Hindawi
Journal of Tropical Medicine
Volume 2021, Article ID 7239291, 8 pages
https://doi.org/10.1155/2021/7239291
Research Article
Evaluation of Leea rubra Leaf Extract for Oxidative Damage
Protection and Antitumor and Antimicrobial Potential
Nibedita Das,1 Sanowar Hossain,2 Jaytirmoy Barmon,3 Shahnaj Parvin,4 Mahadi Hasan,4
Masuma Akter,4 and Ekramul Islam 4
1
Rajshahi Medical College, Rajshahi 6100, Bangladesh
Department of Pharmacy, Pabna University of Science and Technology, Pabna 6600, Bangladesh
3
Department of Pharmacy, Varendra University, Kazla, Rajshahi, Bangladesh
4
Department of Pharmacy, University of Rajshahi, Rajshahi 6205, Bangladesh
2
Correspondence should be addressed to Ekramul Islam; ekram@ru.ac.bd
Received 24 June 2021; Revised 6 September 2021; Accepted 20 September 2021; Published 6 October 2021
Academic Editor: Jianbing Mu
Copyright © 2021 Nibedita Das et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background. The leaves of Leea rubra contain an abundance of phenolic constituents and have medicinal uses as antipyretic and
diaphoretic agents and are also used in the treatment of stomach ache, rheumatism, arthritis etc. In spite of the traditional uses,
data on the scientific evaluation of the plant are not sufficient. So, the present study was designed to evaluate the protective role of
the extract against oxidative damage to DNA and human erythrocytes as well as antitumor and antibacterial activities against
some resistant bacteria. Methods. The protective activity of the ethyl acetate fraction (EAF) of the extract was investigated by
evaluating the inhibition of oxidative damage of pUC19 plasmid DNA as well as hemolysis and lipid peroxidation damage to
human erythrocytes induced by 2,2′-azobis-2-amidinopropane (AAPH). Antitumor activity was assessed by evaluating the
percentage inhibition of cell growth, morphological changes of Ehrlich’s ascites carcinoma (EAC) cells, and hematological
parameters. Antimicrobial activity was determined by the disc diffusion method against different resistant microorganisms.
Results. EAF effectively inhibited AAPH-induced oxidative damage to DNA because it can inhibit the transformation of the
supercoiled form of plasmid DNA to open circular and further linear form. The oxidative hemolysis caused by AAPH in human
erythrocytes was inhibited by EAF extract in a time-dependent manner, and the production of malondialdehyde (MDA) was
significantly reduced, which indicates the prevention of lipid peroxidation. In antitumor assay, 76% growth of inhibition of EAC
was observed compared with the control mice (p < 0.05) at a dose of 100 mg/kg body weight. Antimicrobial activity was evaluated
against two pathogenic resistant microorganisms (Escherichia coli and Pseudomonas aeruginosa), and the highest antimicrobial
activity was observed against Pseudomonas spp. Conclusion. EAF may have great importance in preventing oxidative damage to
DNA, erythrocytes, and other cellular components as well as can be a good candidate in cancer chemotherapy and treating
infectious diseases caused by antibiotic-resistant bacteria.
1. Introduction
Aromatic and medicinal plants are sources of diverse nutrient and non-nutrient molecules, many of which display
antioxidant, anticancer, and antimicrobial properties that
may protect the body against both cellular oxidation reactions and pathogens. The assembly of oxidants may be a
typical event related to aerobic metabolism. When oxygen is
supplied in excess or its reduction is insufficient, reactive
oxygen species (ROS) or free radicals such as superoxide
anions, hydroxyl radicals, and peroxide are generated [1].
Accumulation of the free radicals in body organs or tissues
can cause oxidative damage to biomolecules such as DNA
and erythrocytes cell, eventually resulting in many chronic
diseases such as inflammatory, cancer, diabetes, aging,
cardiac dysfunction, and other degenerative diseases [2].
Erythrocytes are highly sensitive to oxidative damage
when exposed to reactive oxygen species (ROS) and hence
are used as a cellular model to study about biomembrane
integrity in relation to oxidative damage [3]. ROS induces
2
DNA damage, as the response to free radicals with DNA
involves strand break, base modification, and cross-linking
of DNA-proteins. On the other hand, DNA damage caused
by oxidative stress to the cell has been reported to be the
main factor to develop degenerative, inflammatory diseases
and accelerate cellular aging or eventually can cause cancer
[4, 5].
In recent times, consumption of plant-derived phytonutrients present in berry crops, herbs, beans, oil seeds, teas,
fruits, and vegetables has markedly increased [6, 7]. Since
medicinal plants are being explored for different therapeutic
agents for thousands of years and still are a major source for
new drugs, searching potential compounds from the plants
or identifying the phytochemical rich food supplements
from the nature is a worthy approach to combat oxidative
stress and diseases caused by such stress [8].
Antimicrobial resistance is becoming a potential threat
for human beings because the number of pathogens that are
resistant to commercial antibiotics is increasing day by day.
On the other hand, there are few hopes and scientists are
struggling for the discovery of new antibiotics. Because
different classes of compounds/extracts of plants have antimicrobial activities and because 60% of the antimicrobial
drugs discovered in the past few decades are of natural
origin, scientists are attracted to do more research on plants
for antimicrobial agents [9].
L. rubra comes from the family of Vitaceae and is also
known as red tree shrub; it is widely found in Australia, the
Malaysia and Thailand rainforest, Bangladesh, China, and
India’s tropical and subtropical forests at low and medium
altitude [10]. Previous investigation on this plant has shown
several therapeutic activities including antimicrobial and
anti-inflammatory activities [11], but the available literature
did not show any antitumor or protective effect against
oxidative damage on the biomolecules by the plant extract.
Hence, the rationale of the present study was to assess the
ethyl acetate fraction (EAF) of the methanolic extract of
L. rubra leaves for its antitumor activity and to verify the
ability to inhibit the oxidative damage to human erythrocytes and DNA and antibacterial activity against resistant
microorganisms.
2. Materials and Methods
2.1.
Chemicals
and
Reagents.
2′-Azobis(2-methylpropionamidine)dihydrochloride (AAPH),
agarose, ethidium bromide, bovine serum albumin, gallic acid,
catechin, homovanillic acid, epicatechin, chlorogenic acid, rutin
hydrate, and quercetin-3-rhamnoside were purchased from
Sigma-Aldrich, USA. pUC19 plasmid DNA was purchased
from Genetix, Bangalore, India. The other chemicals utilized in
the tests were of analytical grade and purchased from the SigmaAldrich and Roche.
2.2. Preparation of Plant Material, Extraction, and Fractionation.
L. rubra leaves were collected from Bandarban, Bangladesh, in the month of September, 2016. The plant was
authenticated by a taxonomist of the Botany Department,
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University of Rajshahi, and was preserved in the Phytochemistry Lab, Department of Pharmacy, University of
Rajshahi, Bangladesh. After washing thoroughly with
distilled water, the leaves were dried, powdered, and
extracted by shaking with methanol and fractionated
sequentially with n-hexane and ethyl acetate. Since the nhexane fraction contains chlorophyll, it was not considered for further experiment. Ethyl acetate extract
(EAF) was filtered through filter paper and dried under
vacuum on a rotary evaporator into a thick residue. Then,
it was stored in a cool and dry place for further studies.
2.3. Collection of Human Erythrocytes. Human erythrocytes
were collected from five healthy volunteers of 18 to 40 years
of age who had not taken any drug for 7 days prior to the
sample collection. Blood was collected at the Medical Center
of University of Rajshahi by an expert physician, preserved
with anticoagulant, and centrifuged, and then erythrocytes
were collected by discarding the supernatant. Collected
erythrocytes were diluted in PBS.
2.4. Assay of the Protective Role against Oxidative Damage.
Reactive oxygen species (ROS) or any oxidizing agent can
damage the cellular components and cause chronic diseases.
So, the capability of the extract (EAF) to neutralize or
scavenge such ROS or oxidizing agents to protect DNA and
erythrocytes as well as prevent lipid peroxidation was
evaluated by the methods described below.
2.4.1. Inhibition of AAPH-Induced Hemolysis of Human
Erythrocytes. The protective activity of EAF against AAPHinduced hemolysis of human erythrocytes was determined
by the method described by Yang et al. [12], with some
modifications. In this test, 50 μl of 5% erythrocyte suspension was mixed well with different concentrations of EAF
and standard ascorbic acid and incubated for 30 min at 37°C.
Then, 2, 2′-azobis-2-amidinopropane (AAPH) at a concentration of 50 mM was added to the above mixture. The
reaction mixture was kept at 37°C for 6 h with frequent
stirring for time-dependent hemolysis. The negative control,
consisting of erythrocytes in a hypotonic buffer (100% hemolysis), was kept under the same conditions as samples.
The samples were centrifuged at 1500 rpm for 10 minutes at
every hour. Then, an aliquot of each supernatant was taken
and diluted in PBS for taking absorbance at 540 nm. The
extent of hemolysis (%) was calculated as the ratio of the
absorbance at 523 nm of the sample to that of the complete
hemolysis sample.
2.4.2. Inhibition of Oxidative Damage on Plasmid DNA.
Transformation of the supercoiled form of plasmid DNA to
open circular and further linear form has been used as an
index of DNA damage. The DNA damage protective activity
of the EAF was assessed using pUC19 plasmid DNA [13].
1 μg of pUC19 plasmid DNA was mixed with two different
concentrations of EAF (20 and 30 μg/ml) and 2 μl of 25 mM
AAPH in PBS (pH 7.4). Then, the mixture was incubated for
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30 min at 37°C and electrophoresed on 2% agarose gel
containing 0.5 μg/ml ethidium bromide. The band intensities were analyzed under trans-illuminating UV light, and
the photo was taken using a gel documentation system.
2.4.3. Inhibition of Lipid Peroxidation. Oxidation of the
unsaturated lipid by ROS or any oxidizing agent can generate malondialdehyde (MDA). So, the capability of the
extract to prevent such oxidation was evaluated by thiobarbituric acid reactive substances (TBARS) assay to calculate the level of MDA according to the method described
by Padmaja et al. [14]. A human erythrocyte suspension,
preincubated with samples at different concentrations, was
used. The standard solution consisted of 500 μl of 20 mM
MDA in 1 ml of TBA. The absorbance was taken at 532 nm
using a spectrophotometer, and the MDA level was determined using the Beer–Lambert law at a molar extinction
coefficient of 156 mM−1·cm−1 and was expressed as pmol/g
Hb.
2.5. Antitumor Activity Assay
2.5.1. Experimental Tumor Model. The Department of
Biochemistry, University of Rajshahi, provided transplantable tumors (Ehrlich’s ascites carcinoma cells) used in this
research. They were maintained in our laboratory by intraperitoneal transplantation into Swiss albino mice.
2.5.2. Determination of Median Lethal Dose (LD50).
Various doses of EAF solution (25, 50, 100, 200, and 400 mg/kg)
were given intraperitoneally in mice to determine the median
lethal dose (LD50). The mortality was recorded after 24 h of
experimental period, and for this antitumor study, 100 mg/kg
dose was selected by the fixed dose method [15].
2.5.3. EAC Cell Growth Inhibition Assay. To assess the cell
growth inhibition properties of EAF, Swiss albino mice were
divided into four groups (n � 5) and Ehrlich’s ascites carcinoma (EAC) cells (1 × 106 cells/mouse) were inoculated
into all mice except those in the normal group intraperitoneally on day 0. Treatments were started after 24 h of
tumor inoculation and continued for 7 days. Group I served
as normal and Group II as control, mice in both groups
received normal saline; Group III mice received EAF at the
dose of 100 mg/kg; and Group IV mice were given bleomycin (standard drug) at the dose of 3 mg/kg. The mice were
sacrificed on the seventh day, and the EAC cells were collected by repeatedly washing with 0.9% saline. Then, the cells
from treated and untreated mice were compared for morphological studies [16].
2.5.4. Morphological Appearance of EAC Cells. The EAC
cells were stained with DAPI (4, 6-diamidino-2-phenylindole) to detect the morphological changes. Visual images
were taken using a fluorescent microscope. Both fluorescent
and optical views were observed.
3
2.5.5. Evaluation of Hematological Parameters. To evaluate
hematological parameters, mice of all groups (four groups;
n � 5) were injected with EAC cells (0.1 ml of 1.6 × 106 cells/
mice) intraperitoneally except the normal group at day 0. After
24 hours of inoculation, normal saline (5 ml/kg/mouse/day) was
administered intraperitoneally to normal (Group I) and EAC
control (Group II) mice, respectively, for 10 days. EAF at
100 mg/kg/mouse/day and belomycin at 3 mg/kg/mouse/day
doses were administered in Group III and Group IV, respectively. On the 12th day, blood was collected from the tail vein
and hematological parameters (hemoglobin, RBC, and WBC)
were measured for each mice of each group [17].
2.6. Evaluation of Antimicrobial Activity
2.6.1. Collection of Drug-Resistant Bacteria. Two drug-resistant bacteria were collected from the Microbiology Department of Rajshahi Medical College and Hospital. These
bacteria were resistant against different types of common
antibiotics. According to the report, Escherichia coli was
resistant to azithromycin, cefuroxime, cefalexin, and cotrimoxazole. Pseudomonas is sensitive to azithromycin and
cephalexin and resistant to cefuroxime, cotrimoxazole,
cefepine, vancomycin, and penicillin. Here, EAF was tested
alone and then given in combination with some antibiotics
(azithromycin, kanamycin, penicillin, amoxicillin, and
cefuroxime) to know its activity against these resistant
bacteria.
2.6.2. Determination of Antimicrobial Activity. The agar disc
diffusion method described by Bonev et al. (2008) and
Razmavar et al. (2014) [18, 19] was used for the determination of the antibacterial activity of the EAF, for which
bacteria were cultured in the nutrient agar medium and each
microorganism (106 cells/ml) was inoculated on the surface
of Mueller–Hinton agar plates. Paper discs (6 mm in diameter) saturated with the extract (50 μl) and standard disc
of kanamycin (30 μg/disc) were placed on the surface of each
inoculated plate. The plates were kept at 4°C for 5 hours and
then at 37°C for 24 hours, after which it was possible to
observe the zone of inhibition. Antibacterial activity was
evaluated by measuring the diameter (mm) of the inhibition
zone around the discs. Cultured bacteria with halos equal to
or greater than 7 mm were considered susceptible to the
tested sample.
2.7. Ethical Consideration. Swiss albino male mice of 3–4
weeks of age, weighing 23–27 g, were collected from the
Animal Research Branch of Jahangirnagar University,
Bangladesh. All animal studies were approved by the Ethical
Committee of the Institute of Biological Sciences, University
of Rajshahi, in accordance with the Guide for the Care and
Use of Laboratory Animals. The protocol for using human
blood cells was approved by the Institutional Animal,
Medical Ethics, Biosafety, and Biosecurity Committee
(IAMEBBC) for Experimentations on Animal, Human,
4
Microbes, and Living Natural Sources at the University of
Rajshahi (reference number: 31/320-IAMEBBC/IBSc).
2.8. Statistical Analysis. The data were analyzed by one-way
ANOVA (analysis of variance) followed by multiple comparisons using Dunnett’s post hoc test using SPSS software
of version 16. All results were represented as mean± standard deviation (SD). Differences at p < 0.05 level were
considered to be statistically significant.
3. Results
3.1. Protective Role against Oxidative Damage. EAF showed
significant protective activity against oxidative damage induced by AAPH to human erythrocytes and DNA as well as
oxidative lipid peroxidation. The result is shown below.
3.1.1. AAPH-Induced Hemolysis Assay in Human
Erythrocytes. Figure 1 shows the protective effect of EAF
against AAPH-induced hemolysis on human erythrocytes.
When AAPH (50 mM) was added to the aqueous suspension
of erythrocytes, time-dependent hemolysis was observed.
But this hemolytic process was inhibited when the EAF (at
concentrations of 50, 100, and 200 μg/ml) was added in a
concentration- and time-dependent manner.
3.1.2. DNA Damage Protection. This assay is based on the
ability of the samples to protect the DNA (PUC19 plasmid)
against the damage caused by peroxy radicals generated by
AAPH. The ability of EAF to protect oxidative damage of
DNA was evaluated by analyzing the band pattern of pUC19
DNA on agarose gel as shown in Figure 2. Lane 1 shows
DNA in the native supercoiled form; whereas in Lane 2,
treated with AAPH, the supercoiled form has been converted into open circular DNA. Addition of EAF in Lane 3
and Lane 4 at concentrations of 30 and 20 μg/ml, respectively, prevented the formation of circular form of plasmid
DNA as like as the standard gallic acid (in Lane 5).
3.1.3. Lipid Peroxidation Inhibition. The protective activity of
EAF was also evaluated by the inhibition of MDA production
from the erythrocyte membrane, induced by AAPH, and the
result is shown in Figure 3. MDA levels for the control-group
erythrocytes were 2.06 ± 0.27, 2.27 ± 0.29, and 2.87 ± 0.15 nmol/
ml, respectively, at 2, 4, and 6 h, increasing to 8.71 ± 0.34,
14.9 ± 025, and 19.02 ± 0.57 nmol/ml, respectively, after incubation with 50 mM AAPH. The addition of AAPH caused timedependent lipid peroxidation of erythrocytes. EAF inhibited
AAPH-induced MDA formation that was 4.99 ± 1.32,
7.73 ± 0.96, and 11.89 ± 1.16 nmol/ml at 2, 4, and 6 h,
respectively.
3.2. Evaluation of Antitumor Activity
3.2.1. Studies on EAC Cell Growth Inhibition. In vivo antitumor activity of EAF against EAC cell-bearing mice was
assessed by viable EAC cells (% inhibition in cell growth).
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Effects of the extract on EAC cells’ growth after tumor
inoculation are shown in Table 1. Treatment with EAF
resulted in a significant reduction of cell growth in vivo. The
percentage of cell growth inhibition was 76.09 ± 3.21% at
dose of 100 mg/kg, whereas it was 85.00 ± 5.2% with the
standard anticancer drug belomycin.
3.2.2. Morphological Changes of EAC Cells. Changes in
morphological characteristics of the EAC cells were evaluated by DAPI staining, for which the cells were collected
after 7 days from both treated (with EAF 100 mg/kg/day)
and nontreated EAC-bearing mice. In the control group
(solvent treated), the nuclei of the EAC cells were round,
regular, and homogeneously stained with DAPI as shown in
Figure 4. At the same time, apoptotic morphologic alterations like membrane and nuclear condensation were also
noted in EAF-treated EAC cells.
3.2.3. Studies on Hematological Parameters. Hematological
parameters of untreated EAC cell-bearing mice showed
significant (P < 0.05) changes when compared with normal
mice (Table 2). The total WBC count was found to increase
with a reduction in the hemoglobin content and total RBC
count. At the same time interval, treatment with EAF could
bring back these altered parameters to almost normal values.
The overall results of this study clearly demonstrated the
antitumor activity of EAF against EAC.
3.3. Antimicrobial Activity. Evaluation of the antibacterial
activity of EAF was tested against two drug-resistant bacteria
E. coli and P. aeruginosa by the disc diffusion method. The
results of this assay are shown in Table 3, where EAF at all
three concentrations showed dose-dependent activity
against both organisms and the maximum zone of inhibition
was 10 mm for E. coli and 11 mm for P. aeruginosa compared
with 6 mm and 12 mm, respectively, for the standard
kanamycin.
Combination of EAF with the standard kanamycin also
showed an increased activity compared with both EAF and
standard alone, where the maximum zone of inhibition for
combined therapy was 10 mm for E. coli and 16 mm for
P. aeruginosa as shown in Table 3.
4. Discussion
The main focus of the study was to investigate the EAF of the
extract of L. rubra leaves for its inhibition capacity against
ROS to prevent the damage caused by such free radicals to
cellular components such as DNA, cell membrane, and
erythrocytes [20], as well as antitumor and antibacterial
properties. Previous phytochemical studies with leaves of
this plant showed an abundance of phenolic constituents
such as flavonoids, leucoanthocyanidins, p-hydroxybenzoic
acid, syringic acid, and gallic acid [21]. So, the presence of
such naturally occurring compounds indicates the potential
of the plant to act against oxidative stress-related damage to
DNA and erythrocytes as well as the capacity to prevent
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5
120
% Hemolysis
100
80
60
40
20
0
1
2
3
4
Time in hour
Control
AAPH
Ascorbic Acid 200 µg/ml
5
6
EAF 200 µg/ml
EAF 100 µg/ml
EAF 50 µg/ml
Figure 1: Time course effect of EAF and ascorbic acid on AAPH-induced hemolysis on erythrocytes. Values are expressed as mean ± SD of
three experiments.
L1
L2
L3
L4
L5
Figure 2: Protective effect of EAF on pUC19 plasmid DNA damage
induced by AAPH. Lane 1: native DNA; Lane 2: DNA + AAPH;
Lane 3: DNA + AAPH + 30 μg EAF; Lane 4: DNA + AAPH + 20 μg
EAF; Lane 5: DNA + AAPH + 30 μg gallic acid.
tumor cell growth. The antimicrobial activity of the plant
extracts is mainly exhibited by secondary metabolites such as
alkaloids, tannins, terpenoids, alkaloids, and flavonoids
[22, 23]. The presence of such compounds in L. rubra in
preliminary screening also made the plant extracts a suitable
candidate for antimicrobial screening.
Protection against oxidative damage to DNA and
erythrocytes was evaluated by AAPH-induced hemolysis
protection in human erythrocytes, inhibition of MDA
production from membrane lipid peroxidation, and
prevention of oxidation-induced cleavage in DNA
strands. The findings of these experiments showed some
promising results where EAF was found to prevent hemolysis in a dose-dependent manner. The maximum level
of MDA after treating with AAPH at 6 h was
19.02 ± 0.57 nmol/ml, which was significantly reduced to
11.89 ± 1.16 nmol/ml after treating with EAF. The gel
electrophoresis image also showed brighter bands of
DNA with increasing concentrations of EAF. So, the
above instances indicate the significant protective role of
the extract and therefore can be used in preventing
chronic diseases due to alteration of cellular components
such as cancer, aging, and neurodegenerative diseases [3].
The findings were similar to the study performed by
Reddy et al. in another species of this plant family, Leea
indica [23].
In the next step, we investigated its role in cell growth
inhibition on EAC-bearing Swiss albino mice. The EAC
cells are experimental tumor models used worldwide in
cancer research [10]. EAC cells collected from EAFtreated mice showed some morphological changes such as
breakage of inner cell membrane, cells shrinkage, chromosomal condensation, and nuclear fragmentation when
observed under a fluorescence microscope, which are also
the important and reliable criteria for judging the potency
of any drug as anticancer agents. These morphological
changes are the hallmark of the apoptosis of EAC cells,
which suppress the tumor development. In the absence of
apoptosis process, abnormal cell proliferation may occur
that can lead to cancer development [24]. On the other
hand, EAC cells were normal in control mice. So, in our
experiment, EAF extract could inhibit the cell growth
along with morphological features of apoptosis. The effectiveness of the extract against EAC cell-bearing mice
has further been verified by monitoring the changes in
hematological and biological parameters. In our study,
reduction in RBC or % hemoglobin in tumor-bearing
mice may occur, which is mainly due to iron deficiency or
hemolytic or myelopathic conditions [25]. The extract
could significantly recover the hemoglobin content; RBC
and WBC cell count indicate the protective action on the
hemopoietic system. All these are measured and are very
important aspects in justifying the effectiveness of EAF in
cancer chemotherapy.
6
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MDA level (pmol/gHb)
25.00
20.00
15.00
10.00
5.00
0.00
2
4
Time in hour
Control
AAPH
Ascrbic Acid 200 µg/ml
6
EAF 50 µg/ml
EAF 100 µg/ml
EAF 200 µg/ml
Figure 3: Level of MDA after treating with EAF and standard ascorbic acid. Values are expressed as mean ± SD of three experiments.
Table 1: Effects of EAF on viable Ehrlich’s ascites carcinoma (EAC) cell growth.
Treatment group
Control
EAF
Bleomycin
Dose (mg/kg/day)
—
100
3
No. of viable EAC cells (×107 cells/ml)
61.14 ± 2.23
14.17 ± 1.97∗
13.78 ± 1.58
% of cell growth inhibition
—
76.09 ± 3.21∗
85.00 ± 5.2
Data are expressed as mean ± SD (n � 5). Analysis of variance followed by LSD and Dunnett’t post hoc test (IBM-SPSS/20); ∗ P < 0.05: significant difference
with respect to EAC control.
(a)
(c)
(b)
(d)
Figure 4: (a-b) Fluorescence and (c-d) optical microscopic observations of EAC cells for control mice and treated mice. (a, b) Fluorescence
microscopic view of control and EAF-treated mice cells, (c, d) the optical microscopic view of control and EAF-treated cells, respectively.
White (a) and red arrows (c) indicate normal cells. Fragmented cells (apoptotic characteristics) were indicated by red and white arrows in (b)
and (d), respectively.
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7
Table 2: Effects of EAF and standard bleomycin on blood parameters of tumor-bearing and normal mice.
Treatment
Normal
EAC + vehicle
EAC + EAF (100 mg/kg)
Belomycin (3 mg/kg)
% of Hb (g/dl)
13.26 ± 0.12
8.41 ± 0.19∗
10.47 ± 0.19t
11.94 ± 1.05
RBC (×109 cells/ml)
5.98 ± 0.27
2.21 ± 0.168∗
3.67 ± 0.156t
4.01 ± 0.11t
WBC (×106 cells/ml)
10.05 ± 0.42
120.7 ± 1.81∗
42.38 ± 1.92t
35.12 ± 1.56t
Data are expressed as mean ± SD for five animals in each group. Analysis of variance followed by LSD and Dunnett’t post hoc test (IBM-SPSS/20); ∗ P < 0.05:
against normal group, and # P < 0.05: against EAC control group.
Table 3: Diameter of zone of inhibition (mm) of EAF against drug-resistant bacteria.
Zone of inhibition (mm)
Bacteria
Kanamycin-30 (standard) EAF (50 μg/disc) EAF (100 μg/disc) EAF (200 μg/disc) EAF (100 μg/disc) + kanamycin-30
E. coli
6
5
8
10
10
P. aeruginosa
12
6
8
11
16
Antibiotic resistance has been a great issue all around the
world from developing to developed world, and it is predicted
that if such trend continues, treatment for infectious diseases
will be more challenging [26, 27]. Several studies have demonstrated the antimicrobial activity of the species of the genus
Leea, where Harun et al. [28] reported significant activity of
L. indica extract to be effective against two organisms, S. aureus
and S. epidermidis; Islam et al. [29] showed a considerable
activity of L. macrophylla against Gram-negative bacteria and
fungi; and Khan et al. [30] reported such activity of L. tetramera
against fungi.
A similar result was found in our study, where the activity
was assessed against two resistant organisms. An increase in the
diameter of the zone of inhibition with an increase in dose
indicates considerable activity of the plant extract against resistant bacteria. The effect was better when used in combination
with the existent antibiotic (kanamycin) and provided a possible
hope to combat antimicrobial resistance.
5. Conclusion
The potential of the EAF of L. rubra leaf extract to prevent
oxidative damage of the cellular components has been found
to be significant. At the same time, its effect on the inhibition
of tumor cell growth was notable, and hence it can be a
potential candidate for further investigation for anticancer
drugs. Activity against resistant organisms was considerable,
but better activity was reported while used in combination
with standard antibiotics.
Data Availability
The data used to support the findings of this study are
available from the corresponding author upon request.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
Acknowledgments
The authors are thankful to the Institute of Biological Science
(IBSc), University of Rajshahi, for its support to conduct the
study. This study was partially supported by the Scientific
Research Projects of Rajshahi University (Project Number:
5/52/RU/Sci.-31/2017-2018) to carry out the experiment.
However, the university did not contribute to manuscript
preparation, editing, approval, and publication processes.
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