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189
Document heading doi:10.1016/S2221-1691(12)60040-9
襃
2012 by the Asian Pacific Journal of Tropical Biomedicine. All rights reserved.
Antibacterial activity of leaves extracts of
Trifolium alexandrinum
Linn.
against pathogenic bacteria causing tropical diseases
Abdul Viqar Khan
1
*
, Qamar Uddin Ahmed
2
, Indu Shukla
3
, Athar Ali Khan
1
1
Department of Botany, Faculty of Life Sciences, Aligarh Muslim University, Aligarh-202002, State of Uttar Pradesh, India
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, International Islamic University Malaysia, 25200-Kuantan, Pahang Darul
Makmur, Malaysia
3
Department of Microbiology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh-202002, State of Uttar Pradesh, India
Asian Pac J Trop Biomed
2012; 2(3): 189-194
Asian Pacific Journal of Tropical Biomedicine
journal homepage:www.elsevier.com/locate/apjtb
*Corresponding author: Dr. Abdul Viqar Khan, Department of Botany, Faculty of Life
Sciences, Aligarh Muslim University, Aligarh-202002, State of Utter Pradesh, India.
E-mail: viqarvicky@gmail.com
Foundation Project: This work was financially supported by SERC Fast Track OYS
Schem, Department of Science and Technology (DST), New Delhi, India [grant No.
304/2004 (SR/FT/L-129/2004)].
1. Introduction
Man has been using plants to cure different diseases
associated with pathogenic bacteria since antiquity.
According to a study conducted by the World Health
Organization ( W HO ) based on publications on
pharmacopoeias and medicinal plants in
91 countries, the
number of medicinal plants is nearly
21 000. Nearly 6-7
thousand species of medicinal plants out of about
17-18
thousand flowering plants
are known to be in use in folk
and officially recognized systems of medicine in
India, i.e.,
Ayurveda, Sidha, Unani and Homeopathy. Such a big figure
of medicinal plants is the highest percentage of flowering
plants in any particular country of the world for the existing
flora of that country.
The wide range of medicinal plants
existing in
India owing to a vast range of agroclimatic
variability is acc
ountable for increased demand of Indian
medicinal plants in the international market in recent
years.
WHO has projected that the global herbal market for
medicinal plants has been estimated bo be worth around
US
$
120 billion which is growing at 7
%
-10
%
every year and it
is likely to increase to more than
US $5 trillion by 2050
[1,2]
.
Plants grown in this region are not systematically tested
for their bi
ological activities in general and antimicrobial
activity in particular.
Exceptional ways to available
antibiotics for disease management have been increasingly
felt due to the increase in the resistance of bacterial
isolates.
This has urgently demanded the requirement
of second and third line drug and plants are considered
potent candidates to overcome such inevitable problems
associated with the
complications of antimicrobial resistant
bacteria
[3-12]
.
Plants from the genus Trifolium have been used in
traditional medicine by many cultures.
In Turkish folk
medicine, for example, some Trifolium species are used for
their expectorant, analgesic, antiseptic properties and are
also used to treat rheumatic aches.
Some species are also
grown as pasture crops for animals in the
Mediterranean.
ARTICLE INFO ABSTRACT
Article history:
Received 15 August 2011
Received in revised form 8 September 2011
Accepted 3 October 2011
Available online 28 March 2012
Keywords:
Trifolium alexandrinum L.
Fabaceae
Antibacterial activity
Pathogenic bacteria
Gram-positive bacteria
Gram-negative bacteria
Tropical disease
Infectious disease
Objective:
To investigate antibacterial potential of Trifolium alexandrinum (T. alexandrinum)
Linn. against seven gram positive and eleven gram negative hospital isolated human pathogenic
bacterial strains responsible for many tropical diseases.
Methods:
Non-polar and polar extracts
of the leaves of T. alexandrinum i.e., hexane, dichloromethane
(DCM), ethyl acetate (EtOAc),
methanol
(MeOH) and aqueous (AQ) extracts at five different concentrations (1, 2, 5, 10 and 15 mg/
m
L) were prepared to evaluate their antibacterial value. NCCL standards were strictly followed to
perform antimicrobial disc susceptibility test using disc diffusion method.
Results:
Polar extracts
demonstrated significant antibacterial activity against tested pathogens.
EtOAc and MeOH
extracts showed maximum antibacterial activity with higher inhibition zone and were found
effective against seve
nteen of the tested pathogens. While AQ plant extract inhibited the growth of
sixteen of the test strains.
EtOAc and MeOH plant extracts inhibited the growth of all seven gram
positive and ten of the gram negative bacterial strains.
Conclusions:
The present study strongly
confirms the effectiveness of crude leaves extracts against tested human pathogenic bacterial
strains causing several tropical diseases.
Since Egyptian clover is used as a fodder plant, it could
be helpful in controlling various infectious diseases associated with cattle as well.
Contents lists available at ScienceDirect
Abdul Viqar Khan et al./Asian Pac J Trop Biomed 2012; 2(3): 189-194
190
The plant, Trifolium alexandrinum (T. alexandrinum) Linn.
(Family: Fabaceae) (common name: Egyptian clover, berseem
clover
) is a fodder plant and is distributed all over Asia,
Europe and USA. Berseem clover is a winter annual legume
with oblong leaflets and hollow stems.
It grows upright and
produces yellowish-white flowers.
Berseem may grow as tall
as
18 to 30 inches and contains from 18 to 28 percent crude
protein, which is equ
al to or better than crimson clover and
alfalfa
[13]
.
Earlier phytochemical investigations of T. alexandrinum
have revealed the presence of terpenoind glycosides, amino
acids and their derivatives, protiens, flavonoids and their
glycosides, isoflavonoids and fatty acids in different parts
of the same plant
[13,14]
. Daily intake of (water, hexane and
ethanolic
) extracts of the flower head of T. alexandrinum
in drinking water for
4 weeks immediately after diabetes
induction in male rats has been reported to cause significant
decrease in glucose and glycated hemoglobin levels and
increase in insulin level.
It also greatly improved the levels
of serum lipid parameters and significantly decreased lipid
peroxidation in addition to increase the hepatic glutathione
(GSH) content significantly
[15]
.
Detection of bioactive compounds i.e., terpenoids, steroids,
flavonoids, amino acids and proteins in appreciable amount
in the leaves extracts of T. alexandrinum in our laboratory
and antimicrobial effect of phenolic extract of another
Trifolium species i.e., red clover
(Trifolium pratense) on the
ruminal hyper ammonia-producing bacterium, Clostridium
sticklandii
[16]
prompted us to evaluate the possible
antibacterial properties of T. alexandrinum in vitro.
Hence,
polar and non-polar extracts of the leaves were explored
ag
ainst eighteen human pathogenic bacterial strains,
responsible for many of the tropical diseases.
Bacteria
included in this study were isolated from the patients
admitted at the
Jawaharlal Nehru Medical College, AMU,
Aligarh, India.
2. Materials and methods
2.1. Plant material
Fresh leaves (10 kg) of T. alexandrinum were collected
during winter from different localities of
Aligarh district,
U.P., India. Voucher specimen numbers (AV24, AV206) of
the pl
ant were deposited in the Herbarium of Department of
Botany, Faculty of Life Sciences, Aligarh Muslim University,
Aligarh-202002, U.P., India.
2.2. Preparation of polar and non-polar extracts
Shade dried plant material (2.7 kg) was pulverized in
an electric grinder.
Powder so obtained was stored in a
dessicator.
500 g plant powder was macerated with 95
%
methyl alcohol
(MeOH) in a round bottom flask at room
temperature for about
24 h. Mother liquor (crude MeOH
extract
) was filtered out and residual plant material was again
macerated with
95
%
MeOH for another 24 h. The process was
repeated four times to ascertain the maximum yield of
MeOH
extract.
The extract was evaporated to dryness at 35
曟
under
reduced pressure using rotary evaporator and finally freeze
dried at -
50
曟
to obtain final yield (64.5 g) of crude methanol
extract which wa
s kept in the chiller at -18
曟
till further
use
[17]
.
Freeze dried methanol extract (64.5 g) was refluxed with
hexane over hot water bath for
5 h. After filtration, the
residual methanol extract was again refluxed with hexane
for another
5 h and filtered. This process was repeated three
times.
Hexane was evaporated under reduced pressure to
obtain hexane soluble extract.
Hexane insoluble fraction of
methanol extract obtained in above process was refluxed
with dichloromethane
(DCM) for 5 h. Thereafter, it was
filtered and refluxed again with
DCM for 5 h and filtered.
The process was repeated three times. DCM was evaporated
under reduced pressure to obtain
DCM soluble extract. DCM
insoluble fraction was refluxed with ethyl acetate
(EtOAc) for
5 h. Thereafter, it was filtered and refluxed again with EtOAc
for
5 h and filtered. The process was repeated three times.
EtOAc was evaporated under reduced pressure to obtain EtOAc
extract.
EtOAc insoluble fraction was refluxed with MeOH
(95
%
)
for 5 h and filtered. The process was repeatedly refluxed
for three times with methanol.
The methanol soluble fraction
was evaporated under reduced pressure to obtain
MeOH
extract, while methanol insoluble
residue was discarded
[12]
.
All resultant extracts were freeze dried at -50
曟
with yields of
hexane extract
(14.5 g), DCM extract (13.0 g), EtOAc extract
(8.5 g) and MeOH extract (25.0 g).
2.3. Preparation of aqueous extract
Shade dried plant material (500 g) was ground to a fine
powder then it was poured with double distilled water,
and left for
72 h at room temperature. The flask was then
refluxed over hot water bath for
5 h and the mother liquor
was filtered.
The distilled water was again added, refluxed
and filtered.
This process was repeated for four times. The
filtrate, thus obtained, was evaporated to complete dryness
under reduced pressure and finally freeze dried at -
50
曟
to give final yield
(37.0 g) of aqueous (AQ) extract which was
stored in labeled sterilized screw capped bottle at -
18
曟
[17]
.
2.4. Phytochemical analysis
2.4.1. Test for carbohydrates (Molish test)
Five mL aqueous solution of each extract was mixed
with few drops of
Molish reagent (alpha naphthol) and
concentrated
H
2
SO
4
was added from side wall of test tube.
Formation of purple coloured ring at junction indicated the
presence of carbohydrates
[17]
.
2.4.2. Test for lipids (Sudan IV test)
Hexane soluble portion of each extract was added in a
test tube and few drops of
Sudan IV were also added. Lipids
stained red when
Sudan IV was added and formed a separate
Abdul Viqar Khan et al./Asian Pac J Trop Biomed 2012; 2(3): 189-194
191
ring which indicated positive test
[17]
.
2.4.3. Tests for proteins and amino acids (ninhydrin test)
Aqueous solution of each extract was heated with
ninhydrin reagent.
Characteristic deep blue or pale yellow
colour due to formation of complex between two ninhydrin
molecule and nitrogen of free amino acid was an indication
of positive test
[17]
.
2.4.4. Tests for steroids and triterpenoids (Libermann-
Bruchard test)
Extracts were evaporated to dryness and extracted with
CHCl
3
and few drops of acetic anhydride followed by
concen
trated H
2
SO
4
from side wall of test tube to the CHCl
3
extract were added. Formation of violet to blue coloured ring
at the junction of two liquid was an indication of positive
test
[17]
.
2.4.5. Tests for flavonoids
Five mL of dilute ammonia solution was added to
a portion of the aqueous filtrate of each plant extract
followed by addition of concentrated
H
2
SO
4
. A yellow
colouration observed in each extract indicated the presen
ce
of flavonoids.
The yellow colouration disappeared on
standing
[17]
.
2.5. Microorganisms
Leaves extracts were tested for possible antibacterial
activity using disc diffusion assay against eighteen human
pathogenic bacteria.
The bacteria were obtained from the
bacterial stock,
Department of Microbiology, Jawaharlal
Nehru Medical College, Aligarh, India. The bacterial cultures
were maintained at
4
曟
on nutrient agar.
2.6. Antibacterial activity (bacterial susceptibility tests)
National Committee for Clinical Laboratory Standards
(NCCLS) were strictly followed to perform antimicrobial disc
susceptibility testing using disc diffusion method to test
plant extracts against test strains
[13]
. Standardized inoculums
(1-2
暳
10
7
CFU/mL 0.5 McFarland standard) were introduced
on the surface of the plates containing
Mueller Hinton agar
(MHA), which was spread evenly with a glass spreader. The
paper discs
(6 mm in diameter; Whatman No. 1 filter paper)
containing
1, 2, 5, 10 and 15 mg/mL plant extracts were dried
and placed aseptically on the agar surface with the help of a
sterile forceps.
Finally paper discs were pressed slightly with
forceps to make complete contact with the surface of the
medium.
Plates were allowed to stand at room temperature
for
30 min and then incubated aerobically at 37
曟
and
examined for the zone of inhibition after
24 h. The zone of
inhibition was measured in diameter.
The experiments were
repeated thrice.
Chloramphenicol (10
毺
g/disc) was used as a
standard drug.
3. Results
0.5 kg pulverized leaves of T. alexandrinum yielded 37.0 g
(7.4
%
)
freeze-dried AQ extract. 0.5 kg pulverized leaves of T.
alexandrinum yielded
64.5 g (12.9
%
)
freeze-dried methanol
extract.
Methanol extract upon successive extraction with
non-polar and polar organic solvents at room temperature
subsequently yielded hexane
(14.5 g, 2.9
%
)
, DCM (13.0 g,
2.6
%
)
, EtOAc (8.5 g, 1.7
%
)
and MeOH (25.0 g, 5.0
%
)
soluble
freeze-dried fractions.
Qualitative analysis of polar and non-polar extracts of
the leaves of T. alexandrinum
L. revealed the presence of
different class of phytochemicals in different proportion
(Table 1). Hexane extract displayed positive results for the
presence of lipids, steroids and terpenoids and negative
for carbohydrates, proteins, amino acids and flavonoids.
DCM extract showed positive results for the presence of
lipids, steroids, terpenoids and flavonoids only.
EtOAc
extract revealed negative tests for proteins only while
MeOH
and
AQ extracts showed absence of lipids, steroids and
terpenoids.
Phytochemical analysis of EtOAc, MeOH and
aqueous extracts explicitly revealed greater abundance for
carbohydrates, proteins, amino acids and flavonoids while
Table 1
Qualitative analysis of the phytochemicals of polar and non-polar extracts of the leaves of T. alexandrinum L.
Leaves extracts Carbohydrates Lipids Proteins Amino acids Steroids Terpenoids Flavonoids
Hexane -
++++
- -
+ ++
-
Dichloromethane -
++++
- -
++ ++ +
Ethyl acetate
+ +
-
+ ++ ++ ++++
Methanol
++
-
+ ++
- -
++++
Water
++++
-
++++ ++++
- -
+
-: negative;
+
: small amount;
++
: average;
++++
: high.
hexane and DCM extracts revealed appreciable amount for
the presence of lipids in the leaves of T. alexandrinum.
In the present study, the investigation of antibacterial
activity of non-polar and polar extracts of the leaves of
T. alexandrinum against seven gram-positive and eleven
gram-negative bacteria was studied using disc diffusion
method.
The data pertaining to the antimicrobial potential
of non-polar
(hexane & DCM) and polar (EtOAc, MeOH
&
AQ) extracts of the leaves of T. alexandrinum were
presented in
Table 2. The results revealed variability in
the inhibitory concentrations of
each extract against given
bacteria.
All extracts presented antimicrobial activity
to at least six of the tested microorganisms.
None of the
extracts of the leaves of T. alexandrinum was active against
Abdul Viqar Khan et al./Asian Pac J Trop Biomed 2012; 2(3): 189-194
192
Pseudomonas aeruginosa. Moderate biological activity
was demonstrated by non-polar extracts as haxane extract
was found to be effective against two of the gram positive
(Staphylococcus albus, Streptococcus haemolyticus) and
four gram negative
(Proteus mirabilis, Salmonella typhi,
Shigella boydii, Shigella dysenteriae
) pathogens only.
Dichloromethane extract inhibited the growth of four gram
positive
(Staphylococcus aureus, Staphylococcus aureus ATCC
25953, Streptococcus haemolyticus, Bacillus subtilis) and
only two of the gram negative
(Edwardsiella tarda, Shigella
boydii
) pathogens. While AQ extract showed no inhibitory
activity against Pseudomonas aeruginosa and Plesiomonas
shigelloides.
In contrast, polar extracts i.e., EtOAc and MeOH extracts
presented the highest activity, i.e., they were able to inhibit
17 (94.44
%
)
types of microorganisms of interest. Moreover,
they also had the highest activity rate against bacteria.
On
the other hand,
AQ extract showed inhibitory activity against
16 (88.88
%
)
types of microorganisms and non-polar extracts
(hexane and DCM) revealed antimicrobial activity against 6
(33.33
%
)
microorganisms of interest only. The antibacterial
activity was more prominent on the gram-positive bacteria
than the gram-negative bacteria.
Gram-positive bacteria
were the most susceptible to growth inhibition by
EtOAc
and
MeOH extracts. The results showed that all the extracts
had variable degree of antibacterial activity and that the
inhibition of bacterial growth was dose
dependent as
inhibitory action of the extracts was found to increase with
the increase of concentration against all bacterial strains
as evidenced by the higher zone of inhibitions at higher
concentration of each extract.
All extracts displayed similar antibacterial activity
compared with positive control
(chloramphenicol, 10
毺
g/
disc
) though at higher concentration. Non-polar (hexane
and
DCM) and AQ extracts up to 1 mg/mL did not exert
any antibacterial activity on all bacterial strains.
The
highest antibacterial activity was observed at
15 mg/
m
L by all extracts against most of the bacterial strains.
EtOAc extract exhibited the highest antibacterial activity
i.e., higher inhibition zones against most of the bacterial
strains in comparison with other extracts of the leaves of T.
alexandrinum
(Table 2).
Table 2
Antibacterial activity of non-polar and polar extracts of T. alexandrinum L. leaves.
Extracts (mg/mL/disc) Zone of inhibition (mm)
Gram positive bacteria Gram negative bacteria
1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 9 10 11
Hexane 1 - - - - - - - - - - - - - - - - -
2 - - 8 - - - - - - - - - - - - - - -
5 - - 11 - 7 - - - - - 8 - - 9 9 7 - -
10 - - 13 - 9 - - - - - 11 - - 13 10 9 - -
15 - - 15 - 14 - - - - - 14 - - 15 12 11 - -
Dichloromethane 1 - - - - - - - - - - - - - - - - - -
2 8 - - - - - - - - - - - - - - - - -
5 11 - - - 7 - 7 - 7 - - - - - 9 - - -
10 13 7 - - 9 - 9 - 9 - - - - - 12 - - -
15 15 10 - - 13 - 13 - 11 - - - - - 13 - - -
Ethyl acetate 1 7 7 8 8 7 8 7 - 8 7 8 8 - - - 9 9 -
2 10 9 11 13 9 11 8 7 9 9 10 10 - 9 9 11 10 8
5
17 15 17 18 13 16 11 11 12 12 11 13 - 14 13 14 16 10
10
20 19 20 20 18 20 17 16 18 18 14 14 - 17 17 17 18 15
15
24 22 22 24 22 24 23 20 23 22 16 16 - 21 20 23 20 18
Methanol 1 8 - 9 - - - - - - - 7 7 - - - - - -
2 9 7 9 9 7 9 8 - 9 7 9 10 - 7 - 8 - -
5 13 9 14 10 12 13 11 7 10 11 14 15 - 11 9 13 9 7
10
16 12 19 14 15 17 14 12 15 15 16 16 - 13 12 15 12 11
15
19 18 21 18 17 20 16 15 18 17 19 18 - 16 15 18 15 12
Aqueous 1 - - - - - - - - - - - - - - - - - -
2 9 - - 9 - - - - - - - - - 7 - 7 - -
5 11 8 9 13 7 8 9 7 8 9 9 9 - 11 - 11 - -
10 15 10 12 17 11 12 13 8 12 13 11 12 - 13 8 13 11 -
15 19 14 15 20 15 17 17 12 16 17 16 15 - 15 12 16 15 -
Chloramphenicol 10
毺
g/disc 18 18 16 - - - 16 18 16 - 16 18 - 16 17 19 18 20
Gram positive bacteria:
1: Staphylococcus aureus; 2: Staphylococcus aureus ATCC 25953; 3: Staphylococcus albus; 4: Streptococcus haemolyticus Group-A;
5: Streptococcus haemolyticus Group-B; 6: Streptococcus faecalis; 7: Bacillus subtilis.
Gram negative bacteria:
1: Escherichia coli; 2: Edwardsiella tarda; 3: Klebsiella pneumoniae; 4: Proteus mirabilis; 5: Proteus vulgaris; 6: Pseudomonas aeruginosa;
7: Salmonella typhi; 8: Shigella boydii; 9: Shigella dysenteriae; 10: Shigella flexneri; 11: Plesiomonas shigelloides.
-: no inhibition.
Abdul Viqar Khan et al./Asian Pac J Trop Biomed 2012; 2(3): 189-194
193
4. Discussion
The discovery of penicillin by Alexander Fleming in 1928 is
a milestone in the history of medicine.
As more antimicrobial
compounds were discovered, it was predicted that infectious
diseases would be eliminated through the use of these
antimicrobials.
Unfortunately, the development of bacterial
resistance to these antimicrobials quickly diminished this
optimism
[7]
. Secondly, there is also reported reduction
in the discovery of new antimicrobial agents globally.
Ethnomedicinal reports have brought to light that our rich
floristic heritage is one of the reliable sources which can
be traced pharmacologically for their possible antibacterial
potential.
It is known that plants bio-constituents have been
a good source of antibacterial agents.
Still many of the plant
species remained unexplored
or under explored
[19]
. Plants
are important sources of potentially useful substances for
the development of new chemotherapeutic agents.
Various
phytochemical compounds which are naturally present in
plants as secondary metabolites have been implicated in
the conferment of antibacterial activities
[20,21]
. The presence
of some of such secondary metabolites in a significant
amount in the investigated part o
f T. alexandrinum may
have conferred the strong antibacterial activity on the
leaves extracts.
In this regard, higher concentration of these
phytochemicals may have been responsible for a higher
degree of inhibition on the bacterial strains.
Previously, in many studies of similar kinds, ethanol and
methanol are used as extractants, however, it could not
demonstrate the greatest sensitivity in yielding antimi
crobial
compounds
[22]
. For this reason in the present study five
extractants based on their different degree of polarity and
solubility, i.e., hexane, dichloromethane, ethyl acetate,
methanol and water were used to obtain maximum active
compounds in the extracts.
It is worth mentioning to note
that a correlation was observed between the extract solubility
and antibacterial activity of different fraction
s. This suggests
that in sequential extraction, maximum antibacterial
compounds were solublized according to their degree of
solubility in polar solvents as
EtOAc and MeOH extracts
displayed the highest antibacterial activity followed by
AQ,
DCM and hexane extracts. These results further confirm that
significant antibacterial compounds are polar in nature as
evidenced by the higher degree of antib
acterial activity of
EtOAc and MeOH extracts of the leaves of T. alexandrinum
in vitro.
All the extracts highly affected the activity of gram
positive bacteria in comparison to gram-negative bacteria.
The greater susceptibility of gram-positive bacteria
towards various plant extracts than that of gram-negative
bacteria was also reported earlier
[23-25]
. The activity of T.
alexandrinum leaves extracts against both gram-positive
and gram
-negative bacteria might indicate the presence of
broad spectrum antimicrobial compounds.
Natural products,
either as pure compounds or as standardized plant extracts,
provide unlimited opportunities for new drug leads because
of the unmatched availability of chemical diversity
[26-47]
.
These results suggest that the bioactive compounds present
in polar extracts have good potential for development of
nov
el antibacterial herbal products.
Results obtained from our study explicitly confirm
antibacterial potential of the leaves of T. alexandrinum.
Polar extracts i.e., EtOAc and MeOH extracts demonstrated
the most pronounced antibacterial activities against bacterial
strains taken into consideration.
Hence, EtOAc and MeOH
extracts of the leaves of T. alexandrinum deserve further
investigations to develop new antibiotics that may
help in
combating several bacterial diseases in tropical countries.
Since this plant is used as a fodder plant, it could be helpful
in controlling various infectious diseases associated with
cattle as well.
This is the first ever report on the antibacterial
potential of the leaves of T. alexandrinum.
Conflict of interest statement
We declare that we have no conflict of interest.
Acknowledgements
Thanks are due to Department of Science and Technology
SERC Division, New Delhi for financial support to the author
Dr. Abdul Viqar Khan.
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