ArticlePDF Available

Characterizing the antioxidant activity of amla (Phyllanthus emblica) extract

Authors:

Abstract and Figures

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.
Content may be subject to copyright.
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, eaq gets con-
verted to hydroxyl radical (.OH) as shown in eq. (2),
which can induce LPO in microsomes.
H2O
-ray
eaq, .OH, H. (1)
N2O + eaq 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
(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
form.
1. Chopra, R. N., Nayar, S. L. and Chopra, I. C., Glossary of In-
dian Medicinal Plants, Council of Scientific and Industrial Re-
search, New Delhi, 1956.
2. Gupta, B. C., The Vanusadhi-darpana, S. C. Auddy & Co, Cal-
cutta, 1908.
3. Rao, T. S., Kumari, K. K., Netaji, B. and Subhokta, P. K., J.
Res. Ayurveda Siddha, 1985, 6, 213214.
4. Ghosh, A., Sharma, A. and Talukdar, G., Food Chem. Toxicol.,
1992, 30, 865869.
5. Levine, M., N. Engl. J. Med., 1986, 314, 892902.
6. Frei, B., England, L. and Ames, B., Proc. Natl. Acad. Sci. USA,
1989, 86, 63776381.
7. Shah, G. M. and Bhattacharya, R. K., J. Biosci., 1982, 4, 263
268.
8. Bhattacharya, A., Chatterjee, A., Ghosal, S. and Bhattacharya,
S. K., Indian J. Exp. Biol., 1999, 37, 676680.
9. Ihantola-Vormisto, A., Summanen, J., Kankaanranta, H., Vuo-
rela, H., Asmawi, Z. M. and Moilanen, E., Plant Med., 1997, 63,
518–524.
10. Santos, A. R., De-Campos, R. O., Miguel, O. G., Cechinel,
Filho V., Yunes, R. A. and Calixto, J. B., Eur. J. Pharmacol.,
1999, 379, 717.
11. Halliwell, B., Free Radic. Res., 1996, 25, 439454.
12. Indian Pharmacopia, Government of India, Ministry of Health
and Family Welfare, The Controller of Publications, Delhi, vol.
1, 1996.
13. Satav, J. G. and Katyare, S. S., Mol. Cell. Endocrinol., 1982, 28,
178–189.
14. Khopde, S. M., Priyadarsini, K. I., Guha, S. N., Satav, J. G.,
Venkatesan, P. and Rao, M. N. A., Biosci. Biotechnol. Bio-
chem., 2000, 64, 503509.
15. Satav, J. G., Katyare, S., Fatterpekar, P. and Sreenivasan, A.,
Biochem. J., 1976, 156, 215223.
16. Sreejayan, N., Rao, M. N. A., Priyadarsini, K. I. and Devasa-
gayam, T. P. A., Int. J. Pharmaceu., 1997, 151, 127130.
17. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall,
R. J., J. Biol. Chem., 1951, 193, 265275.
18. Spinks, J. W. T. and Woods, R. J., An Introduction to Radiation
Chemistry, Wiley, New York, 1990, p. 243.
19. Buege, J. A. and Aust, S. D., Methods Enzymol., 1978, 52, 302
310.
20. Sum, M. and Zigman, S., Anal. Biochem., 1978, 90, 8189.
21. Chevion, S., Roberts, M. A. and Chevion, M., Free Radic. Biol.
Med., 2000, 68, 860870.
22. Chevion, S., Or, R. and Berry, E. M., Mol. Biol. Int., 1999, 47,
1019–1027.
23. Chevion, S., Chevion, M., Chock, P. B. and Beecher, G. R., J.
Med. Food, 1999, 2, 111.
24. Guha, S. N., Moorthy, P. N., Kishore, K., Naik, D. B. and Rao,
K. N., Proc. Indian Acad. Sci. (Chem. Sci.), 1987, 99, 261271.
25. http//genome.cornell.edu/botony/about Phytochem db.html.
26. Fridovich, I., Arch. Biochem. Biophys., 1986, 247, 1–11.
27. McCord, J. M. and Fridovich, L., J. Biol. Chem., 1969, 244,
6049–6055.
28. Kamat, J. P., Boloor, K. K., Devasagayam, T. P. A. and Kesa-
van, P. C., Curr. Sci., 1999, 77, 286289.
29. Miller, N. J., Sampson, J., Candeias, L. P., Bramely, P. M. and
Rice-Evans, C. A., FEBS Lett., 1996, 384, 240242.
ACKNOWLEDGEMENTS. We thank Dr T. Mukherjee, Head, RC
& CD Division, BARC, Mumbai for support.
Received 25 January 2001; revised accepted 27 April 2001
... The Phyllanthus emblica extract used in the present study enhances the ability of mitochondria to perform oxidative phosphorylation and synthesize ATP in hepatocytes that have been exposed to free radicals. This leads to the maintenance of mitochondrial activity in the hepatocytes, and the mitochondria in hepatocytes can produce sufficient amounts of ATP for use by the hepatocytes, allowing them to maintain normal metabolism (Khopde et al., 2001). ...
... Phyllanthus emblica is known for its high vitamin C (ascorbic acid) and polyphenol contents. To evaluate its antioxidant activity, Khopde et al. (2001) tested the ability of aqueous Phyllanthus emblica extracts to inhibit γ-radiation-induced lipid peroxidation (LPO) and superoxidase dismutase (SOD)-induced damage in rat liver mitochondria. For the LPO experiment, an aqueous solution of Phyllanthus emblica extract was used, and irradiation was performed at different time intervals. ...
Article
The objective of this study was to prepare a medicinal formulation with the extract of Phyllanthus emblica to repair liver damage. The hepatocyte cell line Hep-G2 was used in the experiment. The experimental sample was prepared by adding 20,000 hepatocytes to each well of a 24-well Seahorse XF24 analyzer special cell culture plate and cultured for 24 hrs. Subsequently, the culture medium from each well was removed. The hepatocytes in the wells were processed according to the conditions of the experimental, control, or comparator group. The oxygen consumption of the hepatocytes in the experimental, control, and comparator groups in the wells of the cell culture plate was measured using the Seahorse XF24 analyzer bioenergy meter. Treating cells with hydrogen peroxide can simulate the intracellular oxidation of free radicals, permitting examination of the mitochondrial activity of cells under oxidative stress. The mitochondrial activity in the hepatocytes was maintained. The mitochondria produced a sufficient amount of adenosine triphosphate (ATP), allowing the hepatocytes to maintain their normal metabolic functions. Owing to the improved synthesis efficiency and capability of triphosphate required for cell damage repair, damaged hepatocytes were able to obtain adequate energy for repair. Thus, liver repair was accelerated, and it returned to its normal condition.
... The fruit juice contains nearly 20 times as much vitamin C as in orange juice. Its other constituents are phenols and tannins containing gallic acid, elegiac acid and glucose which prevent oxidation of vitamin C. It has been observed that ascorbic acid and other polyphenols present in natural extracts of aonla show superior antioxidant activity and counteracting the toxic effects of metal salts in hepatic and renal toxins than equivalent amount of pure ascorbic acid (Roy et al., 1991, Khopde et al., 2001 [18,13] . Aonla fruits are used in traditional Indian systems of medicines, like ayurvedic and unani, due to their therapeutic values (Agarwal and Chopra, 2004) [1] . ...
... The fruit juice contains nearly 20 times as much vitamin C as in orange juice. Its other constituents are phenols and tannins containing gallic acid, elegiac acid and glucose which prevent oxidation of vitamin C. It has been observed that ascorbic acid and other polyphenols present in natural extracts of aonla show superior antioxidant activity and counteracting the toxic effects of metal salts in hepatic and renal toxins than equivalent amount of pure ascorbic acid (Roy et al., 1991, Khopde et al., 2001 [18,13] . Aonla fruits are used in traditional Indian systems of medicines, like ayurvedic and unani, due to their therapeutic values (Agarwal and Chopra, 2004) [1] . ...
Article
Full-text available
Attempts were made to analyze the effect of pre-treatments on juice recovery and ascorbic acid content of juice extracted from aonla fruits at College of Food Processing Technology and Bio-energy, Anand Agricultural University, Anand, Gujarat. Aonla fruits were given pre-treatment of blanching either by hot water treatment at different temperatures and time intervals (i.e. 85-100 °C and 2-8 min); or steam blanching at different time intervals (2-8 min). The steam blanching of aonla fruits for 2 minutes resulted in more quantity of juice (51%) with higher ascorbic acid content (698 mg/ 100 ml of juice. The maximum juice recovery (76%) was given by screw type juice extractor compared to other juice extraction machines. The screw type juice extractor was operated at four different rotational speeds (90, 180, 270 and 360 rpm). The maximum juice recovery (74%) was at 90 rpm. The optimized conditions obtained for aonla juice extraction were steam blanching for 2 minutes, pulverization of the shreds and juice extraction using screw type juice extractor with the speed of rotation of 90 rpm.
... Although ruminants can synthesize vitamin C in the liver and it is not considered to be an essential nutrient for healthy cattle, a large reduction in plasma vitamin C concentration was reported in lactating cow with artificially induced mastitis (Weiss et al. 2004) [48] . Khopde et al. (2001) [17] reported that ascorbic acid and other polyphenols present in the natural formulation of amla showed much superior antioxidant activity compared to their equivalent amounts in pure isolated form. ...
... Although ruminants can synthesize vitamin C in the liver and it is not considered to be an essential nutrient for healthy cattle, a large reduction in plasma vitamin C concentration was reported in lactating cow with artificially induced mastitis (Weiss et al. 2004) [48] . Khopde et al. (2001) [17] reported that ascorbic acid and other polyphenols present in the natural formulation of amla showed much superior antioxidant activity compared to their equivalent amounts in pure isolated form. ...
Article
Full-text available
Embilica officinalis or Indian gooseberry is perhaps the single most often mentioned herb in "Charak Samhita", the Ayurvedic medicine literature (500 BC). Ayurveda, which is the oldest health system in the world, appreciates and uses amla to treat a host of diseases and promote positive health. Thirty two animals positive for subclinical mastitis were randomly divided into four different groups containing 8 animals each and supplemented with various doses of deseeded fresh amla. Changes in milk yield, fat%, solids not fat, total solids and total bacterial count were studied. Supplementation of amla @ 250 gram and 200 gram increased the milk yield by 14.58% and 14.28%, significantly decreased total bacterial count but did not have any significant changes in fat%, total solids and solid not fat, although an increase in fat was recorded. Supplementation at 150 gram too increased milk yield, decreased total bacterial count but the changes were statistically non significant. In unsupplemented group, an increase in somatic cell count, total bacterial count and decline in milk yield was recorded indicative of development of mastitis. Thus, amla can be used as an alternate to conventional therapy and can be supplemented to cattle in routine feeding especially in areas where it is surplus.
... They have been shown to have antioxidant and anti-inflammatory properties and have been shown to be anti-diabetic in alloxan treated diabetic rats (Sabu et al. 2002). They also inhibit γ-radiation-induced lipid peroxidation in rat liver microsomes and superoxide dismutase (SOD) damage in rat liver mitochondria (Khopde et al 2001). The fruit has significance in Asian medicine, in the treatment of various diseases. ...
... Phyllanthus emblica (Amla) fruit is an Indian tree that is rich in vitamin C, minerals and antioxidants (15,16) and is an antibacterial (17) and lowers blood lipids, protects the liver, and eliminates indigestion and biliary disorders. Amla can help digest and expel excess moisture from the gastrointestinal tract by preventing the rise of fumes and strengthening the gastric cardia, and preventing the reflux of stomach contents and acid into the esophagus. ...
... 41 The phenolic compound and aqueous extracts of Āmla (E. officinalis) were found to have anti-inflammatory 42 and antioxidant activity, 43 respectively. In a carrageenaninduced inflammation model, Mohan et al discovered that an ethanolic extract of Kishnīz (C. ...
Background: Despite there being advanced treatment options, psoriasis remains an incurable and recurring disease. Noteworthy scholars of Unani (Greco-Arab) medicine have proposed many drugs and formulations for psoriasis but the scientific evidence on the same is scarce. Hence, trial formulations were selected for the study. Primary study objectives: This study was designed to evaluate the efficacy and safety of two herbal Unani formulations, Ma΄jūn Mundī and Qairūtī Karnab, in the management of chronic plaque psoriasis (CPP). Methods/design: This open-label, single-arm clinical trial was conducted on 33 participants, of whom 30 completed the 12-week treatment course. Setting: This study was conducted at the Central Research Institute of Unani Medicine (CRIUM), Hyderabad, Telangana, India, from 01 August 2018 to 25 May 2019. Participants: Participants of any gender aged 18 to 65 years with clinically diagnosed CPP and psoriasis area severity index (PASI) ≥ 10% were included in the trial. Interventions: The participants received 5 g of Ma΄jūn Mundī (a semisolid preparation) orally, twice daily with water, followed by the topical application of Qairūtī Karnab (a homogenous paste) to cover the lesions over 12 weeks. Outcome measures: The primary outcome measure was the change in PASI determined pre- and post-trial in terms of mean and percentage reduction. Secondary outcome measures were changes in patient global assessment (PGA) on a 100 mm visual analog scale, investigator global assessment (IGA) on a 6-point scale, and subjective parameters including erythema, induration, scaling, and itchiness. Results: The analysis revealed a significant reduction in the PASI score, with 12 subjects (40%) achieving PASI 75 and 3 subjects (10%) achieving PASI 90. Significant improvements were also observed in secondary outcome measures with no adverse events. Conclusion: The findings of the study indicate that the trial formulations exhibit a notable anti-psoriatic effect without any adverse effects. The formulations are worthy of further evaluation as an alternative treatment for CPP.
... Although ruminants can synthesize vitamin C in the liver and it is not considered to be an essential nutrient for healthy cattle, a large reduction in plasma vitamin C concentration was reported in lactating cow with artificially induced mastitis (Weiss et al., 2004). Khopde et al. (2001) reported that ascorbic acid and other polyphenols present in the natural formulation of E.O. showed much superior antioxidant activity compared to their equivalent amounts in pure isolated form. ...
Research
Full-text available
The present study describes in-vitro efficacy of Emblica officinalis against Methicillin Resistant S. aureus mastitis. Diffusion technique was used to assess in-vitro efficacy of Emblica officinalis. Zone of inhibition was measured and used to compare the in-vitro efficacy. The zone ranged between 10-13 mm with maximum zone of 13 mm observed in 200 and 225 mg/ml DMSO disc, followed by 12 mm in 175 and 150 mg/ml DMSO disc, 11 mm in 125 mg/ml DMSO disc and 10 mm in 100 mg/ml DMSO disc. The results indicate that the sensitivity pattern for Emblica officinalis at 200 & 175 mg/ml DMSO concentration and was comparable with the standard antibiotics in Methicillin sensitive S. aureus. In Methicillin resistant S. aureus isolates, the zone of inhibition was in the order Oxytetracycline (15mm) followed by Emblica officinalis-200 (13 mm) and Methicillin, ampicillin, gentamicin, ofloxacin were resistance.
... Furthermore, topical application of Terminalia chebula cream protects rats against developing psoriasis as a result of UVB exposure (Pai et al., 2020). Other ingredients Ā mla Khushk (Phyllanthus emblica) and Rewand (Rheum australe) have anti-inflammatory (Muthuraman et al., 2011) and antioxidant activities (Khopde et al., 2001;Hu et al., 2014), which appear to contribute to the reduction of psoriatic lesions. ...
Article
Ethnopharmacological relevance Psoriasis, despite modern therapeutic options, is incurable and recurrent. In Unani (Greco-Arab) medicine, many medications and formulations have been prescribed by eminent scholars for conditions clinically similar to psoriasis, though empirical evidence is sparse. Hence, the experimental formulations Itrīfal Shāhtra and Marham Ḥina were chosen to be compared to the standard therapies PUVAsol and petrolatum for their safety and efficacy. Materials and methods This open-label, randomized control clinical trial was conducted on 66 male and female participants with chronic plaque psoriasis, ranging in age from 18 to 65 years. In each group, 33 participants were block randomized to either receive test formulations or control drugs for 12 weeks. The Unani group received oral Itrīfal Shāhtra (a semisolid paste) and topical MarhamḤina (an ointment) twice daily, and the control group received oral 8-methoxypsoralen and topical petroleum jelly for local application. Participants of both groups were advised to get daily sunlight exposure for 5–15 min. The primary outcome measure was the change in psoriasis area and severity index (PASI) assessed at each visit. Secondary outcome measures were patient global assessment on a 100 mm VAS applied at baseline and after 12 weeks of treatment and change in subjective parameters including erythema, induration, scaling, and itching, assessed on a 5-point scale at every visit. Hemogram, LFTs, RFTs, CXR, ECG, urine, and stool tests were all assessed at baseline and after treatment for the safety of the drugs. Results The per-protocol analysis was done on 25 participants in each group. The mean ± SD of the psoriasis area severity index (PASI) significantly decreased from 27.88 ± 12.01 and 23.61 ± 9.79 at baseline to 5.01 ± 4.59 and 9.85 ± 7.16 after completion of the trial therapies in both Unani and control groups, respectively. Also, the test formulations outperformed the control drugs on clinically significant endpoints, PASI 50 and PASI 75, with all 25 participants achieving PASI 50 and 76% achieving PASI 75. Conclusion The trial formulations, ItrīfalShāhtraand MarhamḤinamay be superior to control drugs PUVAsol and petrolatum in terms of safety, efficacy, and tolerability in the treatment of chronic plaque psoriasis. Thus, the Unani formulations may further be evaluated in a well-designed multicentric superiority trial with an adequate sample size.
... In the last few decades there is an increasing production and consumption of herbal products as medicines/food additives due to their various health benefits , minimal side effect compared to allopathic medicines and excellent antioxidant properties [1][2][3][4][5][6][7][8]. Since antioxidants play an important role in the maintenance of health and prevention of diseases, these natural herbal medicines are getting popularized both in developing and developed countries. ...
Article
Full-text available
One of the important component in herbal extracts responsible for their medicinal effects is antioxidants like phenolics, flavonoids, tannins, etc. The herbs Emblica officinalis(EON), terminalia chebula(TMC) and terminalia bellerica(TMB) and triphala(TRP) are extensively used as potent ayurvedhic medicines. A knowledge on their antioxidant capacitites is required for effective ayurvedic formulations. In the present study the most reliable analytical tool viz, cyclic voltammetry (CV) was used to analyse the antioxidant capacities of the aqueous-methanolic extracts of these herbs. Voltammograms were recorded on a potentiostat using 10 ml capacity cell having glassy carbon, Pt and Ag/AgCl as working, counter and reference electrodes respectively, and 0.1MKCl as supporting electrolyte for the potential range 0-0.8 to1.0 V at a scan rate of 100mV/s. CV of these herbs showed anodic peaks at0.40 to 0.45 V(Peak1) and 0.5 to 0.55 V(Peak 2).The anodic currents increased with the concentration of herbal extracts. For 100µL of each extract, the antioxidant capacities increased in the order TRP<EON<TMC<TMB and these were compared with those of ascorbic acid and catachol taken as reference antioxidants. The study revealed that CV is a promising and simple analytical tool for rapid screening and measuring the antioxidant capacities of various herbs without any chemical reagent.
Research
Full-text available
Amla (Emblica officinalis) is well known for its minerals and vitamins and is one of the rich sources of vitamin C. We analyzed the effects of inlet temperature of spray dryer and maltodextrin percentage on the functional properties of amla powder. Maltodextrin is used as encapsulation material due to the higher retention potential of bioactive compounds in amla. The juice of amla prepared from freshly obtained amla fruit is subjected to spray drying with different inlet temperatures (160℃, 170℃, and 180℃) and encapsulated with maltodextrin in different concentrations as 16%, 20%, and 24%. Physicochemical parameters such as vitamin C content, antioxidant activity, total phenol content, and proximate composition were analyzed in the spray-dried amla powder. Powder properties like particle size, water activity, water solubility index, and color value were also analyzed. Therefore, from our results, we suggest that the amla powder obtained with 160℃ as inlet temperature and 24% maltodextrin concentration could be suitable to produce amla fruit beverages.
Article
Full-text available
Reactive oxygen-derived species are produced in cells under physiological conditions and in response to stress. Among the various antioxidant systems responsible for protection against these species, the low-molecular-weight antioxidants (LMWA), such as ascorbate, play an important role. Cyclic voltammetry (CV) has been proposed as a tool for quantitation of the total antioxidant capacity of plasma. It has also been shown that biological oxidation potentials, as determined from the anodic current waves of the CV tracings, are specific characteristics of the various LMWA components, and that the amplitude of each wave can be used for quantitation of the specific component. The adaptation of CV for evaluation of the total antioxidant capacity of edible plants is demonstrated here. The area under the anodic current wave is proposed as a better indicator for the content of LMWA, compared with the amplitude. This distinction could prove valuable when more than a single molecule contributes toward a specific anodic wave and when the identities of the components of a wave are not known. Vegetables and fruits that are commonly consumed in the U.S. diet were used. They were extracted with either water, aqueous acetic acid (30%), or a mixture of water, acetic acid, and acetonitrile (40:30:30). The LMWA contents were evaluated by CV. In three to five steps the LMWAs were completely extracted from the edible foods, and their amounts were translated into equivalents of ascorbate.
Article
Radiation is one of the physical agents that induce oxidative stress. Exposure of rat liver mitochondria to high doses of 60Co γ-rays (45-600 Gy) results in the loss of activity of superoxide dismutase (SOD). Presence of caffeine, even in micromolar amounts, during exposure prevents loss of SOD activity. Caffeine, at a concentration of 1 mM also showed protection against radiation-induced inhibition of two other mitochondrial enzymes, namely succinate dehydrogenase and cytochrome c oxidase. The observed radioprotective ability of caffeine may be due to its ability to scavenge the reactive oxygen species generated by radiation and to inhibit radiation-induced membrane damage, as assessed by lipid peroxidation and protein oxidation.
Article
Pretreatment of male Wistar rats with L-ascorbic acid results in a decrease in thein vivo covalent binding of benzo(a)pyrene to hepatic nuclear DNA.In vitro formation of this adduct is also found to be low in liver slices and in liver nuclei of pretreated rats. No inhibition of the adduct formation is, however, observed when benzo (a) pyrene and exogenous DNA are incubated with liver microsomes isolated from ascorbic acid treated rats.It appears that the presence of ascorbate in the cellular or subcellular environment is essential for its inhibitory action.
Article
1. Heavy and light mitochondrial fractions obtained by differential centrifugation were further characterized with respect to their lipid composition and synthesis and protein profiles, as seen by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 2. The light mitochondrial fraction was rich in total lipids, phospholipids and cholesterol. The cardiolipin content, however, was low. 3. Rates of [3H]glycerol incorporation into phospholipids of heavy mitochondria and microsomal fractions were almost identical. On the other hand, incorporation into the individual phospholipids in light mitochondria was about 4-6 times higher. Incorporation into cardiolipin of light mitochondria was about 10-fold higher than in the heavy mitochondria. 4. Analysis of protein profiles by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis showed that the pattern obtained for the light mitochondria was similar to that for heavy mitochondria. However, the light fraction was relatively poor in high-molecular-weight proteins and rich in low-molecular-weight proteins. The microsomal protein profile was altogether different. 5. The significance of these findings is discussed in relation to mitochondrial biogenesis.
Article
Aqueous extracts of Phyllanthus emblica L. fruit and an equivalent amount of vitamin C were administered orally by gavage to laboratory-bred Swiss albino mice for 7 days in order to evaluate the protection afforded by the two extracts against clastogenic effects of different doses of caesium chloride (CsCl) on bone marrow cells of Mus musculus in vivo. Both pretreatments significantly reduced the frequency of chromosome aberrations induced by CsCl given at three different doses, indicating that vitamin C, an essential component of P. emblica extract, was the effective agent in protecting against the clastogenicity of the metal salt.
Article
The antioxidant activity of tannoid active principles of E. officinalis consisting of emblicanin A (37%), emblicanin B (33%), punigluconin (12%) and pedunculagin (14%), was investigated on the basis of their effects on rat brain frontal cortical and striatal concentrations of the oxidative free radical scavenging enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), and lipid peroxidation, in terms of thiobarbituric acid-reactive products. The results were compared with effects induced by deprenyl, a selective monoamine oxidase (MAO) B inhibitor with well documented antioxidant activity. The active tannoids of E. officinalis (EOT), administered in the doses of 5 and 10 mg/kg, i.p., and deprenyl (2 mg/kg, i.p.), induced an increase in both frontal cortical and striatal SOD, CAT and GPX activity, with concomitant decrease in lipid peroxidation in these brain areas when administered once daily for 7 days. Acute single administration of EOT and deprenyl had insignificant effects. The results also indicate that the antioxidant activity of E. officinalis may reside in the tannoids of the fruits of the plant, which have vitamin C-like properties, rather than vitamin C itself.