Characterizing the antioxidant activity of amla (Phyllanthus emblica) extract

Abstract

Amla is well-known for its rich vitamin C (ascorbic acid) and polyphenol contents. To assess its antioxidant activity, we examined aqueous amla extract for its ability to inhibit γ-radiation-induced lipid peroxidation (LPO) in rat liver microsomes and superoxide dismutase (SOD) damage in rat liver mitochondria. For the LPO experiment, amla extract was added as its aqueous solution; and irradiation was carried out at different time intervals. The extent of LPO was measured in terms of thiobarbituric acid reactive substances. It was observed that the amla extract acts as a very good antioxidant against γ-radiation-induced LPO. Similarly, it was found to inhibit the damage to antioxidant enzyme SOD. The antioxidant activity of the amla extract was found to be both dose- and concentration-dependent. The amount of ascorbic acid in amla was standardized by HPLC and titrimetric methods and was found to be 3.25 to 4.5% w/w. However in microsomes containing this composition of pure ascorbic acid alone, no inhibition in LPO was observed. Cyclic voltammetry of the amla extract was carried out to estimate the ascorbic acid equivalents, which was found to be 9.4% w/w of amla. This value was found to be in agreement when compared with the reactivity of both amla and ascorbic acid towards ABTS.- radical, a stable free-radical. Based on these results it is concluded that amla is a more potent antioxidant than vitamin C.

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RESEARCH ARTICLE
CURRENT SCIENCE, VOL. 81, NO. 2, 25 JULY 2001 185
Characterizing the antioxidant activity of amla
(Phyllanthus emblica) extract
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†,*,#
†Chemistry Group, and ‡Radiation Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India
**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
acid) and polyphenol contents. To assess its antioxi-
dant activity, we examined aqueous amla extract for
its ability to inhibit γγ-radiation-induced lipid peroxi-
dation (LPO) in rat liver microsomes and superoxide
dismutase (SOD) damage in rat liver mitochondria.
For the LPO experiment, amla extract was added as
its aqueous solution; and irradiation was carried out
at different time intervals. The extent of LPO was
measured in terms of thiobarbituric acid reactive
substances. It was observed that the amla extract
acts as a very good antioxidant against γγ-radiation-
induced LPO. Similarly, it was found to inhibit the
damage to antioxidant enzyme SOD. The antioxidant
activity of the amla extract was found to be both
dose- and concentration-dependent. The amount of
ascorbic acid in amla was standardized by HPLC
and titrimetric methods and was found to be 3.25 to
4.5% w/w. However in microsomes containing this
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
ascorbic acid equivalents, which was found to be
9.4% w/w of amla. This value was found to be in
agreement when compared with the reactivity of
both amla and ascorbic acid towards ABTS.– radical,
a stable free-radical. Based on these results it is con-
cluded that amla is a more potent antioxidant than
vitamin C.
PLANT and plant products are being used as a source of
medicine since long. Among the most important con-
stituents of edible plant products, low molecular weight
antioxidants are the most important species. It is known
that consumption of fruits and vegetables is essential
for normal health of human beings. Vegetarian diet can
reduce the risk of cancer, atherosclerosis, etc. Phyllan-
thus emblica, also known as amla, has been used in Ay-
urveda, the ancient Indian system of medicine. It has
been used for treatment of several disorders such as
common cold, scurvy, cancer and heart diseases1–4. It is
*For correspondence. (e-mail: mittaljp@magnum.barc.ernet.in)
believed that the major constituent responsible for these
activities is vitamin C (ascorbic acid). Ascorbic acid
shows antioxidant, anti-inflammatory and antimutagenic
properties5–7. It is a very effective free-radical scaven-
ger. However, there are some in vivo studies indicating
that antioxidant activities of amla cannot be attributed
to ascorbic acid alone and that the overall effect is due
to other polyphenols such as ellagic acid, gallic acid,
tannins, etc.8–10. It is in fact reported that autoxidation
of ascorbic acid can actually increase free-radical pro-
duction11. In the present paper, we studied the effect of
aqueous amla extract on the γ-radiation-induced lipid
peroxidation (LPO) in rat liver microsomes and inhibi-
tion of superoxide dismutase (SOD) enzyme. Attempts
have also been made to understand the role of ascorbic
acid and the antioxidant equivalents in its activity.
Materials and methods
Thiobarbituric acid (TBA), butylated hydroxytoluene
(BHT), ascorbic acid and epinephrine were obtained
from Sigma Chemicals. All the other reagents were of
analytical reagent grade. Nitrous oxide (N2O) gas, ob-
tained from Indian Oxygen Ltd, Mumbai was of IOLAR
grade purity.
Preparation of amla extract
Fresh fruits were freed from foreign matter like dust or
other organic matter. The cleaned raw material was then
commuted to reduce its size. The commuted raw mate-
rial was extracted with the extraction medium and con-
verted into powder form. Since the extract is
hygroscopic, enough care was taken while handling the
sample.
Method of estimation of ascorbic acid
Sample solution equivalent to 0.2 mg ascorbic acid/ml
was prepared in water containing 3% w/v metaphos-

RESEARCH ARTICLE
CURRENT SCIENCE, VOL. 81, NO. 2, 25 JULY 2001 186
phoric acid, added to increase the stability of ascorbic
acid. It was titrated against standard 2,6-dichlorophenol
indophenol (2,6-DCPIP) solution of concentration
0.5 mg/ml, until the pink colour developed completely.
The operation was repeated with a blank solution omit-
ting the sample being examined. From the difference,
the ascorbic acid in each mg of sample was calculated
from the ascorbic acid equivalent of the standard DCPIP
solution12. The ascorbic acid content in the amla extract
was determined to be 4.465% or 44.65 mg/g of amla.
Ascorbic acid content of amla extract was also esti-
mated by HPLC (Spectra Series, P 100) with UV detec-
tor at 265 nm, C-18 column and 5% v/v methanol in
0.01 M KH2PO4 as the mobile phase, at a flow rate of
1 ml/min. Sample solution (0.2%) was prepared in the
mobile phase. Chromatogram of the sample showed a
peak at 2.60 min retention time and was assigned to
ascorbic acid, as standard solution of ascorbic acid also
gave a peak at 2.64 min (chromatogram not shown).
From the peak area the ascorbic acid content was calcu-
lated to be 3.25% or 32.5 mg/g of amla.
Isolation of microsomes and mitochondria
Rat liver mitochondria and microsomes were isolated
from liver of male albino wistar strain rats (180–200 g)
as described earlier13,14. Animals were killed by decapi-
tation, livers were quickly removed and washed with
isolation medium (ice-cold 0.25 M sucrose containing
10 mM Tris-HCl, pH 7.4). A 10% liver homogenate was
made in isolation medium. Mitochondria were isolated
by differential centrifugation, washed twice with
10 mM phosphate buffer at pH 7.4 and suspended in the
same buffer. Microsomes were isolated from mitochon-
dria-free supernatant by differential centrifugation15,16.
They were washed twice with 10 mM phosphate buffer
(pH 7.4) and suspended in the same buffer. All opera-
tions were carried out at 0–4°C. The protein was esti-
mated by the Lowry method17. During the experiments,
microsomes/mitochondria were diluted with pH 7.4
phosphate buffer. For studying the effect of amla ex-
tract or ascorbic acid, aqueous solutions at pH 7.4 were
prepared just before the experiment added, to the miro-
somes/mitochondria and diluted to get the required con-
centration expressed as µg/ml of the microsomal
solution.
γ -Radiolysis
Steady state γ-radiolysis was carried out using 60Co
source with a dose rate of 7.4 Gy/min, measured by
standard Fricke dosimetry18. LPO was studied in N2O-
purged microsomal solution at pH 7.4. γ-radiolysis of
aqueous solution generates primary radicals as given in
eq. (1). Under N2O saturated condition, e–aq gets con-
verted to hydroxyl radical (.OH) as shown in eq. (2),
which can induce LPO in microsomes.
H2O
-ray
e–aq, .OH, H. (1)
N2O + e–aq → N2 + .OH + OH– (2)
Lipid peroxidation in the presence and the absence of
amla extract or ascorbic acid was studied as follows:
Two sets of sealed vials, one containing normal micro-
somes diluted to 2 ml at a protein concentration of 0.4–
0.6 mg per ml and the other containing microsomes
with amla extract/ascorbic acid with the same dilution
and protein concentration were prepared and vortexed.
Dilution was done with pH 7.4 phosphate buffer. N2O
purging was done by passing N2O gas through the mi-
crosomal suspension for two or three minutes, in such a
way that some dissolved oxygen would still remain in-
side. Both sets were irradiated for different time inter-
vals. For blank correction, identical sets were prepared
to see the extent of LPO in absence of irradiation. The
extent of LPO was estimated in terms of thiobarbituric
acid reactive substances (TBARS) as follows: At regu-
lar intervals, 0.5 ml of microsomal suspension from the
respective vial of both the sets was removed and added
to the TBA reagent (TBA reagent: 15% w/v tri-
chloroacetic acid, 0.375% w/v TBA, 0.25 N hydrochlo-
ric acid, 0.05% w/v BHT) and heated for 20 min at
80°C in a water bath. After centrifuging, the precipitate
was removed and the absorbance of the supernatant was
measured at 532 nm (ε532 = 1.56 × 105 M–1 cm–1)19 to
calculate TBARS.
Estimation of superoxide dismutase enzyme activity
Effect of amla on protection of γ-radiation-induced
damage to SOD was studied in rat liver mitochondria.
Mitochondria suspended in oxygenated phosphate
buffer equivalent to 2 mg protein/ml were taken in glass
vials and exposed to a total dose of 570 Gy, both in the
presence and absence of amla extract. For control ex-
periment, identical glass vials were prepared and the
activity was calculated in the absence of radiation. SOD
levels in control and irradiated samples were esti-
mated20. Briefly, 1 ml solution contains sodium carbon-
ate buffer (50 mM, pH 10), 5 mM epinephrine and
40 µg mitochondrial protein. The rate of auto-
oxidation of only epinephrine standard was initially
followed by monitoring its absorbance at 320 nm, spec-
trophotometrically. Similarly, the absorbance at 320 nm
was also monitored in unirradiated and irradiated
mitochondria samples under identical conditions. The
difference in the absorbance of epinephrine stand-
ard and that in mitochondria sample was used
to calculate the enzyme activity. A difference in the

RESEARCH ARTICLE
CURRENT SCIENCE, VOL. 81, NO. 2, 25 JULY 2001 187
absorbance of 0.033 at 320 nm is defined as 1 unit of
SOD20.
Estimation of antioxidant capacity of amla extract
and its components by cyclic voltammetry and
pulse radiolysis
Antioxidant capacity of amla extract was estimated in
terms of mg equivalents of ascorbic acid per gm of amla
using cyclic voltammetry methodology21–23. The cyclic
voltameter used for these studies was obtained from
Ecochemie Autolab, model PGSTAT 20. Three-
electrode system was employed with Ag/AgCl as the
reference electrode, a glassy carbon electrode as work-
ing electrode and a platinum wire as a counter elec-
trode. The cell contains 25 ml of sample solution and
0.1 M KCl. pH was adjusted to 7 using phosphate
buffer. Cyclic voltammetry tracings were recorded from
– 0.25 V to 1.2 V at a scan rate of 50 mV/s.
The antioxidant capacity with respect to pure ascorbic
acid was also estimated by determining the reactivity
towards ABTS.–. These studies were carried out using
pulse radiolysis technique, the details of which are de-
scribed elsewhere24. Typically 50 ns electron pulses
from a 7 MeV linear electron accelerator were used for
the pulse radiolysis studies and the reaction was moni-
tored by the kinetic spectrophotometry.
Results and discussion
Figure 1, curves a and b shows the change in TBARS
formation as a result of LPO in microsomes in the
200 300 400 500 600
0
2
4
6
8
10
12
(c)
(b)
(a)
TBARS (nmole/mg prot.)
[Amla] (µg/ml)
TBARS (nmole/mg prot.)
Absorbed dose (Gy)
0100 200
2
3
4
5
Figure 1. Effect of amla extract on γ-radiation-induced lipid per-
oxidation. Microsomes in N2O-purged buffer (pH 7.4) were exposed
to different doses using 60Co source and lipid peroxidation was as-
sayed in terms of TBARS; (a) normal microsomes, (b) 24 µg/ml amla
extract, (c) 192 µg/ml amla extract. (Inset) Effect of concentration of
amla on .OH-induced lipid peroxidation at an absorbed dose of
444 Gy. Error bars indicate mean variation of two independent ex-
periments.
absence and presence of 24 µg of amla extract/ml of
microsomal solution, after exposing it to γ-radiation for
different time intervals corresponding to the total ab-
sorbed doses of 148, 296, 444 and 592 Gy. It can be
seen that the peroxidation increased with increasing
dose absorbed, but in the presence of amla extract it
decreased, suggesting inhibition of .OH radical-induced
LPO by the amla extract. The effect is more pronounced
at a low dose than at a high dose. At a dose of 296 Gy,
the protection by amla extract is 65%, while at 592 Gy
the protection is only 40%. This experiment was re-
peated by increasing the amla content significantly al-
most by 8 times (Figure 1, curve c), which gives 93%
protection at 296 Gy and 40% at 592 Gy. These results
suggested that the membrane-protecting ability of amla
extract is dependent on the concentration or the amount
of the extract given. To determine the IC50 value (the
amount of amla extract required to inhibit LPO by 50%)
for the amla extract, we followed the LPO at constant
dose of γ-radiation, changing the amount of amla extract
in the range 20–240 µg/ml of microsomal solution. The
inset of Figure 1 shows the effect of varying content of
amla extract on LPO at an absorbed dose of 444 Gy. It
can be seen that the protection rendered by amla extract
increased with increasing amount up to 100 µg/ml. Fur-
ther increase in amla extract did not show any signifi-
cant protection. From this figure, the IC50 value was
estimated to be 30 µg/ml.
Since the ascorbic acid content in amla is about 4.5%
(the highest value estimated by titration was used), we
felt it was necessary to know the role of ascorbic acid
on LPO. Thus, 4.5% of 24 µg/ml of amla corresponds to
1.1 µg/ml of ascorbic acid. At this concentration of
ascorbic acid, the LPO in microsomes was studied at
different γ-radiation doses under the conditions as seen
with amla extract and no inhibition in LPO was ob-
served. However, only very high concentration of
ascorbic acid showed significant protection. Thus, at an
ascorbic acid concentration of 6.2 µg/ml of microsomal
solution, which corresponds to 25.4% of amla extract,
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.
Protection of superoxide dismutase enzyme by
amla extract
Superoxide radicals (O2–.) have been implicated in sev-
eral pathological disorders and are responsible for ele-
vated oxidative stress26. SOD catalyses the
decomposition of O2–. to give H2O2 and O2 and there-
fore acts as one of the important antioxidant enzymes27.

RESEARCH ARTICLE
CURRENT SCIENCE, VOL. 81, NO. 2, 25 JULY 2001 188
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-
zyme protein or its increased consumption by the exces-
sive generation of reactive oxygen species28. We have
tested the SOD activity in rat liver mitochondria under
irradiation conditions, both in the presence and absence
of amla extract. Figure 3 shows the bar chart indicating
the initial level of SOD in the control, unexposed to
irradiation and after exposure to an absorbed dose of
570 Gy. Compared to the control, upon irradiation,
there is a reduction in the SOD activity by 72%. In the
same figure is given the bar chart for SOD levels in mi-
tochondria containing 24 and 192 µg of amla extract/ml
of mitochondria solution and exposed to the radiation.
At low levels of amla extract, the protection in SOD is
less; however at 192 µg/ml, the SOD level is equivalent
to that of the control. This experiment shows that amla
extract acts as a very good antioxidant by scavenging
the reactive oxygen species and protects the antioxidant
enzymes like SOD required for the cellular defence.
Cyclic voltammetric estimation of the antioxidant
capacity
Cyclic voltammetry method as suggested by Chevion et
al.21–23 was used to estimate the total antioxidant capac-
ity. Here, the potential of the working electrode is
scanned from an initial value of – 0.25 V to a final
value 1.2 V. Initially, the voltammograms were re-
corded for aqueous solutions of ascorbic acid at varying
concentration from 0.1 to 1.2 × 10–3 M at pH 7, which
(Figure 4, inset, a) shows a peak at 350 mV correspond-
ing to the oxidation potential of ascorbic acid. The area
under the curve and the peak current were measured at
different concentrations of ascorbic acid and were found
to increase linearly with increasing ascorbic acid con-
centration. Figure 4 shows the linear plot of peak area
against concentration of ascorbic acid, which is used as
a calibration curve to estimate antioxidant capacity of
amla extract. The inset of Figure 4 shows the voltam-
mogram for the aqueous solution of amla extract
(408 µg/ml) and ascorbic acid (0.25 mM). The cyclic
voltammetry signal for amla extract shows a peak at
317 mV (Figure 4, inset, b). The peak is shifted by
~ 30 mV compared to ascorbic acid. Such shifts were
also noticed by Chevion et al.21 in several natural tis-
sues and formulations and were attributed to the pres-
ence of other low molecular weight antioxidants. From
the area and the peak current and using the linear plot,
the oxidizable equivalents were found to be 94 ± 6 mg/g
of amla extract. This suggested that the total antioxidant
capacity in terms of the ascorbic acid equivalents is
94 mg/g of amla extract, which is ~ 9.4%. This value
appears very different from that estimated by the HPLC
and titrimetric methods, indicating that the
0
2
4
(6.2
µ
g/ml Vit. C)
(60 µg/ml amla)
(Normal)
TBARS (nmoles/mg prot.)
Figure 2. Inhibition of γ-radiation-induced lipid peroxidation as-
sessed in terms of TBARS in the absence and presence of amla ex-
tract and ascorbic acid corresponding to 10% of amla extract. Error
bars indicate mean variation of two independent experiments.
Figure 3.
Protection of superoxide dismutase activity by amla
extract. SOD level of mitochondria (a) without irradiation, (b) e
x-
posed to 570 Gy dose of γ-radiation. Loss in SOD activity was mea
s-
ured in the absence and presence of two different concentrations of
amla extract. Error bars indicate mean variation of two independent
experiments.

RESEARCH ARTICLE
CURRENT SCIENCE, VOL. 81, NO. 2, 25 JULY 2001 189
antioxidant capacity is not only due to ascorbic acid, but
that other components such as polyphenols are also re-
sponsible.
Estimation of oxidizing equivalents by the
reactivity towards ABTS–.
Reactivity with ABTS.– radicals can also be used to es-
timate the antioxidant activity of natural compounds29.
For this, ABTS–. radicals were generated after the radio-
lysis of N2O-saturated aqueous solutions containing
2 mM ABTS2–, 0.05 M NaN3 at pH 7.
N3– + .OH → N3. + OH–, (3)
N3 + ABTS2– → ABTS–. + N3– (4)
ABTS–. + Amla/ascorbic acid
→ ABTS2– + (amla).+/(ascorbic acid).+. (5)
Here, the .OH radicals produced by water radiolysis
(eqs (1) and (2)) react with N3– to produce N3. radicals
(eq. (3)), which in turn oxidize ABTS2– to produce
ABTS–. (eq. (4)), absorbing at 600 nm. In the absence of
any additive, it does not show any decay even in the
time-scale of seconds (Figure 5 a), but increased in the
presence of both ascorbic acid and amla (Figure 5 b and
c), respectively. In presence of 1 × 10–4 M ascorbic acid
(19.2 µg/ml), ABTS.– radical decays with the rate con-
stant of 6.15 ± 0.15 × 103 s–1. This pseudo first-order
decay constant is indicative of the total reactivity of
ABTS–. towards the substrate. The reactivity is the
product of the bimolecular rate constant for the reaction
0200 400 600 800 1000
0.00
0.03
0.06
0.09
(c)
(b)
(a)
∆ O.D.
Time (µs)
Figure 5. Absorption-time profiles showing the decay of ABTS–.
radical at 600 nm (a) in the absence of any additive, (b) in the pres-
ence of 204 µg/ml of amla extract, and (c) in the presence of
19.2 µg/ml of ascorbic acid.
and the concentration of the substrate. Earlier, from
cyclic voltammetry, we estimated that ascorbic acid
equivalents as 94 mg/g of amla. For the ascorbic acid
concentration of 1 × 10–4 M, equivalent amount of amla
extract containing 9.4% ascorbic acid therefore corre-
sponds to 204 µg/ml. At this amla content, we tested
reactivity of amla extract with ABTS.– under similar
conditions, which also showed a total reactivity of
6.23 ± 0.15 × 103 s–1. This further confirmed that the
ascorbic acid equivalents determined by cyclic voltam-
metry are in very good agreement with the reactivity
parameter for ABTS–..
Conclusions
Amla or Phyllanthus emblica is known since ancient
times for its medicinal value and is commonly used in
Ayurvedic medicine. It is also believed to be a rich
source of vitamin C and is being considered as a good
replacement for vitamin C. However, in the medical
field amla is not as popular as ascorbic acid. In this pa-
per, our efforts are to show that amla is a more powerful
antioxidant than ascorbic acid. Our results showed that
amla extract inhibits radiation-induced lipid peroxida-
tion in microsomes and SOD in mitochondria. Amla
extract being water-soluble, may scavenge the free radi-
cals responsible for initiating LPO. However, ascorbic
acid alone does not account for all these antioxidant
activities. The ascorbic acid content estimated by ti-
trimetry and HPLC gives 4.5% and 3.25% respectively,
whereas the total ascorbic acid equivalents estimated by
cyclic voltammetry and reactivity to ABTS–. radical
indicate a value around 9.4%. This suggests that other
polyphenols, which are also present in amla and which
are capable of scavenging oxidizing radicals are respon-
-0.5 0.0 0.5 1.0
0
2
4(b)
(a)
Current*106 (A)
Potential (V)
0.3 0.6 0.9 1.2
1
2
3
4
5
Peak area *10
5
(A X V)
Vitamin C (mM)
Figure 4. Peak ar
ea of cyclic voltammogram was plotted against
corresponding concentrations of vitamin C (ascorbic acid). 0.1 M
phosphate buffer was used for adjusting the pH to 7 and 0.1
M KCl
was used as a supporting electrolyte. (Inset) Cyclic voltammetry
traces of (a) 0.25 mM of vitamin C corresponding to 40 µ
g/ml, and
(b) 408 µg/ml of amla extract. Signals were recorded from –
0.25 to
1.2 V with the scan rate of 50 mV/s.

RESEARCH ARTICLE
CURRENT SCIENCE, VOL. 81, NO. 2, 25 JULY 2001 190
sible for its enhanced antioxidant activity. Even at ele-
vated ascorbic acid concentration of 9.4%, ascorbic acid
alone does not show as much protection as amla extract,
which is shown as bar graphs in Figure 2 giving the
extent of inhibition of LPO. This suggests that ascorbic
acid and other polyphenols present in the natural formu-
lation of amla show much superior antioxidant activity
compared to their equivalent amounts in pure isolated
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ACKNOWLEDGEMENTS. We thank Dr T. Mukherjee, Head, RC
& CD Division, BARC, Mumbai for support.
Received 25 January 2001; revised accepted 27 April 2001