Evaluation of the antioxidant activity of syzyzium cumini leaves

1. Presented By:
Md. Abdullah-Al-Masud
B -110606011
Evaluation of the Antioxidant
Activity of Syzygium cumini Leaves

2. Zhi Ping Ruan 1,2, Liang Liang Zhang 1,2 and Yi Ming Lin 1,2,*
1) Key Lab of Ministry of Education for Coast and Wetland Ecosystems, Xiamen 361005; P.R. China; E-mails:
rzp20012001@yahoo.com.cn (Z-P. R.); zhll20086@gmail.com (L-L. Z.)
2) Department of Biology, School of Life Sciences, Xiamen University, Xiamen 361005; P.R. China
Author to whom correspondence should be addressed; E-mail: linym@xmu.edu.cn; Tel.: (+86) 592 2187657.
Received: 22 August 2008; in revised form: 10 October 2008 / Accepted: 13 October 2008 /
Published: 16 October 2008
Evaluation of the Antioxidant Activity of Syzygium cumini
Leaves

3.  Objectives of the study
 Introduction
 Plant Description
 Antioxidant system
 Material and Methods
 Collection and Extraction procedure
 Antioxidant activity
 Total phenolic and Total flavonoid assay
 2,2-diphenyl-1-picrylhydrazyl(DPPH) assay
 Ferric reducing antioxidant power (FRAP) assay
 HPLC analysis of phenolic compound
 Result and Discussion
 Conclusion
Contents

4. The present investigation is undertaken by utilizing Syzygium cumini leaves with
following objectives.
 Extraction of leaves with methanol.
 Find the antioxidant property.
 To determination of total phenolic and total flavonoid content.
 To determine free radical scavenging ability on 2,2-diphenyl-1-picrylhydrazyl(DPPH).
 To determine ferric reducing antioxidant power(FRAP) assay.
Objective of the study

5.  Syzygium cumini is an important medicinal plant
belonging to the family myrtraceae, having a lot of
pharmacological properties and are extensively used
to treat various health ailments.
 S.Cumini is slow growing tree, medium to large
sized up to height 30 meter and can live up to 100
year.
 It’s Bengali name ‘kala jam’
Introduction

6. Plant Description
Taxonomical classification
Kingdom: Plantae
Division: Tracheophyta
Class: Magnoliopsida
Order: Myrtales
Family: Myrtaceae
Genus: Syzygium
Species: Syzygium cumini
Phytochemistry(Chemical composition)
 Anthocynins
 Glucoside
 Ellagic acid
 Isoquercetin
 Kaempferol
 Myricetin
 Acetyl oleanolic acid
 Triterpenoid
 Mineral salt
 Vitamin C

7. S.cumini seeds
 Astringent
 Anti-diabetes
 Anti-glycemic
 Mild digestive
S.cumini leaves
 Astringent
 Antiemetic
 Antihemorrhagic
 Antidiabetes
 Antihyperglycenic
S.cumini Fruits
 Mild astringent
 Digestive stimutent
 Liver stimulent
 Anti inflammatory
S.Cumini barks
 Digestive
 Astringent
 Carminative(prevent flalulence)
 Anthelmintic(expel parasite out of
the body)
 Antibacterial
 Anti-dysentery
 Mild Antihemorrhagic
MEDICINAL PROPERTIES

8. Antioxidant
 Antioxidant is a molecule that inhibits the oxidation of the other
molecules.
 Oxidation is a chemical reaction that transfers the electrons
from the substance to an oxidizing agent.
 These oxidation reactions can produce free radical which starts
the chain reaction and causes damage and death to the cell.
 Antioxidant can terminate the chain reactions by removing the
free radicals and inhibit the oxidation reactions.
 They do this oxidizing themselves , so these are often called as
reducing agents such as thiol , ascorbic acid, polyphenols etc.

9. Materials and Methods
Chemicals and Plant materials
 Methanol
 Ethyl acetate
 Chloroform
 N-hexane
 Vitamin C(Vit. C)
 Butylated hydroxyanisole(BHA)
 Tannic acid
 Ferulic acid
 Rutin
 Catechin
 TPTZ(2,4,6-tripyridryl-S-triazine)
 DPPH(2,2-diphenyl-1-picrylhydrazyl)
 Milli-Q water
 HPLC grade CH3CN
 Mature leaves of S.cumini

10. Leaf sample(120g)
crushed or cut small
Placed in a closed
vessels
Macerating in methanol
Liquid strained off
Allow them to stand 7 days
room temp.in a dark
cabinet(shaking occasionally)
Clarified by filtration
Evaporation the solvent by
rotary evaporator
Methanolic extract(ME)was
further fractionated through
solvent-solvent partitioning to
obtain different fractions
 The ME and its four
water(WtF)-37.33%
 Ethyl acetate(EaF)-5.33%
 Chloroform(CfF)-4.01%
 N-hexane(HxF)-24.67%
Extraction procedure

11. ANTIOXIDANT ACTIVITY
 Various antioxidant activity methods have been used in food and natural products to monitor and
compare.
 In the present research program , in vitro antioxidant activity was determined using following
methods.
# Total phenolic and total flavonoid assay.
# 2,2-diphenyl -1- picrylhydrazyl(DPPH) free radical scavenging ability.
#Ferric reducing antioxidant power(FRAP) assay.
# HPLC analysis of phenolic compounds.

12. PRUSSIAN BLUE ASSAY FOR TOTAL PHENOLS
Reagent
 0.1M FeNH4(SO4)2 in 0.10M HCL
1) Dilute concentrated HCl to 10 M by bringing
8.3mL of the concentrated acid to 1L with distilled
water.
2) Make the ferric ammonium sulfate by dissolving
48.2g of the dodecahydrate salt. FeNH4(SO4)2
.12H2O in 1L of the 0.10 M HCl . This will make a
pale yellow solution .
 0.008M K3Fe(CN)6 Dissolve 2.63g of potassium
ferricyanide in 1L of distilled water. This will
make a yellow solution.
Method
2 min After
Dispense 0.10mL sample into a 125mL Erlenmeyer flask+
50.0mL distilled water
Add 3.0mL FeNH4(SO4)2 and swirl. Addition should be
timed, 1 min intervals are convenient.
Adding ferric ammonium sulfate , start time (1min intervals)
additions 3.0 ml K3Fe(CN)6 Swirl
Add ferricyanide.read absorbance at 720 nm making
reading at 1 min interval
Include solvent only blank. Standardize against 0.01M
tannic acid.

13. TOTAL FLAVONOID ASSAY METHOD
.
The methanolic extract(2mg)+ 2ml distilled water +NaNO2(0.3ml, 5%)
After 6 min at 250C
Added Al(NO3)3 (0.3ml , 10%)
Treated with NaOH (4 ml,1M),diluted to volume (10ml) With 50% ethanol solution
After 6 min
After a thorough mixing the absorbance at 500nm was read.
Rutin was used as the standard and the total flavonoid content was express as rutin
equivalents(RE) mg/g dry weight of the extract

14. 2,2-DIPHENYL-1-PICRYLHYDRAZYL(DPPH) ASSAY
Each extract (0.1ml, 15-250g/ml) in methanol + methanol solution(3ml) +
DPPH(0.004%,w/w)
Mixture was shaken vigorously
Left to stand for 30min in the dark
Then the scavenging ability was calculated ability using the following equation scavenging ability(%)=[(A517
of control - A517 of sample) / A517 of control ]×100
Three replicates were carried out.
Measuring the absorbance at 517nm against a blank

15. FERRIC REDUCING ANTIOXIDANT POWER (FRAP)ASSAY
FRAP reagent – 10 mM TPTZ(2,4,6-tripyridyl-S-triazine) solution (2.5ml)+40mM HCl+ 20 mM
FeCl3(2.5mL)+0.3 M acetate buffer (pH 3.6,25ml)
3ml of FRAP reagent, prepared freshly + 0.1 ml of test sample or methanol(for the reagent blank)
Incubation at 250 for 10 min
 The FRAP values, expressed in mmol ascorbic acid equivalents (AAE)/g sample in dry
weight were derived from a standard curve.
The absorbance of reaction mixture was measured spectrophoto - metrically at 593 nm

16. HPLC ANALYSIS OF PHENOLIC COMPOUND
 The contents of phenolic compounds in leaf extracts of
S.cumini were determined by HPLC , performed with
an Agilent 1100 diode array detector system equipped
with a quaternary pump.
 The analysis were carried out on a Hypersil ODS
column (4.6mm × 250 mm, 2.5 m ) column.
 Extracts were filtered through a 0.45m filter before
use.
 Gradient B in A according to the elution profile 0-3 min
2% B(isocratic), 3-20 min 2%-25%B(linear gradient),
20-25min 25-35% B (linear gradient), (A) water (0.05%
TFA) (B) CH3CN (0.05% TFA);
 Flow rate 1mL/min
 Volume injected 10 L, temperature 220C; detection 280
nm.

17. RESULTS AND DISCUSSION
Total phenolic and total flavonoid content
 Phenols are very important plant constituents because of their radical scavenging ability due to their
hydroxyl groups. The phenolics content may contribute directly to the antioxidative action.
 It has been suggested that polyphenolic compounds have inhibitory effects on mutagenesis and
carcinogenesis in humans.
 The total phenolic content in the methanolic extract of S.cumini was 610 ± 9.03 mg/g while the flavonoid
content was 451 ± 9.85 mg/g .
 This results demonstrate that flavonoids represent the main group of phenolic compounds in S.cumini leaves.
Phenolic compounds Flavonoid compounds

18. RESULTS AND DISCUSSION
DPPH free radical- scavenging assay
 The DPPH free radical is a stable free radical, which has been widely accepted as a tool for estimating free radical-
scavenging activities of antioxidants . The percentages of remaining DPPH in the presence of the ME and its fractions
at different concentrations are shown in table 1
 The proportion of the remaining DPPH decreased
slightly with the WtF and CfT. Great decreases in a
concentration–dependent manner of the remaining
DPPH in ME and EaF indicated that with the exception
of HxF, S. cumini extracts possess potent free radical-
scavenging activity.
 By comparing ME and its active fractions, the free
radical-scavenging activities followed the order :
EaF ≈ ME> CfF ≈ WtF > HxF

19. RESULTS AND DISCUSSION
Ferric reducing antioxidant power (FRAP)
 The FRAP assay is based on the ability of antioxidants to reduce Fe3+ to Fe2+ in the presence of
TPTZ, forming an intense blue Fe2+ TPTZ complex with an absorption maximum at 593 nm.
The absorbance decrease is proportional to the antioxidant content.
 All fractions showed high ferric reducing power
with increasing concentration, with exception
HxF
 The FRAP value, expressed in ascorbic acid
equivalents, was used to determine the
antioxidant ability of the different extract in
present study.
 The FRAP values for the S.cumini leaf extracts
were high, ranging from 2.32±0.02 to
3.12 ± 0.07 mmol AAE/g(Table 2)
 In addition , in a comparison of the ferric
reducing power of the ME and its active
fractions with BHA, the reducing power of all
extracts were lower than that of BHA
(4.66mmol AAE/g)

20. RESULTS AND DISCUSSION
Correlation between total phenolic content
antioxidative function
 The free radical scavenging activity and reducing power
of the methanolic extract were significantly related to
their total phenolic content( RDPPH= 0.9930,
RFRAP=0.9998) (Fig-1)
 The methanolc extract exhibited the highest radical
scavenging activity and ferric reducing power with the
grearest amount of phenolic content.
 The presence of polyphenolic compounds in methanolic
extract of S.cumini might be responsible for this high
antioxidant activity.

21. RESULTS AND DISCUSSION
Determination of phenolic compounds
 Phenolic compounds, such as quertin, rutin, nargin, caffeic acid gallic acid and chlorogenic acid are very important
plant constituents because of their antioxidant activities.
 The concentrations were determined by calculating the HPLC peak areas which are proportional to the amount of
analytes in a peak and presented as the mean of three determinations which were highly repeatable. Figure 3a shows
the chromatogram of authentic standards of catechin and ferulic acid.
 The result from the chromatograms indicated that EaF contained the highest content of catechin and ferulic acid. As
shown in Figure 4 contained approximately 7-fold more catechin and about 9-fold more ferulic acid than ME .By
comparing the different fractions , the content of catechin and ferulic acid decreased in the same order of EaF
>CfF>WfF≈ME>HxF, and the rank order of ME and CfF was different according to their antioxidant potency and
free radical-scavenging ability,

22. CONCLUSION
 Total phenolic content in methanolic extract of S.cumini leaves was 610.32±9.03 mg/g
while the flavonoid content of the methanolic extract was 451.50±9.85mg/g.
 The five extracts of S.cumini leaves have potent antioxidant activity according to the
DPPH and FRAP assays.
 The HPLC data indicated that S.cumini leaf extract contained phenolic compounds, such
as ferulic acid and catechin.
 A significant linear relationship between antioxidant potency, free radical- scavenging
ability and the content of phenolic compounds of leaf extract supported this observation.

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