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Research article Rapports De Pharmacie Vol.

2 (4), 2016, 304-308


ISSN: 2455-0507
DISCERNING THE ANTIOXIDANT AND MICROKINETIC POTENTIAL OF
EUCALYPTUS VIMINALIS LABILL EXTRACTS
*Fuloria Shivkanya, Subramaniam Ganesheni, Ying Teoh Suah, Vijpuri Narmadevi,
Fuloria Neeraj Kumar, Sikarwar Singh Mukesh, Kaveti Balaji, Sundram Karupiah,
Vijayabalan Shantini.
Faculty of Pharmacy, AIMST University, Semeling Bedong, Malaysia-08100

ABSTRACT
The present study was aimed to determine the antioxidant and microkinetic activity of methanolic extract
of leaves of Eucalyptus viminalis labill. The Eucalyptus viminalis labill leaves (EV1) and stem (EV2)
methanolic extracts were prepared by hot percolation (Soxhlation). EV1 and EV2 extracts were subjected
to aluminium trichloride assay for determination of flavonoids; Folin-Ciocalteu colorimetry for estimation
of phenolic content; DPPH and FRAP assay for evaluation of antioxidant potential; and agar well
diffusion and 96 well ELISA reader method for discerning of the antibacterial potential. The EV2 extract
possess high phenolic content (5.17 mg GAE equivalent/gm dried wt.) and flavonoid content (20.46 mg
Rutin equivalent/ gm dried wt.). During In vitro antimicrobial evaluation EV2 and EV1 displayed
significant inhibitory activity against E. Coli. Present study concludes that stem extract (EV2) of
Eucalyptus viminalis labill possess high total phenolic and total flavonoids contents compared to leaves
extract (EV1). Both EV1 and EV2 extracts possess DPPH radicals scavenging activity, reducing potential,
and high antibacterial extracts. Present study supports Eucalyptus viminalis labill methanolic extract
potential for antimicrobial and antioxidant activity.
Keywords: Eucalyptus viminalis, Total phenolic content, Flavonoid content, Antioxidant activity, Growth
curve
INTRODUCTION
Over the past three decades, there was an Hence, present study was aimed to determine the
exponential increment in the use of herbal total phenolic and flavonoid content, antioxidant
remedies in several medical conditions [1]. (by DPPH and FRAP) and antibacterial potential
Investigation supports the role of reactive oxygen of methanolic extract of leaves and stems of
species (ROS) and reactive nitrogen species (RNS) Eucalyptus viminalis.
in oxidative stress mediated damage of cellular MATERIAL AND METHODS
structures [2]. Oxidative stress causes Chemicals:
accumulation of free radicals and roots several Aluminium chloride, Methanol, potassium
pathophysiological processes, such as cancer, ferricyanide, trichloroacetic acid, gallic acid (GA),
diabetes, cardiovascular and neurodegenerative rutin (RU), and FolinCiocalteus reagent, 1,1-
diseases [3]. Antioxidants display free radical Diphenyl-2-picrylhydrazyl (DPPH), Sodium
scavengers activity stimulates immune defence carbonate, Ascorbic acid, Mueller Hilton media.
system and lowers the risk of degenerative All chemical reagents were of analytical grade.
diseases, by preventing and repairing damages Collection of plant material:
caused by ROS and RNS. The plant material was collected from the premise
Eucalyptus viminalis labill (family: myrtaceae) of AIMST University, Malaysia. The taxonomic
leaves are reported to possess volatile oil identification of plant was confirmed, and
constituents like 1, 8-cineole, pinene, eudesmol, herbarium was submitted to Pharmacognosy
and tannins. Eucalyptus viminalis labill plant is Department, Faculty of Pharmacy (Herbarium
also reported for antiviral, antibacterial, voucher specimen Accession number
antirheumatic activities [4]. Although to the best AIMST/FOP/05).
of researcher's knowledge, validation of phenolic, Preparation of extracts:
flavonoid content, antioxidant and antibacterial The leaves and stem parts of Eucalyptus viminalis
activities of Eucalyptus viminalis labill is still an plant were separately collected and dried under
uninvestigated and unpublished area of research. shade at room temperature, and later powdered
Address for correspondence: using mechanical blender. The leaves and stem
Dr. Shivkanya Fuloria, extracts of Eucalyptus viminalis labill were
Department of Pharmaceutical Chemistry, prepared by hot percolation method. The hot
Faculty of Pharmacy,AIMST University, percolation method involved, placement of
Bedong- Semeling, Kedah, Malaysia 08100 powdered material in Soxhlet apparatus and
Email: shivani2jaju@gmail.com extraction with with methanol at 45-55C for 10-
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Fuloria Shivkanya et al,: Microkinetic potential of eucalyptus viminalis labill extracts

12 h. The obtained extracts were concentrated on Where,


rotavapor, dried and stored in desiccator for Ac = absorbance of control (DPPH free radical
further study. without the addition of test solution)
Determination of total phenolic content: As = sample absorbance (absorbance of DPPH
The total phenolic content of methanolic extract of free radical after addition of test solution)
leaves (EV1) and stem (EV2) was measured using Ferric reducing power assay:
the FolinCiocalteu reagent method. Briefly, each Each extract EV1 and EV2 was evaluated for
100 l of extract of leaves (EV1) and stem (EV2) ferric reducing power using an assay described in
were mixed with 3 ml distilled water (in method. To 1 ml of different concentration of each
triplicate). To this solution 100 l of Folin- EV1 and EV2 extract, 2.5 ml of 200 mM
Ciocalteaus reagent (10%) and 2.0 ml of sodium phosphate buffer (pH 6.6) and 2.5 ml of 1%
carbonate (7.5%) were added. The mixture was potassium ferricyanide were added and incubated
kept in dark for 2 hours before measuring the at 50oC for 20 min. Later, to each extract EV1 and
absorbance at 750 nm. The extracts were EV2 mixture, 2.5 ml of 10% trichloroacetic acid
quantified for total phenolic content using standard was added and mixtures were centrifuged at 3,000
curve of Gallic acid. The concentration of EV1 rpm for 10 min. About, 2.5 ml of supernatant layer
and EV2 ranged from 0.1-0.5 g/ml (R2 = 0.998). of each EV1 and EV2 extract solution was diluted
The obtained results were expressed as mg gallic with 2.5 ml of water and was shaken with freshly
acid equivalent (GAE)/100g plant materials [5]. prepared 0.5 ml of 0.1% ferric chloride and the
Determination of total flavonoid content: absorbance of the reaction mixtures was measured
The extracts EV1 and EV2 were subjected to at 700 nm. The final results were expressed as mg
determination for total flavonoid contents using ascorbic acid equivalent/g of dry weight.
Aluminium chloride method. To 1 ml of each Growth curves:
extract, 0.7 ml of 5 % sodium nitrate was added in As per the procedure described in literature with
triplicate. Subsequently, each extract solution was minor modifications, the growth curve was
mixed with 10 ml of 30% ethanol for five minutes, determined for each extract EV1 and EV2. To each
and further mixed with 0.7 ml of 10% aluminium well of a sterile 96-well microtiter plate containing
chloride. After six minutes, to the extract different concentration of each extract (100
solutions, 5 ml of 1 mol/L sodium hydroxide was L/well, triplicate) EV1 and EV2, a 100 L of
added. Later, solutions were diluted with ethanol bacterial inoculum was added. The two wells
(25 ml, 30%). The extract solutions were allowed containing bacterial suspension with no drug
to stand for 10 minutes and subjected to (growth control) and two wells containing only
measurement of absorbance at 450 nm using UV media (background control) were included in this
spectrophotometer. A standard calibration curve of plate. For the study, gentamicin was used as
rutin concentration 10-100 g/ml (r2 = 0.999) was positive control and each assay was repeated in
generated, that assisted in quantification of total triplicate. Later, ODs were measured for 24 h at
flavonoid content. Total flavonoid content was 35C using a multidetection microplate reader
calculated and expressed as mg of rutin equivalent (Bio-red, USA) at 600 nm and were automatically
(RU)/g of dry weight. recorded every 15 min for each well.
DPPH assay: The turbidimetric growth curves were obtained
The extracts EV1 and EV2 were evaluated for depending on the changes in the OD of bacterial
their antioxidant potential using DPPH assay. growth for each drug concentration and the drug-
Briefly, to different concentrations of 2.5 ml of free growth control.
each extract EV1 and EV2, 1 ml of 0.3 mM of an In order to determine crude extract MICs using
alcoholic solution of DPPH was added and each spectrophotometric kinetic assay, the growth
mixture was incubated in dark place for 30 percentage for each drug concentration was
minutes at room temperature. The extract solutions calculated with the following equation:
were subjected to measurement of absorbance at
517 nm using UV spectrophotometer. A standard
graph was plotted using different concentrations of
BHT as standard and methanol as blank. Later, the
scavenging ability of each extract EV1 and EV2
solution against DPPH radical was determined For each well, and inhibition percentage values
using following equation: were calculated. MIC50 was calculated from the
graph plotted between concentration and inhibition
( )
[6].

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Fuloria Shivkanya et al,: Microkinetic potential of eucalyptus viminalis labill extracts

Table 1: Total phenolic content and total flavonoid content of Eucalyptus viminalis
Total Flavonoid Content (mg rutin /100 Total Phenolic content (mg GAE equivalent gm
gm) dried weight)
EV1 EV2 EV1 EV2
14.27 20.46 4.94 5.17

Table 2: Antioxidant activity of Eucalyptus viminalis by DPPH method


Anti-oxidant Activity (%)
Concentration g/ml)
BHT EV1 EV2
10 83.70 88.01 60.25
20 93.49 95.08 71.29
40 95.27 94.37 77.61
60 95.58 93.12 90.13
80 95.76 91.94 95.47
100 96.18 92.59 96.02

Table 3: Absorbance of Eucalyptus viminalis for FRAP


Concentration (g/ml) Ascorbic acid EV1 EV2
25 0.62 0.23 0.43
50 1.04 0.23 0.44
100 1.16 0.27 0.54
200 1.15 0.35 0.70
400 3.25 0.55 1.10

Table 4: MIC of leaves extract (EV1)


Bacteria B. subtilis E. faecalis S. aureus E.coli K .pneumoniae
MIC(g/ml) 519.03 12.96 78.12 6.88 22.42

Table 5: MIC of stem (EV2)


Bacteria B. subtilis E. faecalis S. aureus E.coli K. pneumoniae
MIC((g/ml)) 84.16 291.00 20.03 5.12 16.88

250

200 EV1 B.subtilis


% Inhibition of growth

EVI E. faecalis
150
EVI S.aureus
EVI E.coli
100
EV2 B. Subtilis

50 EV2 E. faecalis
EV2 S. aureus
0 EV2 E.coli
0 50 100 150 200 250 300 350 400 450 500 550
Concentration (g/ml)

Figure 1: Microkinetic assay to determine percent inhibition of growth Vs concentration


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Fuloria Shivkanya et al,: Microkinetic potential of eucalyptus viminalis labill extracts

RESULTS AND DISCUSSION the drug using delta ODs in a shorter time than the
Present study, involved determination of total reference method (approximately 16-20 h) in this
phenolic and flavonoid content of methanolic assay.
extracts of leaves and stem prepared by hot In our study the methanolic stem extract showed
percolation method. Several studies documented comparatively higher antioxidant activity that of
that phenolic compounds are significantly the leaves extract, which is in accordance with the
responsible for antioxidant properties [7]. total phenolic and flavonoid content of the
Flavonoids act as antioxidants due to their ability extracts. In this study, a microbroth kinetic system,
to reduce free radical formation and to scavenge which provides kinetic data throughout the
free radicals. Phenolic compounds have powerful incubation period, was used to determine
antioxidant properties like prevention of free antibacterial activity. The results obtained in
radicals induced oxidative damage as well as present study suggest that Eucalyptus vimillais
different anticarcinogenic, vasodilatory, labill may be a potential plant to be explored for
hypoglycemic and neuroprotective effects [8] Plant its anti-inflammatory and anticancer activity.
materials rich in phenolics and flavonoids, are CONCLUSION
increasingly being used in the food industry for The present study concludes that Eucalyptus
their antioxidative properties and healthbenefits viminalis labill is a potential source for natural
[9]. The total phenolic content of each extract was antibacterial and antioxidant agents. Furthermore,
determined using Folin Ciocalteu method and phytochemical analysis is essential for the
expressed in terms of gallic acid equivalent (data isolation of bioactive molecules from the plant.
given in table 1). Among both extract EV2 ACKNOWLEDGMENTS
displayed highest phenolic content. The total The authors are thankful to AIMST University,
flavonoid content of each extract was determined Malaysia for providing necessary facilities and
using aluminium chloride method. Total flavonoid funding for the present study.
content was expressed as mg quercetin equivalent
(QE) per gram dry extract weight (data given in REFERENCES
table 1) [1] L Subedi, S Timalsena, P Duwadi, R Thapa,
The antioxidant property of the crude extracts was A Paudel, K Parajuli. Antioxidant activity
investigated and compared by various biochemical and phenol and flavonoid contents of eight
assays like DPPH, FRAP assays. In the present medicinal plants from Western Nepal. J
study, both extracts were investigated for Tradit Chin Med, 34: 584-590(2014).
antioxidant activity using two different methods. [2] N Saeed, MR Khan, M Shabbir. Antioxidant
Both extracts displayed significant activity and activity, total phenolic and total flavonoid
results data are given in table 2 and 3. contents of whole plant extracts Torilis
The MIC value of leaves and stem extracts against leptophylla L. BMC Complement. Altern
different microorganisms was reported to be in Med, 12: 221-221 (2012).
g/ml (data given in table 4 and 5). The tested [3] MM Rahman, MB Islam, M Biswas. In vitro
extracts of Eucalyptus viminalis labill stem and antioxidant and free radical scavenging
leaves possessed antibacterial activity against both activity of different parts of Tabebuia pallida
Gram positive and Gram negative bacteria. The growing in Bangladesh. BMC Res Notes, 8:
antibacterial activity of stem extract was found to 621(2015).
be comparatively higher than that of leaf extract. [4] MT Maghsoodlou, N Kazemipoor, J
The leaves extract exhibited highest antibacterial Valizadeh, M Falak Nezhad Seifi, N
activity with against E. coli (6.88 mg/ml) and Rahneshan. Essential oil composition of
lowest activity against B. Subtilis. The growth for Eucalyptus microtheca and Eucalyptus
each strain in each well with different viminalis. Avicenna J Phytomed, 5(6): 540-
concentration monitored from 0 hr to 24 hr and 552(2015).
growth curve was obtained. [5] MZM Salem, HM Ali, NA El-Shanhorey, A
Using the microbroth kinetic assay, MIC50 Abdel-Megeed. Evaluation of extracts and
values of Eucalyptus vimillais were determined in essential oil from Callistemon viminalis
the 24 hr depicted in figure 1. In this study, a leaves: Antibacterial and antioxidant
microbroth kinetic system, which provides kinetic activities, total phenolic and flavonoid
data throughout the incubation period, was used to contents. Asian Pac J Trop Med, 6: 785-
determine antibacterial activity. We measured the 791(2013).
ODs of bacterial growth kinetically in each well at [6] B Taye, M Giday, A Animut, J Seid.
selected time intervals and calculated the MICs of Antibacterial activities of selected medicinal

307
Fuloria Shivkanya et al,: Microkinetic potential of eucalyptus viminalis labill extracts

plants in traditional treatment of human [8] T Ghosh, TK Maity, M Das, A Bose, D


wounds in Ethiopia. Asian Pac J Trop Dash. In vitro antioxidant and
Biomed, 1(5): 370-375(2011). hepatoprotective activity of ethanolic extract
[7] N Amessis-Ouchemoukh, K Madani, PLV of Bacopa monnieri L. aerial parts. Iranian J
Fale, ML Serralheiro, MEM Araujo. PhrmacoTherapu, 6(1): 77-85(2007).
Antioxidant capacity and phenolic contents [9] R Chirinos, R Pedreschi, H Rogez, Y
of some Mediterranean medicinal plants and Larondelle, D Campos. Phenolic compound
their potential role in the inhibition of contents and antioxidant activity in plants
cyclooxygenase-1 and acetylcholinesterase with nutritional and/or medicinal properties
activities. Ind Crops Prod, 53: 6-15 (2014). from the Peruvian Andean region. Indu
Crops Prod, 47:145-152 (2013).

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