromatogram of the sample showed a purging was done by passing ^ gas throug
peak at 2.60 min retention time and was assigned to crosomal suspension for tw0 or three minu
ascorbic acid, as standard solution of ascorbic acid also way that some dissolved oxygen would st
gave a peak at 2.64 min (chromatogram not shown). side Both sets wefe jrradiated for differen
From the peak area the ascorbic acid content was calcu- ya,s For b,ank correctioilj identica, sets
lated to be 3.25% or 32.5 mg/g of amla. t0 see the exten{ of Lpo jn absence of irr
extent of LPO was estimated in terms of thiobarbituric

Isolation of microsomes and mitochondria acid reactive substances (TBARS) as follows: At reg
lar intervals, 0.5 ml of microsomal suspension from the
Rat liver mitochondria and microsomes were isolated respective vial of both the sets was removed and added
from liver of male albino wistar strain rats (180-200 g) to the TBA reagent (TBA reagent: 15% w/v tri
as described earlier13'14. Animals were killed by decapi- chloroacetic acid, 0.375% w/v TBA, 0.25 Ν hydrochlo
tation, livers were quickly removed and washed with ric acid, 0.05% w/v BHT) and heated for 20 min
isolation medium (ice-cold 0.25 Μ sucrose containing 80°C in a water bath. After centrifuging, the precipitat
10 mM Γπ'ί-HCl, pH 7.4). A 10% liver homogenate was was removed and the absorbance of the supernatant w
made in isolation medium. Mitochondria were isolated measured at 532 nm (£532= 1.56 χ 105 M'1 cm4)19 to
by differential centrifugation, washed twice with calculate TBARS.
10 mM phosphate buffer at pH 7.4 and suspended in the

same buffer. Microsomes were isolated from mitochon- Estimation ofsuperoxide dismutase enzyme activity
aria-free supernatant by differential centrifugation ' .
They were washed twice with 10 mM phosphate buffer Effect of am[a Qn protection of r.radiation.jnduced
(pH 7.4) and suspended in the same buffer. All opera- damage tQ SOD was studied in rat ,iver mitochondria.
tions were earned out at 0-4 C. The protein was esti- Mitochondria suspended in oxygenated phospha
mated by the Lowry method . During the experiments, buffer equivalent t0 2 mg
protein/ml were taken in glass
microsomes/mitochondria were diluted with pH 7.4 vials and exposed t0 a total dose of 570 Gy, both in the
phosphate buffer. For studying the effect of amla ex- presence and absence of am,a extract For contro, εχ_
tract or ascorbic acid, aqueous solutions at pH 7.4 were periment) identica, ,ass yia)s were d and the
prepared just before the experiment added, to the miro- actiyity was caIculated in the absence of radjatjon S0
somes/mitochondria and diluted to get the required con- ,eyels in contro, and irradiated
samples were esti
centration expressed as pg/ml of the microsomal mated2o Briefly; } m, solutjon contains so(Jium carbon
so ution. ate buffer (50 mM, pH 10), 5 mM epinephrine and
40 pg mitochondrial protein. The rate of auto
γ-Radio lysis oxidation of only epinephrine standard was initially
followed by monitoring its absorbance at 320 n
Steady state y-radiolysis was carried out using 60Co trophotometrically. Similarly, th
source with a dose rate of 7.4 Gy/min, measured by was also monitored in uni
standard Fricke dosimetry18. LPO was studied in N20- mitochondria samples under id
purged microsomal solution at pH 7.4. y-radiolysis of difference in the absorban
aqueous solution generates primary radicals as given in ard and that in mitocho
eq. (1). Under N20 saturated condition, e~aq gets con- to calculate the enzyme activ
186 CURRENT SCIENCE, VOL. 81, NO. 2, 25 JULY 2001

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 RESEARCH ARTICLE

absorbance of 0.033 at 320 nm is defined as 1 unit of absence and presence of 24 μg of am

SOD20. microsomal solution, after exposing it to y-radiation for
different time intervals corresponding to the total ab
„ . . /·„··. χ ι sorbed doses of 148, 296, 444 and 592 Gy. It can be
Estimation of antioxidant capacity of amla extract , , .. . . . , .
, . , i t/ . ι seen that the peroxidation increased with increasing
and its components by cyclic voltammetry and r , ·
dose absorbed, but in the presenc
pulse radiolysis
decreased, suggesting inhibition of ΌΗ radical-induce
LPO by the amla extract. The effect is more pronounced
Antioxidant capacity of amla extract was estimated in
at a low dose than at a high dose. At a dose of 296 Gy
terms of mg equivalents of ascorbic acid per gm of amla , , . . ,„
° 21-23 iL. ,· the protection by amla extract is 65%, while at 592 Gy
using cyclic voltammetry methodology ~ . The cyclic
voltameter used for these studies was obtained from the protection is only
Ecochemie Autolab, model PGSTAT 20. Three peated by increasing t
most by 8 times (Figu
electrode system was employed with Ag/AgCl as the
protection at 296 Gy a
reference electrode, a glassy carbon electrode as work- , , , .
... , suggested that the membrane-protecting ability of amla
ing electrode
^ Qvtropt t c and a platinum
donanHnnt r\η wire as a counter elec-
tha nnnoontrotirtn . ,tba
/-»»■ , , nmnnnt
. /
extract is dependent on the concentration or the amount
trode. The cell
contains 25 ml of sample s
of the extract given. To determine the IC50 value (the
0.1 Μ KCl. pH was adjusted to 7 using
amount of amla extract required to inhibit LPO by 50%)
buffer. Cyclic voltammetry tracings were rec
-0.25 V to 1.2 V at a scan rate of 50 mV/s
for the amla extract, we followed the LPO at constant
-ι , · dose of y-radiation, changing the amount of amla extract
The antioxidant capacity with respect to pure ascorbic . , ' ™
., , , I j - ι . . in the range 20-240 ug/ml of microsomal solution. The
acid was also estimatedJby determining the reactivity , r _r , ,
J moat r\ t hi γτιιιώ I ο h /λιι;ο the» e» I laot /-» + unrmtifT /*r\ntant rxt
inset of Figure 1 shows the effect
towards ABTS'". These studies were carried out using
amla extract on LPO at an abso
pulse radiolysis technique, the details of which are de- , , , . ,
., , , , 24 L - ,, , , can be seen that the protection rendered by amla extract
scribed elsewhere . Typically 50ns electron pulses . ., . . ,' , , ^
r , , , r increased with increasing amount up to 100 tig/ml. Fur
from a 7 MeV linear electron accelerator were used for , . , , ,
therwas
the pulse radiolysis studies and the reaction increase
moni in amla extract did not show any
cant protection. From this figure, the IC50 v
tored by the kinetic spectrophotometry.
estimated to be 30 pg/ml.
Since the ascorbic acid content in amla is abou
Results and discussion (the highest value estimated by titration was used), we
felt it was necessary to know the role of ascorbic acid
Figure 1, curves a and b shows the change in TBARS on LPO. Thus, 4.5% of 24 μg/ml of amla corresponds to
formation as a result of LPO in microsomes in the 1.1 pg/ml of ascorbic acid. At this concentration of
ascorbic acid, the LPO in microsomes was studied at
different y-radiation doses under the conditions as seen
ο 5 with amla extract and no inhibition in LPO was ob
1 Ja)
served. However, only very high concentration of
Q

" ? 4
ω
\ ascorbic acid showed significant protection. Thus, at an
Ε3
\ ascorbic acid concentration of 6.2 pg/ml of microsomal
ω2
cc
■—■ ■
/ (b) solution, which corresponds to 25.4% of amla extract,
0 100 200
" I
[Amla] (μς/πιΙ) the TBARS were inhibited down to 28% at 444 Gy
(Figure 2). Other known important constituents of amla
extract are polyphenolic substances such as gallic acid
and ellagic acid25. It was not possible to determine their
composition as it was not easy to separate them from
the amla extract.
1 ' 1 1

200 300 400 500 600

Absorbed
Absorbed dosedose
(Gy) (Gy) Protection of superoxide d
amla extract
Figure
Figure 1. Effect
1. Effect of amla
of amla extract extract on y-radiation-induced
on y-radiation-induced lipid per lipid per
oxidation.
oxidation.Microsomes
Microsomes in in
N20-purged
NaO-purged buffer (pH 7.4)
buffer (pHwere
7.4) exposed r, ·, ,. , , , ,. , .
were exposed
to
to different
different doses using
doses 60Co source
using and lipidand
60Co source peroxidation was as- Superoxide
lipid peroxidation was as radicals (O2 ) have been imp
sayed
sayedinin
terms of TBARS;
terms (a) normal
of TBARS; microsomes,
(a) normal (b) 24 pg/ml
microsomes, (b) amla eral pathological
24 pg/ml amla disorders and are res
extract, (c)
extract, (c)192
192pg/ml
pg/ml amla
amla extract.
extract. (Inset)
(Inset) Effect
Effect of concentration
of concentration of oxidative stress26. SOD
of vated
amla on
amla on OH-induced
OH-inducedlipid
lipidperoxidation
peroxidation at at
anan absorbed
absorbed dosedose
of ,of
... . ττ ^ ,.
444
444 Gy.
Gy. Error
Error bars
barsindicate
indicatemean
meanvariation of of
variation two independent
two ex- ex
independent decomposition of O2 to g
periments.
periments. fore acts as one of the important antioxidant enzymes27.
CURRENT SCIENCE, VOL. 81, NO. 2, 25 JULY 2001 187

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RESEARCH ARTICLE

(a) (a) (a) (b)

m
ϋ (b)
Ρ(b)
2 2
11 11
-

Ο
ΡΡ 11
JOΈ
^ ° ίτ· ίο
— J5
πι Ε c
§
Is 5
1 I™ §ro Ε
Ε
II i a
Έ
Β
I Ε τ
Ε
η — -if
5
s 3
CM

3 §
Ο
SB· cm σ>

Figure
Figure 2. Inhibition of r-radiation-induced
2. Inhibition lipid peroxidation as- ...
of /-radiation-induced lipid
sessed
sessed in terms
in termsof TBARSofin TBARS Figure
the absence in andthe 3.
presence
absenceProtectio
of amlaandex-pre
F,S
extract. SOD level o
tract
tract andand ' ascorbic
ascorbic
0 nrvoor acid
tn Π,, corresponding
acid corresponding
Anco nf to 10%
Λ/rn^ ofΤamla
ο» ηη to 10%
extract.
λοο .η ΟΓΛΤΛ Error
r. of am
extra
bars
bars indicate
indicatemean meanvariation posed
posed
variation of of to
two to570
two 570Gy Gy
independentdose dos
independent of e
expe y
ured
ured in inthethe absence
absence and p
amla
amla extract.
extract. Error Error
bars i
experiments.
During irradiation, SOD activity initially increases to
combat oxidative stress and starts decreasing at very
high doses, either due to the direct damage of the en- , , „ ,r T . .
, , . value 1.2 V. Initially, the voltammograms were re
zyme protein or its increased consumption by the exces- . , r , , , . . ,
ig , corded for aqueous solutions of ascorbic acid at varying
sive generation of reactive oxygen species . We have . _ _ , , „ „ :. f
6 concentration from 0.1 to 1.2 x 10 3 Μ at pH 7, which
tested the SOD activity in rat liver mitochondria under
(Figure 4, inset, a) shows a peak at 350 m
irradiation conditions, both in the presence and absence
ing to the oxidation potential of ascorbic
of amla extract. Figure 3 shows the bar chart indicating
under the curve and the peak current wer
the initial level of SOD in the control, unexposed to different concentrations of ascorbic acid and were found
irradiation and after exposure to an absorbed dose of
to increase linearly with increasing ascorbic acid con
570 Gy. Compared to the control, upon irradiation,
centration. Figure 4 shows the linear plot of peak area
there is a reduction in the SOD activity by 72%. In the
against concentration of ascorbic acid, which is used as
same figure is given the bar chart for SOD levels in mi- ,.. . ... .
... ;.. „. , c . w , a calibration curve to estimate antioxidant capacity of
tochondria containing 24 and 192 pg of amla extract/ml . . /
amla extract. The inset of Figure 4 show
of mitochondria solution and exposed to the radiation.
mogram for the aqueous solution of amla extract
At low levels of amla extract, the protection in SOD is
(408 pg/ml) and ascorbic acid (0.25 mM). The cyclic
less; however at 192 pg/ml, the SOD level is equivalent
voltammetry signal for amla extract shows a peak at
to that of the control. This experiment shows that amla
317 mV (Figure 4, inset, b). The peak is shifted by
extract acts as a very good antioxidant by scavenging
. ·...··. ~ 30 mV compared to ascorbic acid. Such shifts were
the reactive oxygen species and protects the antioxidant , . 21 · ■ . ·
ι, 1 j. , ... , r also noticed by Chevion et αι. in several natural tis
enzymes like SOD required for the cellular defence. . P ,
sues and formulations and were a
ence of other low molecular weig
Cyclic voltammetric estimation of the antioxidant the area
capacity the oxidizable equivalents were found to be 94 ± 6 mg/g
of amla extract. This suggested that the total a
Cyclic voltammetry method as suggested by Chevion et capacity in terms of the a
a/.21-23 was used to estimate the total antioxidant capac- 94 mg/g of amla extract
ity. Here, the potential of the working electrode is appears very different from
scanned from an initial value of - 0.25 V to a final and titrimetric methods, indicating that the

188 CURRENT SCIENCE, VOL. 81, NO. 2, 25 JULY 2001

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 RESEARCH ARTICLE

> <4

W//
x 4 Ο
Γ 2
< c
ω

3 0
- Ο

-0.5 0.0 0.5 1.0

Potential
Potential (V) (V)
© 2 -

i—

03

co 1 -

ω
Q_

Ο 200
200400
400 600
600 800
800 1000
1000
0.3 0.6 0.9 1.2

Vitamin C
Vitamin C (mM)
(mM) Time Time
(με)
Time (μβ)
(με)
V IlCll I III I y I I 11 V I J

Figure
Figure 4. Peak area
4. of cyclic voltammogram
Peak area ofwas plotted against
cyclic voltammogram
Figure 5. Absorption-time profileswas showing plotted
the decay of ABTS"against F
corresponding
corresponding concentrations concentrations
of vitamin C (ascorbic acid). 0.1
ofΜradical
vitamin
at 600 nm (a) in C (ascorbic
the absence of any additive,acid). 0.1 Μ rad
(b) in the pres
phosphate
phosphate buffer
buffer was used was
for adjusting the pHused for
to 7 and 0.1 Μ KCl adjusting
ence of 204 pg/ml of the pHand
amla extract, to 7 the
(c) in and 0.1
presence of Μ KC
was
was used used
as a supporting
as aelectrolyte.
supporting (Inset) Cyclicelectrolyte.
voltammetry19.2 μg/ml of (Inset)
ascorbic acid.Cyclic voltammetry 19.2
traces
tracesof (a) 0.25
ofmM (a)
of vitamin
0.25 CmM corresponding
of vitamin
to 40 pg/ml, andC corresponding to 40 pg/ml, and
(b)
(b)408 408
pg/ml of
pg/ml
amla extract.
of Signals
amla were recorded
extract.from - 0.25
Signals
to were recorded from - 0.25 to
1.2 V with the scan rate of 50 mV/s.
1.2 v with the scan rate of 50 mV/s. and the concentration of the substrate. Earlier, from
cyclic voltammetry, we estimated that ascorbic aci
antioxidant capacity is not only due to ascorbic acid, but equivalents as 94 mg/g of amla. For th
that other components such as polyphenols are also re- concentration of 1 x 10" M, equivalent am
, extract containing 9.4% ascorbic acid therefore corre
sponsible. °
sponds to
reactivity
Estimation of
reactivity towards ABTS~' 6.23 + 0.15 χ 103 s-1. This further confirmed that the
ascorbic acid equivalents determined by cyclic volta
Reactivity with ABTS' radicals can also be used to es- metry are in very good agreement with the
timate the antioxidant activity of natural compounds29. parameter for ABTS-'.
For this, ABTS-' radicals were generated after the radio
lysis of N20-saturated aqueous solutions containing
Conclusions
2 mM ABTS2-, 0.05 Μ NaN3 at pH 7.

N3- + ΌΗ —> N3' + OH- (3) Amla or Phyllanthus emblica is known si

times for its medicinal value and is commonly used
N3 + ABTS2- —> ABTS-' + N3- (4) Ayurvedic medicine. It is also believed
source of vitamin C and is being considered as a go
ABTS-' + Amla/ascorbic acid replacement for vitamin C. However, in the
-A ABTS2- +' (amla)'+/(ascorbic acid)'+. (5) fieId amla is not as P°Pular as ascorbic acid'
per, our efforts are to show that amla is a more power
Here, the ΌΗ radicals produced by water radiolysis antioxidant than ascorbic acid. Oui resu
(eqs (1) and (2)) react with N3- to produce N3' radicals amla extract inhibits radiation-induce
(eq. (3)), which in turn oxidize ABTS2- to produce ^on 'n microsomes and SOD in mitocho
ABTS-' (eq. (4)), absorbing at 600 nm. In the absence of extract being water-soluble, may scaveng
any additive, it does not show any decay even in the ca's responsible for initiating LPO. How
time-scale of seconds (Figure 5 a), but increased in the acid alone does not account for all th
presence of both ascorbic acid and amla (Figure 5 b and activities. The ascorbic acid content
c), respectively. In presence of 1 χ 10-4 Μ ascorbic acid trimetry and HPLC gives 4.5% and 3.25%
(19.2 pg/ml), ABTS'- radical decays with the rate con- whereas the total ascorbic acid equivalents
stant of 6.15 ± 0.15 x 103 s-1. This pseudo first-order cyclic voltammetry and reactivity to
decay constant is indicative of the total reactivity of indicate a value around 9.4%. This suggests
ABTS-' towards the substrate. The reactivity is the polyphenols, which are also present in a
product of the bimolecular rate constant for the reaction are capable of scavenging oxidizing radic

CURRENT SCIENCE, VOL. 81, NO. 2, 25 JULY 2001 189

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RESEARCH ARTICLE

sible for its enhanced antioxidant activity. Even at ele

13. Satav, J. G. and Katyare, S. S., Mol. Cell. Endocrinol., 1982, 28,
178-189.
vated ascorbic acid concentration of 9.4%, ascorbic acid
14. Khopde, S. M., Priyadarsini, Κ. I., Guha, S. N., Satav, J. G.,
alone does not show as much protection as amla extract, Venkatesan, P. and Rao, Μ. Ν. Α., Biosci. Biotechnol. Ble
which is shown as bar graphs in Figure 2 giving the chern.. 2000, 64, 503-509.
extent of inhibition of LPO. This suggests that ascorbic 15. Satav, J. G., Katyare, S., Fatterpekar, P. and Sreenivasan, Α.,
acid and other polyphenols present in the natural formu Biochem. J., 1976, 156, 215-223.

lation of amla show much superior antioxidant activity 16. Sreejayan, N„ Rao, Μ. Ν. Α., Priyadarsini, Κ. I. and Devasa
gayam, Τ. P. Α., Int. J. Pharmaceu., 1997, 151, 127-130.
compared to their equivalent amounts in pure isolated 17. Lowry, Ο. H., Rosebrough, N. J., Farr, A. L. and Randall,
form.
R. J„ J. Biol. Chem., 1951, 193, 265-275.
18. Spinks, J. W. T. and Woods, R. J., An Introduction to Radiation
Chemistry, Wiley, New York, 1990, p. 243.
1. Chopra, R. Ν., Nayar, S. L. and Chopra, I. C., Glossary of In 19. Buege, J. A. and Aust, S. D., Methods Enzymol., 1978, 52, 302
dian Medicinal Plants, Council of Scientific and Industrial Re 310.
search, New Delhi, 1956. 20. Sum, M. and Zigman, S., Anal. Biochem., 1978, 90, 81-89.
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190 CURRENT SCIENCE, VOL. 81, NO. 2, 25 JULY 2001

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