Antioxidant activity of methanolic extract of emblica fruit (Phyllanthus emblica L.) from six regions in China

1. Deepa kumari karn
B pharm 6th sem
Department of pharmaceutical sciences
8/3/2012 1
Antioxidant activity of methanolic extract of
emblica fruit (Phyllanthus emblica L.) from
six regions in China

2. INTRODUCTION
Phyllanthus emblica belong to family
Euphorbiaceae
Widely distributed in subtropical and
tropical areas of China, India, Indonesia,
and Malaysia
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3. Emblica fruits are reported to have
hypolipidemic,hypoglycemic,
hepatoprotective activities
Widely used as antimicrobial,
antitumor, anti-inflammatory agent and
improve the metal induced clastogenic
effect
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4. Phenolic compound and flavanoids
present in the emblica fruits show
antioxidant activity
In the present experiment the
methanolic extract of emblica fruit from
six different regions in China were
evaluated
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5. MATERIALAND METHODS
Plant material:-
Fresh fruits of emblica were gathers
from six regions in China
Guangzhou
Huizhou
Liuzhou
Chuxiong
Putian8/3/2012 5

6. The weather of the sampling regions
were:-
Sub tropical to monsoon for
Liuzhou and Chuxiong
Subtropical oceanic climate for
Guangzhou, Huizhou, Putian
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7. Tropical oceanic for Haikou
The fruits were stored at 4ºC till
analysis
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8. Chemicals:-
Nitroblue tetrazolium (NBT)
1,1-diphenyl-2-picrylhydrazyl
(DPPH)
Phenazine methosulfate (PMS)
Catechin
 Quercetin
Ferrozine
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9. All the chemical were purchased from
sigma chemicals Co. (St. Louis, MO,
USA)
All other chemical and reagents were
of analytical grade
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10. Preparation of methanolic extract:-
Fresh emblica fleshes were air dried in
an oven at 40ºC for 8h
Pulverized to 0.2 to 0.4 mm particle in
the cutting mill
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11. Powder 10 g was extracted for 24 h
with 100ml of methanol in a glass
conical flask using a shaker at 25º C
 Filtered through 0.45µ filter paper
Residue was then extracted twice with
100ml methanol
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12. Combined methanolic extract were
concentrated at 40ºC, using a rotary
evaporator under low pressure
Residue was freeze- dried and then
stored in amber color container at 4ºC
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13. Determination of total phenolic,
flavonoids and proanthocyanidin
content:-
The total phenolic content was
determined by the Folin- Ciocalteu
method:-
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Sodium 3,4-dioxo-3,4-
dihydronaphthalene-1-sulfonate

14. Extract +methanol
Folin Reagent+ sodium bicarbonate
Opaque sample is obtained
O D of blue sample is measured at
756nm
Total phenolic content was expressed as
mg gallic acid equivalent/g dry basis
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15. Flavonoid contents were measured
using a modified colorimetric method:-
Extract solution+ distilled water+
sodium nitrate
10% aluminum chloride
After 6min sodium hydroxide was
added and mixture was diluted with
distilled water
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16. The absorbance was measured at 510
nm
Compared with standard curve of
quercetin
The final amount of flavonoid was
expressed as quercetin equivalent/ g
dry basis
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17. Content of proanthocyanidins was
determined by the procedure of Sun et
al:-
Extract + vanillin-methanol solution
+ Hcl
After absorbance was determined at
500nm
Result was expressed as Catechin
eq/g dry basis
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18. Determination of DPPH radical
scavenging activity:-
The free radical scavenging was
determined by the method of Shimada et
al and Yang et al
Various concentration of methanolic
extract 200, 100,80,60,40,20 µg/ml
was prepared
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19. Extract solution+ methanolic solution
containing DPPH radical
Shaken vigorously and maintained
for 30 min in dark
Absorbance was measured at 517nm
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20. Determination of reducing power:-
Determined according to method of
Oyaizce
Extract solution+ phosphate buffer
+potassium ferricyanide
Incubated at 50ºC for 20m
Trichloroacetic acid was added to the
mixture
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21. Above mixture+ distilled water+
0.1% ferric chloride
After 10 min absorbance was
measured at 700nm
Increased absorbance indicated high
reducing power
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22. Determination of superoxide anion
radical scavenging activity:-
The assay for superoxide anion radical
scavenging activity was based on the
riboflavin-light NBT system
Phosphate buffer+ riboflavin+ PMS+
NBT+ methanolic extract of different
concentration
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23. Incubate for 20 min
Absorbance was measured at
560nm
The absorbance of control
was determined by replacing the sample
with methanol
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24. Determination of hydroxyl radical
scavenging activity:-
Assayed by the method of Halliwell
and Gutteridge
2-deoxyribose+ Potassium
phosphate buffer+ permixed EDTA +
hydrogenperoxide + extract solution
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25. Add ascorbate and incubate for 1 h at
37ºC
Add TBA+ NaOH+ TCA
Heat for 15m on boiling water bath and
then cooled
Absorbance measured at 532nm
Absorbance of control was determine
by replacing sample with methanol
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26. Inhibition of lipid peroxide formation
induced by Fe++ ascorbate system:-
Lipid peroxidation assay of rat
microsomal was carried out as reported
earlier by Sabu and Kuttan
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27. Extract solution at various
concentration+ rat liver homogenate+
Hcl buffer+ ascorbic acid + ferrous iron
Incubate for 1h at 37ºC
Absorbance of organic layer was
measured at 532nm
Result of test is compared with that of
control sample without extract solution
% of inhibition was determined
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28. Ability of chelating ferrous iron:-
Measured by the ferrous iron-ferrozine
complex method
Various concentration of methanolic
extract+ Fecl2 +ferrozine
Incubate for 10 min at 25±2ºC
Absorbance was measured at 256nm
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29. Absorbance of control was determined
by replacing the sample with methanol
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30. Statistical analysis:-
Data were presented as mean ±
standard deviation
Statistical analyzer was performed
using a one way analysis of variance
Difference were considered
significant of at P<0.05
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31. The EC50 values were calculated by
liner regression analysis
Results were calculated by employing
the statistical software (SPPS, version
13.0, SPSS Inc., Chicago, USA)
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32. RESULT AND DISCUSSION
Extractive yields:-
The extraction yields of these samples
from six various regions varied from
21.0% to 39.4%
The data of extractive yields value is
given below
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34. Total phenolic, flavonoids and
proanthocyanidin content:-
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35. Various stage of maturation and
growing condition such as a temperature
and rainfall affect the content of
phenolic compound
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36. DPPH radical scavenging activity:-
DPPH is a stable nitrogen centered
free radical
Its color changes from violet to
yellow when is reduced by either the
process of hydrogen or electron
donation
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37. 8/3/2012 37

38. Superoxide anion radical scavenging
activity:-
Although superoxide anion is a weak
oxidant, it gives rise to the generation of
powerful and dangerous hydroxyl radical
as well as singlet oxygen
Both of which contribute to the
oxidative stress
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40. Hydroxyl radical scavenging activity:-
Hydroxyl radical is extremely
reactive in biological system
Capable of damaging biomolecule
of the living system
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42. Determination of capacity of lipid
peroxidation inhibition:-
Lipid peroxidation can inactivate
cellular component and plays an
important role in oxidative stress in
biological system
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45. Ability of chelating ferrous iron:-
Ferrous ion, commonly existed in food
system is well known as an effective pro-
oxidant
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48. Reducing power:-
Reducing power has been used as one
of important antioxidant capability for
medicinal herbs
The reducing power of emblica extract
is dose dependent
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50. Correlation with free radical
scavenging activity, antioxidants
activity and phenolic content:-
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51. CONCLUSION
Huizhou sample had higher content of
total phenolic, flavonoids and
proanthocyanidins than other sample
The Chuxiong and Huizhou samples
exhibited quite strong antioxidant and
radical scavenging activities in
comparison to the reference compound
BHM and quercetin
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52. The results indicated that regions
could leads to significant differences
both in the content of bioactive
compounds and their bioactivities
The phenolic component might be the
major active component responsible for
the strong antioxidant activity
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53. Thus emblica fruits can be a source of
plant antioxidants, with a potential use in
food, cosmetic and pharmaceutical fields
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54. REFERENCE
8/3/2012 54
Liv Xiaoli, Zhao Mouming et al
(2008), Antioxidant activity of
methanolic extract of emblica fruit
(Phyllanthus emblica L) from six
regions in China, Journal of Food
composition and analysis 21,219-228

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