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in vitro Evaluation of Leaves and Fruits of Elaeagnus latifolia L. for Antioxidant and Antimicrobial Activities

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Abstract

In this work, we have studied the antioxidant and antimicrobial potential of leaves and fruits of Elaeagnus latifolia L. Antioxidant activities were conducted by using hydrogen peroxide radical scavenging and DPPH method. Total phenolic and flavonoid content was measured by using gallic acid and quercetin equivalent. in vitro Antibacterial activity of these plants was examined against five pathogenic bacteria. Highest DPPH scavenging activity was shown by methanolic extract of the flower of Elaeagnus latifolia with an IC50 value of 144.64 ± 0.25 µg mL⁻¹ in comparision to standard ascorbic acid with an IC50 value of 29.39 ± 7.11 µg mL⁻¹. Highest hydrogen peroxide radical scavenging activity was shown by the leaves of Elaeagnus latifolia with an IC50 value of 444.59 ± 3.77 µg mL⁻¹, whereas IC50 value of standard ascorbic acid is 279.89 ± 1.81 µg mL⁻¹. The leaves of Elaeagnus latifolia showed highest total phenolic and flavonoid content with 61.15 ± 1.23 µg mL⁻¹ and 15.12 ± 0.125 µg mL⁻¹, respectively. The result of antimicrobial study showed that the plant is potent against the tested organism which is comparable to standard drug ofloxacin. From the findings, it can be interpreted that the plant could be utilized as natural source of antioxidant in food processing or in medicine industry.
ASIAN JOURNAL OF CHEMISTRY
ASIAN JOURNAL OF CHEMISTRY
https://doi.org/10.14233/ajchem.2018.21430
INTRODUCTION
Antioxidants are the substances which delay, prevent or
even inhibit the oxidation of oxidizable substrate/system in
which they are present, by inhibiting the propagation or initiation
of oxidative chain reactions [1]. They reduce themselves, thus
acts as an antioxidant. Oxidizable substrate means very nearly
everything found in the living cells counting proteins, lipids,
DNA and carbohydrates [2]. Antioxidants possess free radical
chain reaction breaking properties thus defend living cells
against oxidative damage. They help in reducing and quenching
of singlet oxygen formation and function as radical scanv-
engers [3-5]. Antioxidant activity is indispensable for life to
neutralize the strongly oxidizing environment present inside
our body. The most interesting point about free radical caused
cell injury is that we can combat against this physiological
pathway, by doing some modifications in our food habits. Generally
antioxidants mainly from fruits and vegetables are regarded
as a richest reservoir of natural antioxidant compounds [6-10].
in vitro Evaluation of Leaves and Fruits of Elaeagnus latifolia L.
for Antioxidant and Antimicrobial Activities
INDUBHUSAN DUTTA1, BARNALI GOGOI2, RUPANJALI SHARMA2 and HEMANTA KUMAR SHARMA2,*
1Centre for Studies in Biotechnology, Dibrugarh University, Dibrugarh-786 004, India
2Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh-786 004, India
*Corresponding author: E-mail: hemantasharma123@yahoo.co.in
Received: 8 May 2018; Accepted: 19 July 2018; Published online: 27 September 2018; AJC-19090
In this work, we have studied the antioxidant and antimicrobial potential of leaves and fruits of Elaeagnus latifolia L. Antioxidant
activities were conducted by using hydrogen peroxide radical scavenging and DPPH method. Total phenolic and flavonoid content was
measured by using gallic acid and quercetin equivalent. in vitro Antibacterial activity of these plants was examined against five pathogenic
bacteria. Highest DPPH scavenging activity was shown by methanolic extract of the flower of Elaeagnus latifolia with an IC50 value of
144.64 ± 0.25 µg mL-1 in comparision to standard ascorbic acid with an IC50 value of 29.39 ± 7.11 µg mL-1. Highest hydrogen peroxide
radical scavenging activity was shown by the leaves of Elaeagnus latifolia with an IC50 value of 444.59 ± 3.77 µg mL-1, whereas IC50 value
of standard ascorbic acid is 279.89 ± 1.81 µg mL-1. The leaves of Elaeagnus latifolia showed highest total phenolic and flavonoid content
with 61.15 ± 1.23 µg mL-1 and 15.12 ± 0.125 µg mL-1, respectively. The result of antimicrobial study showed that the plant is potent
against the tested organism which is comparable to standard drug ofloxacin. From the findings, it can be interpreted that the plant could
be utilized as natural source of antioxidant in food processing or in medicine industry.
Keywords: Elaeagnus latifolia L., Antioxidant activity, Antimicrobial activity.
Asian Journal of Chemistry; Vol. 30, No. 11 (2018), 2433-2436
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Due to extensive misuse of antimicrobial drugs, microbial
resistance problem is gradually growing over the decades. As
a result the antibiotics are becoming less effective which cause
havoc in global healthcare worldwide. Therefore, proper research
should be done to minimize the spreading and development
of resistance and to create newer options for treatment of micro-
organisms. From historic times herbs have been a valuable source
of therapeutically potent natural molecules for maintaining
human health. Even the pharmaceutical companies focused
on drug discovery from natural sources. A large number of medi-
cinal plants are claimed to be useful in bacterial diseases in all
traditional system of medicine and folklore [11-13]. Therefore,
a comprehensive study of such plants should be investigated
to produce a drug lead. As a part of search for new biological
activity of a plant extract, preliminary bioscreening were
performed to study the antimicrobial activity of methanolic
extracts of leaf and fruit of Elaeagnus latifolia L.
The plant Elaeagnus latifolia L. is a thorny scandent shrub.
Shoots are reddish brown colored with scales. Spines are
present in stems and branches. Leaves are 3-9 cm long and 2-5
cm broad, elliptic to obovate or elliptic-lanceolate, acute or
rounded at base. The leaves are green coloured and charac-
teristic silvery-white at the beneath while the flowers are silver
coloured. The fruits are green at early stage and dark pink/orange
colour at ripening. Seeds are 1.8 cm long, erect, hard and shining.
It flowers during September-December and the light pink
coloured fruits are harvested during March-April month. They
are swamps and grow at the elevations of 1500 metres in the
Himalayas. In North East India, it is found in Naga hills (Naga-
land state), Sibsagar, Lakhimpur (Assam state); Khasi, Garo
and Jaintia hills of Meghalaya state upto elevation of 1500 m
altitude [11-13].
The fruit is acidic in nature and eaten as raw or cooked.
They contain vitamins and minerals, flavonoids and a good source
of essential fatty acids while the flowers are used as astringent.
The plant is investigated as a good source of food plant inhibiting
growth of cancer cells [14,15]. This current research commu-
nication deals with the study of antimicrobial and antioxidant
activity of leaves and fruits of Elaeagnus latifolia L.
EXPERIMENTAL
The plant Elaeagnus latifolia L. was collected from Moidamia
village, North Lakhimpur, India in the month of February,
2017. The plant was identified by Botanical survey of India,
Eastern Regional Circle, Shillong. A herbarium specimen No.
ID-HS-BT-PS-PL-E was sent to BSI eastern regional Centre,
Shillong for identification of the plant species. After collecting
the fruits and leaves, these were allowed to dry for 10-15 days
in shady area under cool condition preventing from sunlight.
Three Gram-positive and two Gram-negative strains of
bacteria were selected for in vitro antibacterial screening. These
strains were obtained from Department of Sciences, Dibrugarh
University, Dibrugarh, India.
Preparation of methanolic extract: The leaves and fruit
of Elaeagnus latifolia L. were dried, grounded in a grinder to
coarse powder and stored in airtight containers to prevent moisture.
The methanolic extract of leaves of Elaeagnus latifolia (MELEL)
and methanolic extract of fruits of Elaeagnus latifolia (MEFEL)
were done by Soxhelation with methanol after pre-treatment
with petroleum ether. The concentrated extract was dried under
vacuum and stored.
Antioxidant activity
DPPH radical scavenging activity: DPPH radical scav-
enging activity of methanolic leaves and fruit extract of Elaeagnus
latifolia was measured with UV at 517 nm. All determinations
were performed in triplicate [16]. The following formula was
used to measure capability of extract to scavenge the DPPH
radical:
ot
o
(A A )
Inhibition of DPPH scavenging activity (%) 100
A
(1)
where, Ao was the absorbance of the control and At was the
absorbance of test/standard.
Hydrogen peroxide radical scavenging activity: The
hydrogen peroxide radical scavenging activity of the extract
was determined as the percentage inhibition of hydrogen peroxide
radical by the extract. The UV absorbance was measured at 230
nm [17]. All determinations were performed in triplicate. Hydrogen
peroxide radical scavenging activity was calculated using the
following formula:
Inhibition of H2O2 free radical scavenging activity (%) =
ot
o
(A A ) 100
A
×(2)
where, Ao was the absorbance of control and At was the absor-
bance of test/ standard. Antioxidant activity of extract was
expressed as IC50 value.
Total phenolic content: In this experiment, gallic acid is
used as a standard phenolic compound. From the standard
curve of gallic acid, phenolic content was measured and
expressed in gallic acid equivalents (GAE) [18]:
T = C × VM (3)
where, T= total phenolic contents (mg g-1) plant extract in gallic
acid equivalent (GAE), C = concentration (mg g-1) of gallic
acid obtained from calibration curve, V = volume of extract
(mL), M = weight (mg) of methanolic plant extract.
Total flavonoid content: In this experiment, quercetin is
used as a standard flavanoid compound. From the standard
curve of quercetin, flavonid content was measured and expressed
in quercetin equivalent by using the standard quercetin graph
[18] and using the formula
T = C × VM (4)
where, T = total flavonoid content (mg g-1) plant extract in
quercetin equivalent (QE), C = concentration (mg g-1) of quercetin
obtained from calibration curve, V = volume of extract (mL),
M = weight (mg) of methanolic plant extract.
Antimicrobial activity: The leaves and fruits extracts were
evaluated against 3 Gram-positive and 2 Gram-negative micro-
organism by the determination of the zone of inhibition. The
zone of inhibition was determined by disk diffusion method
[13,19] using Muller-Hinton agar, procured from Hi-media
Laboratories, Mumbai, India.
Determination of zone of inhibition: The antibacterial
sensitivity tests were performed by Kirby-Bauer disc diffusion
method.
Preparation of seed organisms: Nutrient broth medium
was used to seed the microorganisms. Medium (5 mL) was filled
in the test tubes were capped with cotton plugs and sterilization
was done by autoclaving. Inoculation was done with a loop of
microorganisms into the liquid broth and incubated at 37 ± 1
ºC and used within 12-18 h.
Preparation of sensitivity plate: The Mueller-Hinton
Agar medium was used to prepare the plates. The media was
autoclaved at 15 psig pressure (121 ºC) for 15 min and it was
poured into glass, flat-bottomed sterilized Petri dishes uniformly.
Preparation of discs containing extracts: The paper discs
of 6 mm diameter were sterilized by autoclaving at 25 psi g
pressure (121 ºC) for 10 min. The extracts were dissolved into
DMSO and in this way that each disc contains 250, 500 and
1000 µg of methanolic extracts of leaf and fruit of Elaeagnus
latifolia. For reference, market supplied standard disk of
ofloxacin (Hi-media), 5 µg per disk was used.
Procedure: Various microorganisms of 0.2 mL were asepti-
cally put over the solid agar medium, which was previously
2434 Dutta et al. Asian J. Chem.
prepared. After some time, previously prepared extracts discs
(individually contain 250-1000 µg), solvent controlled discs
and standard ofloxacin discs were placed aseptically on sensitivity
plates. The plates were then put for 24 h incubation period at
37 ± 1 ºC. The clear zone of inhibition on agar plate was measured
and calculated.
RESULTS AND DISCUSSION
Methanolic extract of the plants showed strong antioxidant
activity by inhibiting DPPH and hydrogen peroxide when
compared with standard ascorbic acid. Antioxidant activity of
extract was expressed as IC50 value. The IC50 values were calcu-
lated by linear regression of plots, where the abscissa represents
the concentration of the tested plant extracts and the ordinate
represents the average percent of scavenging capacity.
The methanolic extract of fruits of Elaeagnus latifolia
(MEFEL) exhibits the strongest DPPH scavenging activity with
an IC50 value of 144.64 ± 0.25 µg mL-1 which is likely to be
comparable with standard ascorbic acid with an IC50 value of
29.39 ± 7.11 µg mL-1 (Tables 1 and 2). In the assay against H2O2,
methanolic extract of leaves of Elaeagnus latifolia (MELEL)
found to be the strongest in hydrogen peroxide scavenging
assay with an IC50 value of 444.59 ± 3.77 µg mL-1 as compared
to standard ascorbic acid with an IC50 value of 279.89 ± 1.81
µg mL-1, respectively (Tables 3 and 4).
TABLE-2
DPPH FREE RADICAL SCAVENGING
ACTIVITY OF STANDARD (ASCORBIC ACID)
Concentration
(µg/mL)
Percentage inhibition
(Mean ± S.E.M) IC50 (µg mL–1)
Ascorbic acid 29.39 ± 7.11
20 59.11 ± 0.18
40 89.16 ± 0.27
60 91.41 ± 2.71
80 92.83 ± 2.18
100 96.17 ± 0.005
TABLE-3
HYDROGEN PEROXIDE SCAVENGING
ACTIVITY OF MELEL AND MEFEL
Percentage inhibition
(Mean ± S.E.M)
IC50
(µg mL–1)
Conc.
(µg/mL) MELEL MEFEL MELEL MEFEL
100 5.83±1.1 2.642±1.3 444.59±3.77 487.31±6.17
200 20.59±1.0 13.28±1.5
300 31.36±0.63 26.63±1.7
400 46.21±0.61 40.32±1.6
500 56.37±0.60 51.05±0.5
TABLE-4
HYDROGEN PEROXIDE SCAVENGING
ACTIVITY OF STANDARD (ASCORBIC ACID)
Concentration
(µg/mL)
Percentage inhibition
(Mean ± S.E.M) IC50 (µg mL–1)
Ascorbic acid 279.89 ± 1.81
100 23.33 ± 0.2
200 39.26 ± 0.28
300 53.49 ± 0.56
400 66.16 ± 0.49
500 82.33 ± 0.47
A significant amount of phenolic and flavonoids compounds
plays a major role in controlling oxidative damages in human
cells. Leaves were a good source of phenols with 61.15 µg mL-1
measured in gallic acid equivalent (GAE) (Table-5). Flavonoid
content was found significantly in the methanolic extract of
leaves which was calculated to be 15.12 µg mL-1 of quercetin
equivalents (QE)/mg of extract (Table-6).
TABLE-5
DETERMINATION OF TOTAL PHENOLIC CONTENT
In gallic acid equivalent (GAE)
(Mean ± S.D.)
Test
MELEL (mg g–1) MEFEL (µg mL–1)
Total phenolic content 61.15 ± 1.23 8.18 ± 0.182
TABLE-6
DETERMINATION OF TOTAL FLAVONOID CONTENT
In quercetin equivalent (GAE)
(Mean ± S.D)*
Test
MELEL (µg mL–1) MEFEL (µg mL–1)
Total flavonoid content 15.12 ± 0.125 6.375 ± 0.128
The results of zone of inhibition of MELEL and MEFEL
along with standard ofloxacin are presented in Table-5. The
result showed that depending upon concentration the metha-
nolic extract showed varied range of antimicrobial activity
against the tested organism which is comparable to standard
drug ofloxacin (Table-7).
Conclusion
Natural antioxidants and antimicrobial agent of plants
origin have greater utilitization as nutraceuticals and phyto-
pharmaceuticals as they have significant impact on human
health and prevention of diseases. The present study scienti-
fically validates the use of these plant extracts in traditional
health practice. The extract showed significant antioxidant and
antimicrobial activity. The result of antioxidant activity is comp-
arable to standard ascorbic acid. Also a noticable amount of
phenolic and flavonoid content is found in the extracts. In the
TABLE-1
DPPH FREE RADICAL SCAVENGING ACTIVITY OF MELEL AND MEFEL
Percentage inhibition (Mean ± S.E.M) IC50 (µg mL–1)
Concentration (µg/mL) MELEL MEFEL MELEL MEFEL
100 36.69 ± 0.18 39.04 ± 0.05 200.39 ± 5.44 144.64 ± 0.25
150 41.78 ± 0.27 50.696 ± 0.273
200 50.6 ± 2.71 60.46 ± 0.22
250 56.35 ± 2.18 74.2 ± 0.97
300 64.36 ± 0.005 84.03 ± 0.40
Vol. 30, No. 11 (2018) in vitro Evaluation of Leaves and Fruits of Elaeagnus latifolia L. 2435
present study, screening of antimicrobial effects of Elaeagnus
latifolia L. was done using selected pathogenic microbes. The
result focussed on the significant antimicrobial activity against
the tested organism which is comparable to standard ofloxacin
effect. The antioxidant and antibacterial activity of methanol
extract may be due to various types of phytochemicals consti-
tuents present in the extract. Thus, it is suggested that this
plant provides health benefits to humans and may be employed
in food processing or in medicine industry.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interests
regarding the publication of this article.
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TABLE-7
RESULT OF THE ANTIMICROBIAL ACTIVITY
Zone of inhibition (mm) (Mean ± SD)
Gram-positive organisms Gram-negative organisms
Extracts and
standard drug B. subtilis S. aureus S. faecalis E. coli P. mirabilis
MELEL (250 µg) 10 ± 1.00 12 ± 1.52 11 ± 1.60 10 ± 2.08
MELEL (500 µg) 12 ± 1.00 11 ± 2.10 14 ± 1.52 13 ± 1.51
MELEL (1000 µg) 15 ± 1.52 17 ± 2.51 19 ± 1.52 18 ± 1.52
MEFEL (250 µg) 10 ± 1.00 12 ± 1.52 13 ± 1.52
MEFEL (500 µg) 13 ± 1.10 15 ± 2.51 16 ± 2.51
MELFL (1000 µg) 18 ± 1.00 17 ± 1.52 17 ± 2.10
Ofloxacin (standard) 28 ± 1.57 35 ± 2.00 27 ± 1.52 29 ± 2.51 25 ± 1.0
DMSO
2436 Dutta et al. Asian J. Chem.
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Elaeagnaceae is a small dicotyledonous plant family that is represented by about 70 to 80 species in three genera. Genus Elaeagnus is one of them and has cosmopolitan distribution covering Asia, North America, Europe, and Australia. The multipurpose folkloric utilizations, pharmacological effects, and chemical compositions of Elaeagnus species make them worth investigating and, Elaeagnus latifolia L. and Elaeagnus umbellata Thunb. are among the most investigated., The objective of this book chapter is to present the reader with a comprehensive summary of the literature on these species.
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Elaeagnus latifolia is an underutilized plant of the Elaeagnaceae family. The extraction of E. latifolia pulp was carried out from 30 to 120 min by different environmentally friendly extraction methods: agitation, percolation, Soxhlet, and ultrasound-assisted extraction with ethanol, and hydrodistillation. The extracts were analyzed for their total phenolic compounds, carotenoids and ascorbic acid contents, and antioxidant activity. The highest yield of extract, of 63.5 g/100 g of raw material (RM), in dry basis (db), total phenolic compounds, of 3.3 mg gallic acid equivalent (GAE)/g extract, and 2.042 mg GAE/g RM, and carotenoids, of 176.6 mg β-carotene equivalent/kg RM, were obtained by Soxhlet for 120 min. All the extracts presented similar antioxidant activity. Therefore, E. latifolia can be a source of compounds of interest to the food industry.
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