Cancer Plus

ORIGINAL RESEARCH ARTICLE

Assessment of antibacterial, anti-inflammatory,

and anti-cancer activities of Melia azedarach L.
leaf extract
Mahendra Chikkamadaiah1 , Dwaraknath Venkatesha2 , Abhirami Dilkalal1 ,
Ravindra Kembalu Narayana3 , Venkatesh Hosur Narayanappa4 ,
and Satish Anandan5*
1
Department of Studies in Botany, Bengaluru City University, Central College Campus, Bangalore,
Karnataka, India
2
Department of Studies in Environmental Science, Tumkur University, Tumkur, Karnataka, India
3
Department of Studies in Botany, Karnataka State Open University, Mysuru, Karnataka, India
4
Research and Development, Miklens Bio-Private Limited, Bengaluru, Karnataka, India
Department of Clinical Nutrition and Dietetics, Sri Devaraj Urs Academy of Higher Education and
5

Research, Tamaka, Kolar, India

Abstract
The purpose of the present study was to evaluate the antibacterial, anti-inflammatory,
and cytotoxic properties of Melia azedarach leaf. The phytoconstituents found in the
leaves were extracted with four different solvent systems based on their polarity index.
*Corresponding author: Antimicrobial activity of selected plant extracts was evaluated using disc diffusion
Sathish Anandan
(satisha@sduaher.ac.in) and micro-broth dilution methods against four different pathogenic bacterial strains.
The ethanolic extract of M. azedarach leaf showed a significant inhibitory effect on
Citation: Chikkamadaiah M,
Venkatesha D, Dilkalal A, the growth of Escherichia coli (15.07 mm) and Staphylococcus aureus (18.23 mm)
Narayana RK, Narayanappa VH, (P < 0.005), followed by Bacillus subtilis and Salmonella typhi. Further, the mechanism
Anandan S. Assessment of of action was confirmed by live/dead cells analysis, which revealed the death of
antibacterial, anti-inflammatory,
and anti-cancer activities of Melia E. coli and S. aureus upon treatment with ethanolic extract. In vivo anti-inflammatory
azedarach L. leaf extract. Cancer test conducted using carrageenan-induced rat paw edema model revealed that
Plus. 2024;6(1):2763. the 300 mg/kg ethanolic extract exhibited a significant anti-inflammatory activity
doi: 10.36922/cp.2763
of 51.78% compared with that of standard drug diclofenac (P < 0.001). Further, the
Received: January 17, 2024 cytotoxicity of ethanolic extract against human hepatocarcinoma cell lines (HepG2)
Accepted: April 30, 2024 was evaluated by MTT assay, and the findings showed a moderate level of toxicity
against the HepG2 cell line with an IC50 value of 540.00 ± 0.6 µg/mL compared
Published Online: May 21, 2024
to doxorubicin. HepG2 cells treated with doxorubicin (2 µg/mL) and ethanolic
Copyright: © 2024 Author(s). extract showed a 2.33- and 1.35-fold increase in p53 gene expression, respectively.
This is an Open-Access article
distributed under the terms of the
Apoptosis activity was measured in terms of DNA laddering, which indicated the late
Creative Commons Attribution stage of apoptosis. The results showed that the extract-treated cell lines induced
License, permitting distribution, DNA fragmentation, which was found to be a potent anti-cancer mechanism in
and reproduction in any medium,
provided the original work is
HepG2 cells. This study corroborated that the leaf extract of M. azedarach is a good
properly cited. source of active phytochemicals, with promising biological activities.
Publisher’s Note: AccScience
Publishing remains neutral with Keywords: Anti-inflammatory; Apoptosis; Carrageenan; HepG2; MTT assay; p53 gene;
regard to jurisdictional claims in
published maps and institutional RT-PCR
affiliations.

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Cancer Plus Bioefficacy of Melia azedarach leaf extract

1. Introduction rat paw edema method, bactericidal properties, and

anticancer activity of the M. azedarach leaf extracts
Herbal drugs are widely regarded as a safer option for
was carried out. Cytotoxicity of the extract was further
consumption as compared to synthetic drugs. Most herbs
confirmed by determining apoptosis level through the
with medicinal properties were recognized and applied
measurement of p53 expression in human hepatocarcinoma
based on traditional knowledge, but validated scientific
(HepG2) cell lines treated with ethanolic leaf extract of
evidence pertaining to their efficacy is lacking. At present,
M. azedarach.
we are not equipped with a proper monitoring system and
adequate knowledge to determine the safe dosage level
2. Materials and methods
of these natural products. These inadequacies serve as
the prime reason why the natural products have yet to be 2.1. Collection of plant materials and extract
integrated in the health-care system.1-3 preparation
The beneficial properties of plants are accounted for Fresh and healthy leaves of M. azedarach were collected
by the phytochemicals they possess. Antibacterial, anti- from a local community in Mysore, Karnataka (11° 30’
inflammatory, and anticancer activities are among the and 18° 30’ N latitude and 74° 15’ and 78° 30’ E longitude)
important biological and medicinal qualities ascribed to based on its ethnobotanical importance. The plant
these phytochemicals. Cancer remains the second leading specimen was authenticated by a taxonomist, and the
cause of death worldwide, with about 18 million new cases herbarium specimen (VsNo-MCbot02) was submitted to
and 9 million cancer-related deaths reported every year as the Department of Studies in Botany, University of Mysore,
per the World Health Organization (WHO).4 Inflammation Mysuru. Leaf materials were thoroughly washed with
is a complex cascade caused by many external factors and running tap water, shade-dried at room temperature, and
eventually changes the functional and phenotypic traits of made into a fine powder, and 100 g of powdered sample
the cells, thereby precipitating the development of cancer. were extracted in 250 mL of petrolium ether, chloroform,
According to WHO, 80% of people living in developing ethyl acetate, and ethanol using soxhlet extractor for 48 h.
nations, including India, depend on herbal medicine to The extracts obtained were subjected to flash evaporation
meet their health-care needs.5 India is one of the countries and stored at 4°C in an airtight glass bottle until further
blessed with numerous medicinal plant resources, and the studies.10
therapeutic potency of such plants has been demonstrated
to check their bioefficacy by several researchers. 2.2. Phytochemical screening
Melia azedarach (Meliaceae) is a deciduous tree species Phytochemical analysis of different leaf extracts of
capable of growing up to 50 feet in height. Touted with M. azedarach was carried out to determine the presence or
medicinal values, M. azedarach has a wide geographical absence of secondary metabolites by following Harborne’s
distribution, commonly found in India, China, Indonesia, standard method (1998).11 The extracts were subjected to
and throughout Southeast Asia. Its leaves are alternate, qualitative preliminary phytochemical screening to detect
bi-pinnately compound; inflorescence is panicles; and the presence of phytochemicals such as carbohydrates,
fruits are berries. M. azedarach has the potential to grow proteins, steroids, flavonoids, tannins, alkaloids, and
and form a dense-thick canopy, restricting the growth of cardiac glycosides.
native vegetation.6 Fruits and leaf litter of this plant have
been proved to potentially increase mineralizable nitrogen 2.3. Antibacterial activity
and increase soil pH in acidic soils.7 Extracts of the plant The evaluation of antibacterial activity of all four solvent
have been used for various medicinal purposes, including extracts was conducted using disc diffusion method (CLSI,
the treatment of viral infections like herpes. Dilute foliar 2012a).12 After being cultured for 24 h, Staphylococcus aureus
extracts combined with triclopy-based herbicides have (MTCC441), Bacillus subtilis (MTCC220), Salmonella
been utilized to control pests such as insects and other typhi (MTCC325), and Escherichia coli (MTCC452) were
worms.8 Of the several compounds isolated from this plant, uniformly swabbed on Petri plates containing nutrient agar
meliacine appears to be the predominant active constituent medium using a sterile glass spreader (1.5 × 108 CFU/mL).
of M. azedarach. Meliacine – a peptide isolated from the Each disc (6 mm) was loaded with 50 µL (5 mg/disc) of
plant’s leaves – exhibits potent activity against herpes extract and placed equidistantly. Streptomycin (10 µg/disc)
simplex type-1 and has also been used as an abortifacient, and the respective solvents served as positive and negative
an antiseptic, a diuretic, an insect repellent.9 controls. The plates were incubated for 24 h at 37°C. The
In the present study, screening of phytochemical, in vivo zone of inhibition was measured using HI Media Zone
anti-inflammatory activity using the carrageenan-induced measuring scale, and the assay was performed in triplicates.

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2.4. Minimum inhibitory concentration (MIC) approved by the animal ethics committee of the University
of Mysore, Manasagangotri, Mysore (animal sanction
The MIC of the leaf extracts was tested using the micro-
order UOM/IAEC/05/2017).
broth dilution method13 coupled with an enzyme-linked
immunosorbent assay (ELISA) multi-plate reader. 2.6.2. Acute toxicity study
About 100 μL of nutrient broth and 10 µL of inoculum
suspension of each test bacterium (1.5 × 108 CFU/mL) The ethanol extract was subjected to determine the toxicity
were added to each well. The extracts were prepared at level of the animals. An acute toxicity study was performed
50 mg/mL concentration, which served as a stock solution. as per the OECD-423 guidelines.15 The animals were
The solvent extract (100 μL) was added to the first well of randomly selected for the toxicity study. The rats were
each row, and two-fold serial dilution was carried out along divided into five groups with six animals each and were
the rows to create a series of extracts with different extract fasted overnight but allowed to drink water. After that,
concentrations ranging from 5 to 0.002 mg/mL. The plates the extract was administered orally at a dose of 5 mg/kg.
then were incubated at 37°C for 24 h. After incubation, Instead of administering extract, a similar quantity of 1%
each well was supplemented with 10 μL of 2,3,5-triphenyl carboxyl methylcellulose solution was administered to
tetrazolium chloride dye at 2 mg/mL and incubated for 1 h, the control group. The rats were kept for observation for
and absorbance was measured at 620 nm using an ELISA 6 days. If no mortality was noted, the process was repeated
multi-plate reader. The concentration at which color had using higher doses such as 50, 300, and 2000 mg/kg.
stopped changing was considered the MIC value. The assay 2.6.3. Carrageenan-induced rat paw edema
was performed in triplicates.
Paw edema was induced in the right hind paw of each rat,
2.5. Bacterial cell morphology assay and the drugs were administered orally as suggested by Singh
The assay was performed to distinguish the morphology of et al.16 In group A, the rats served as controls treated with
the live and dead bacterial cells after treatment with extract 100 μL of 0.9% drug suspension in saline. The rats in group B
at MIC value using the method described by Li et al.14 About were administered with standard anti-inflammatory drug
diclofenac sodium (Novartis India Ltd.) at 40 mg/kg (p.o.),
100 µL of bacterial cell suspension (1.5 × 108 CFU/mL)
and the rats in groups C, D, and E were orally administered
was treated with 50 µL of selected leaf extracts (100 µg)
with 100, 200, and 300 mg/kg of ethanolic leaf extract of
and incubated at 37 ± 2°C for 24 h. The untreated cells
M. azedarach, respectively. After an hour of administration
and streptomycin-treated cells served as controls. After
of drugs, 0.1 ml of 1% w/v carrageenan (Sigma Aldrich
incubation, the mixture was centrifuged at 5000 rpm
Chemical Co.) solution in normal saline was injected into
for 5 min at 4°C, and the pellet was washed thrice with
the subplantar tissue of the right hind paw of each rat. The
phosphate-buffered saline. Each obtained sample was mixed
paw volume was measured using a plethysmometer, before
with a fluorescent dye solution of ethidium bromide (EB)
and after injection of carrageenan at different time intervals
and acridine orange (AO) at a 1:1 ratio and incubated for
of 1 h, 2 h, and 3 h. The percentage of edema induced was
30 min. Later, 5 μL of these samples were mounted on
assessed in the drug- and extract-treated rats and was
microslide for observing the dead and live cells under a
compared with the control and the inhibitory effects were
Carl-Zeiss Fluorescence Microscope (Lawrence and Mayo,
studied. The comparative strength of drugs was determined
Germany) at 40× magnification. The absorbance was taken
by assessing the percentage of inflammation inhibition
for the green fluorescence dye at 480/500 nm and the red
they achieved, and the percentage inhibition was calculated
fluorescence dye was read at 490/635 nm. The former stains
using the formula in the following:
the bacteria with both intact as well as damaged membranes,
and the latter stains only the cells with damaged membranes.
=
Percent of inhibition (%)
( C − T ) ×100 (I)
2.6. Anti-inflammatory activity C

2.6.1. Experimental animals Where T represents the difference in increased paw volume
after the administration of test drugs, and C represents the
Healthy adult Wistar rats (120–150 g), regardless of their
difference in increased volume in the control group.
gender, were housed in polypropylene cages, maintained
at room temperature of 22 ± 2°C under the conditions of 2.7. In vitro cytotoxicity assay
12-hour light-dark cycles and 40–60% humidity. The rats
were given standard rodent diet and water ad libitum. The 2.7.1. Cell culture
experiment was conducted based on the ethical guidance HepG2 cell line was procured from National Centre for
for the care and use of laboratory animals in research as Cell Sciences, Pune, India, and maintained in Dulbecco’s

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Cancer Plus Bioefficacy of Melia azedarach leaf extract

Modified Eagle Medium (DMEM) supplemented with 2.7.3. DNA fragmentation studies
10% inactivated fetal bovine serum (FBS), penicillin DNA fragmentation study was carried out using the
(100 µg/mL), streptomycin (100 µg/mL), and amphotericin method proposed by Vandghanooni et al.18 cHepG2 cells
B (5 µg/mL) under humidified air of 5% CO2 at 37°C until (3 × 106/mL) were seeded onto 60 mm Petri dishes and
confluent. cultured for 24 h at 37°C in an atmosphere of 5% CO2.
2.7.2. Cytotoxicity assay Afterward, the cells were washed with media and treated
with 2 µg/mL doxorubicin (standard drug) and ethanolic
HepG2 cells maintained in DMEM were treated with
extract at IC50 value; the cells were incubated for 24 h at
ethanol extract of different concentrations (62.5, 125, 250,
37°C and 5% CO2. At the end of the incubation period, the
500, and 1000 µg/mL) to evaluate the cytotoxic effects of
G Bioscience apoptotic DNA ladder kit (Thermo Scientific,
the ethanolic extract on the HepG2. The MTT assay was
USA) was used to isolate the cancer cell’s chromosomal
carried out to analyze the cytotoxicity of the HepG2 cells
DNA as per the instructions of the kit. For three hours,
after 24 h of incubation as described by Mosmann.17 A the DNA samples were run at 5 V/cm on a 2% agarose
microplate reader with 96 wells was used to detect the gel electrophoresis. The gel was imaged and visualized
optical density at 570 nm, and the percentage of toxicity using the UVP gel documentation system (Thermo Fisher
was calculated using the formula in the following: Scientific, USA).
Percent of growth inhibition (%)
2.8. Statistical analysis
Mean OD of individual test group
=100 − × 100 (II) The data of triplicate values are expressed as mean ±
Mean OD of control group
 standard deviation. The statistical differences among the
The mRNA levels of p53 were determined using a test groups were analyzed using a one-way analysis of
semi-quantitative reverse transcriptase-polymerase chain variance coupled with a post hoc Duncan’s multiple range
reaction (RT-PCR). HepG2 cells were cultured on 60 mm test. The statistical analyses were performed using SPSS
Petri dish and cultivated in DMEM supplemented with Version 18.0 (SPSS Inc., Chicago, IL, USA). Differences
with P < 0.05 are regarded as statistically significant. The
FBS and amphotericin for 24 h. The dish was filled with
IC50 value was determined using a logarithmic equation,
the ethanolic extract of desired concentration (at IC50
i.e., Y = M(x) + C. Here, Y = 50, and M and C values were
value) and incubated for 24 h. Total cellular RNA was
derived from the viability graph.
extracted from untreated (control) and treated cells using
TRI Reagent (Sigma Aldrich, Germany) in adherence 3. Results
with the manufacturer’s protocol. cDNA was synthesized
from the extracted RNA using a reverse transcriptase kit 3.1. Preliminary phytochemical analysis
(Thermo Scientific, USA) according to the manufacturer’s The preliminary phytochemical screening of leaf extracts
instructions. The reaction mixture (20 µL) was then of M. azedarach showed the presence of different classes
subjected to PCR for amplification of p53 cDNAs using of phytochemicals. Among the different solvent extracts
precisely designed primers obtained from Eurofins India, tested, ethanolic extract showed a maximum number
and the housekeeping gene GAPDH was co-amplified with of phytoconstituents followed by ethyl acetate extract,
each reaction as an internal control reference. The optimum while the least number of phytochemicals were noted
thermal cycling conditions for PCR are as follows: 95°C for in petroleum ether and chloroform extract. The tested
5 min, followed by 30 cycles of denaturation at 95°C for extracts showed the presence of carbohydrates, proteins,
30 s, annealing at 65°C for 30 s, and extension at 72°C for steroids, flavonoids, tannins, alkaloids, and cardiac
1 min. This was followed by a final extension at 72°C for glycosides (Table 1).
10 min.
3.2. Antibacterial activity and MIC
During the first strand synthesis, oligo dT primers were
Four different solvent extracts of M. azedarach leaf were
used. For second strand synthesis, the product generated
subjected to antibacterial activity testing against E. coli,
has a size of 371 bp. The sequences of primers used in this
B. subtilis, S. typhi, and S. aureus. The overall results of the
experiment are as follows:
tested extracts and their antibacterial activity and MIC
• Forward: 5’-CTGAGGTTGGCTCTGACTG are depicted in Table 2. Our results showed that the four
TACCACCATCC-3’ solvent ethanolic extract effectively inhibited the growth of
• Reverse: 5’-CTCATTCAGCTCTCGGAACATC tested bacterial strains and the largest zone of inhibition in
TCGAAGCG-3’ E. coli (15.07 ± 1.54 mm) and S. aureus (18.23 ± 0.57 mm),

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as shown in Figure 1A and E. Similarly, ethyl acetate significant bactericidal activity because of the presence
extract also demonstrated inhibitory effect on pathogenic of a higher number of phytochemicals and this can be
growth, but no such activity was observed in chloroform attributed to several bioefficacy mechanisms.
and petroleum ether extracts. The MIC assay showed
maximum growth inhibition in ethanol and ethyl acetate 3.3. Bacterial cell morphological studies
extracts with a range of 0.625 mg/mL to 1.25 mg/mL, while The maximum growth inhibition observed in E. coli
streptomycin showed an MIC value of 0.0781 mg/mL. and S. aureus was used as a reference to study the
The present study indicates that ethanolic extract exhibits morphological changes that occurred during the process of
inhibition by live/dead cells backlight assay. The ethidium
Table 1. Preliminary phytochemical analysis of various bromide and acridine orange dyes were used to stain the
solvent leaf extracts of M. azedarach bacteria: ethidium bromide can only enter the damaged
bacterial cell through the ruptured cell membrane, while
Phytochemicals Tests Extracts
acridine orange adheres to both live and dead cells. The
PE CL EA ET
bacterial strains treated with ethanolic extracts along with
Carbohydrates Molisch’s test ‑ ‑ + +
control were observed under fluorescence microscopy.
Fehling’s test + + + + The staining results revealed the orange staining of
Barford’s test + ‑ ‑ ‑ extract- and streptomycin-treated cells, indicative of cell
Proteins Biuret test ‑ ‑ ‑ ‑ death (Figure 1B and 1F), and the reddish staining of
Million’s test ‑ + ‑ + the cells (Figure 1D and 1H), suggesting rupture of the
Ninhydrin test ‑ + + +
cell membrane. Meanwhile, untreated cells appeared in
green fluorescence, indicating normal growth without
Steroids Salkowski’s test ‑ + + +
any abnormalities or damage in the cell membrane
Liebermann–Burchard test ‑ ‑ ‑ + (Figure 1C and 1G). The fluorescence intensity of the dead
Test for triterpenoids + ‑ + + and live cells in all the treatments is depicted in Figure 1I.
Flavonoids Shinoda test ‑ + + + Taken together, this study indicates the antibacterial
Alkaline reagent test ‑ ‑ ‑ + efficacy of the ethanolic leaf extract of M. azedarach.
Zinc HCl test ‑ + ‑ ‑
3.4. Anti-inflammatory activity
Tannins Ferric chloride test + + ‑ +
In vivo anti-inflammatory activity of M. azedarach
Alkaloids Mayer’s test ‑ ‑ ‑ ‑
ethanolic leaf extract was tested by carrageenan-induced
Dragendorff ’s test ‑ ‑ ‑ ‑
paw edema method. Initially, the toxicity level of the
Hager’s test + ‑ + + ethanolic leaf extract was tested on the rats up to a dose
Cardiac Killer–Killian’s test ‑ ‑ ‑ ‑ of 2000 mg/kg of extract, and the extract did not cause
glycosides Raymond’s test ‑ ‑ ‑ ‑ any mortality even at higher dosage. Hence, the extract
Baljet test + ‑ + + was considered safe for administration. The ethanolic leaf
Note: ‘+’: Indicates presence; ‘‑’: Indicates absence.
extract at dosage of 100, 200, and 300 mg/kg exhibited
Abbreviations: PE: Petroleum ether; CL: Chloroform; EA: Ethyl acetate; significant anti-inflammatory activity in all the tested rats
ET: Ethylene; M. azedarach: Melia azedarach. (P < 0.001). There is a significant decrease in the edema

Table 2. Antimicrobial activity of various solvent leaf extracts of M. azedarach

Pathogens Zone of inhibition (mm) Minimum inhibition concentration (mg/L)

Extracts Extracts
PE CL EA ET Standard PE CL EA ET Standard
E. coli ‑ ‑ 8.10±0.49c 15.07±1.54b 23.41±0.18a ‑ ‑ 1.25 0.625 0.0781
S. aureus ‑ ‑ 10.44±0.15c 18.23±0.57b 22.32±1.09a ‑ ‑ 1.25 0.625 0.0781
B. subtilis ‑ ‑ ‑ 14.23±0.85 b
18.23±0.74 a
‑ ‑ ‑ 0.625 0.0781
S. typhi ‑ ‑ 8.23±0.44c 11.74±0.23b 19.53±0.66a ‑ ‑ 1.25 0.625 0.0781
Notes: Values are represented as means±SE for three independent replicates. Represented as significantly different value according to Dunken
a,b,c

Multiple test, P<0.05.

Abbreviations: PE: Petroleum ether; CL: Chloroform; EA: Ethyl acetate; ET: Ethanol; M. azedarach: Melia azedarach; E. coli: Escherichia coli; S. aureus:
Staphylococcus aureus; B. subtilis: Bacillus subtilis; S. typhi: Salmonella typhi.

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Cancer Plus Bioefficacy of Melia azedarach leaf extract

A B C D

E F G H

Figure 1. Antibacterial and live/dead cell backlight assay. (A and E) Zone of inhibition in Escherichia coli and Staphylococcus aureus. (B and F) Dead cells
after exposure to extract at 50 mg/L concentration. (C and G) Live cells (negative controls). (D and H) Dead cells after being treated with streptomycin at
10 mg/L concentration. (I) Fluorescence intensity and the percentage ratios of live/dead cell distribution of E.coli and Staph. aureus. The cells were viewed
under the magnification of ×40 using florescence microscope.

volume of rats administered with extract after 3 h, when 3.6. Analysis of apoptosis-related gene expression
compared to the control group (Table 3). The extract The upregulation of p53 gene expression on the HepG2 cells
at 300 mg/kg exhibited significant anti-inflammatory after being exposed to ethanolic extract of M. azedarach
activity of 51.78 % in carrageenan-induced rat paw edema at a concentration of 540 µg/mL for 24 h was detected by
(Figure 2). In addition, the results showed that the extract means of qRT-PCR (Figure 5A and B). Precisely, the cells
inhibited inflammation in treated rats in a dose-dependent treated with doxorubicin (2 µg/mL) and ethanolic extract
fashion. showed an upregulation of p53 gene expression by 2.33 and
1.35 fold, respectively, whose levels were much higher in
3.5. Cytotoxic effect of M. azedarach leaf extract on
comparison to the untreated control.
HepG2 cell line
The MTT test was used to assess the cytotoxic effects of 3.7. DNA fragmentation detection assay
ethanolic extract on the HepG2 cells. Half maximum Apoptosis was further evaluated by measuring the DNA
inhibitory concentration (IC50) was computed from laddering as a result of DNA fragmentation, which is
the dose-response curve of 62.5, 125, 250, 500, and indicative of the late stage of apoptosis. In this study,
1000 µg/mL concentration. The cytotoxicity of the HepG2 cells treated with doxorubicin (2 µg/mL) and
ethanolic extract is depicted in Figure 3, and it was found ethanolic extract exhibited DNA laddering, as shown in
that there was a significant difference (P < 0.05) between Figure 5C.
different concentrations of the tested sample. The IC50
value of ethanolic extract on the HepG2 cells after a 24-h 4. Discussion
exposure was determined as 540.00 ± 0.6 µg/mL. Further, In the current study, we successfully unveiled the presence
using phase contrast inverted microscope, the bacterial cell of various secondary metabolites in M. azedarach leaf
morphology after being treated with ethanolic extract and extracts by means of phytochemical screening. Ethanolic
control-treated cells was observed (Figure 4). extract boasts a huge amounts of phytochemicals such

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Table 3. Anti‑inflammatory activity of leaf ethanolic extract of M. azedarach at different hours in carrageenan‑induced paw
edema model

Groups Dose of extract, p.o. (mg/kg) Change in paw thickness (mm) mean±SD
1st h 2nd h 3rd h
Group A, (saline; negave control) ‑ 0.766±0.07 e
0.818±0.08 e
0.99±0.05a
Group B (diclofenac sodium, positive control) 40 0.461±0.05d 0.431±0.01d 0.372±0.02b
Group C 100 0.679±0.03 c
0.667±0.02 c
0.627±0.04e
Group D 200 0.645±0.02b 0.610±0.04b 0.584±0.01d
Group E 300 0.636±0.05 a
0.586±0.01 a
0.477±0.01c
Notes: Values are represented as means±SE for three independent replicates. a,b,c,d,e: Represented as significantly different according to Dunken Multiple
test P<0.05.
Alphabets with the same letter show that there is no significant difference. Alphabets with different letter show that there is significant difference.
Abbreviation: M. azedarach: Melia azedarach.

Figure 3. Cytotoxic properties of ethanolic leaf extracts of Melia

azedarach and percentage of cell viability against HepG2 cells at different
concentrations.

Figure 2. Anti-inflammatory effect of leaf ethanolic extracts of Melia Furthermore, we also found that ethanolic extract
azedarach and percent inhibition of the carrageenan-induced paw edema was highly toxic and deadly for E. coli and S. aureus, as
in different time intervals. confirmed using a fluorescent viability probe (Live/dead@
Backlight assay). Our results are in agreement with the
as carbohydrates, proteins, steroids, flavonoids, tannins, previous reports of De CC Pinto et al.,20 who found that
alkaloids, and cardiac glycosides, followed by ethyl Annona muricata extract could effectively damage the
acetate extract. These phytochemicals are known to play cell membrane of Gram-positive and Gram-negative
a crucial role in health-care domain. Through the disc bacteria. In general, bacterial cells that have lost the
diffusion assay, we found that the growth of the tested plasma membrane integrity and are unable to maintain an
bacterial strains such as E. coli, B. subtilis, S. typhi, and electrochemical potential are considered dead.21 Although
S. aureus was effectively suppressed by ethanolic leaf the bacterial potential of M. azedarach has been reported
extract of M. azedarach, as compared to its ethyl acetate previously, this study showed that tested extract showcases
extract, whose inhibitory effects on growth were exerted a broad spectrum of antibacterial activities against both
in a dose-dependent manner. No activity was observed Gram-negative and Gram-positive bacteria in a dosage-
in petroleum ether and chloroform extracts. Significant dependent manner.
bactericidal activity was observed in ethanolic leaf extract, Carrageenan-induced acute inflammation is one of
which contains a variety of phytochemicals compared the widely adopted in vivo tests for screening the anti-
to the remaining solvent extracts, as confirmed in the inflammatory activity of natural products. Induced
preliminary phytochemical screening. Similar results have inflammation can be observed in two phases: the early
been reported by Gaggia et al.,19 who reported that the stage that features intense inflammation and the later
aqueous leaf extract of chinaberry exhibited antimicrobial phase that results in a gradual increase in the swelling of
activity against certain phytopathogenic bacteria. the rat paw edema.22 The initial inflammatory response

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A B C

D E F

Figure 4. Morphological changes in HepG2 cells as observed under phase contrast inverted microscope at 24 h: (A) Untreated cells, (B) cells treated with
Melia azedarach ethanolic extracts at 62.25 µg/mL, (C) at 125 µg/mL, (D) at 250 µg/mL, (E) at 500 µg/mL, and (F) at 1000 µg/mL. The cells were viewed
under the magnification of ×10.

B C

Figure 5. Effect of Melia azedarach ethanolic extract at 500 µg/mL concentration on the mRNA levels of p53 in HepG2 cells. Total RNA was isolated from
treated and control samples, and the gene expression was analyzed using reverse transcription polymerase chain reaction (A and B). DNA laddering was
visualized in HepG2 cells upon treatment with ethanolic extract at 500 µg/mL concentration for 24 h (C).

begins within an hour of the carrageenan injection, in the first few hours upon administration. This indicates
influenced partly by the injection trauma and also by the no inhibition of histamine and serotonin. In the late hours,
presence of histamine and serotonin components. In the after 3 h, the inhibition of paw edema was increased up to
present study, there is no significant inhibition of paw 51.78 %. This increase can be caused by the responsiveness
edema: the ethanolic leaf extract of M. azedarach at 100, of carrageenan-induced paw edema to cyclooxygenase
200, and 300 mg/kg concentrations led to only 11.32%, inhibitors. Carrageenan-induced rat paw edema model
15.84%, and 17.10% of paw edema inhibition, respectively, can be employed to assess the impact of non-steroidal anti-

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Cancer Plus Bioefficacy of Melia azedarach leaf extract

inflammatory agents that chiefly target the cyclooxygenase the development of chemotherapeutic drugs based on
responsible for the excessive prostaglandin synthesis.23 In herbs.
the present study, the results align well with earlier reports
The HepG2 cells treated with ethanolic leaf extract
on anti-inflammatory effects of M. azedarach leaf extract.24
showed evidence of DNA fragmentation, a sign of
The ethanolic leaf extract also showed strong cytotoxic confirmed apoptosis. However, no fragmentation was
impacts on the HepG2 cells. Doxorubicin is frequently observed in the control cells. A possible sign that DNA
used to serve as the standard drug for comparison with replication is being inhibited because of inter-nucleosomal
potential anti-cancer drug candidates because of its cleavage caused by apoptosis is DNA molecule breakdown.
well-known pro-apoptotic effect. In the present study, Previous research has demonstrated that Avicennia marina
the viability of HepG2 cells noticeably decreased with extract is an apoptotic agent against MDA-MB 231 cells,
an increase in extract concentration. This particular resulting in DNA fragmentation.30
finding does not concur with a study by Zeng et al.,25 who
discovered that a compound isolated from M. azedarach 5. Conclusion
leaves effectively reduced the viability of the similar cell M. azedarach leaf extracts possess a broad spectrum of
line. This result discrepancy is due to potential variations phytochemicals with a promising bactericidal effect against
in phytochemical content influenced by extraction human pathogenic bacteria and with moderate anti-
procedures, leaf maturity, solvent choice, and dosage used inflammatory and anti-cancer properties against HepG2
or duration of exposure to cells. Another study conducted cancer cell lines. Further, the leaf extract also induced
by Zhang et al.26 proved that M. azedarach extracts are rich apoptosis in MCF-7 cells, probably mediated through p53
in bioactive chemicals, primarily limonoids, phenolics, expression. This study lends credence to the concept that
and oxygenated triterpenoids, which are considered ethanolic leaf extract can be employed in some forms of anti-
potential compounds with high therapeutic values against cancer therapy. Our findings indicate that M. azedarach leaves
malignant cells. are a potential reservoir of natural bioactive compounds,
Apoptosis is regarded as the pharmacodynamics providing an invaluable source of novel biomolecules with
endpoint of anticancer drug therapy as it prevents anti-inflammatory and anti-cancer properties.
cancer from developing resistance to chemotherapy.27
Furthermore, apoptosis prevents the inflammatory Acknowledgments
response caused by necrosis through self-dismantling of The authors would like to thank the University Grant
individual cell components. Thus, apoptosis of cancer cells Commission, New Delhi, and also the University of Mysore
will not cause harmful effects to the adjoining normal cells. for providing the necessary laboratory facility.
In this study, the qRT-PCR method was used to investigate
molecular pathways by inducing apoptosis with ethanolic Funding
leaf extract of M. azedarach. Following treatment with The study is supported by the University Grant Commission,
ethanolic leaf extract, p53 expression was increased. Major Research Project (No.: F.No.37- 460/2009[SR]).
Based on our results, we postulate that ethanolic leaf
extract upregulates the expression of p53, which causes Conflict of interest
p21 protein accumulation, thereby leading to cell cycle
The authors declare that they have no competing interests.
arrest through apoptosis induction in HepG2 cells. Our
results are consistent with several studies that showed how Author contributions
p53 and Bcl-2 function to induce apoptosis.28 The tumor
suppressor gene p53 also plays a vital role in apoptosis. Conceptualization: All authors
In fact, the majority of medications used to treat cancer Investigation: All authors
patients nowadays elicit antitumor effects by reducing Writing–original draft: All authors
tumor growth in a p53-dependent manner. Joray et al.29 Writing–review & editing: All authors
reported that p53 tumor suppressor is necessary for the
efficient execution of the apoptosis following treatment
Ethics approval and consent to participate
with a cytotoxic limonoid derived from M. azedarach. The experiment was conducted based on the ethical
In this study, the ethanolic leaf extract was cytotoxic to guidance for the care and use of laboratory animals in
HepG2 cells and also upregulated p53 expression in these research as approved by the animal ethics committee of the
cells. These findings are in support of M. azedarach extract University of Mysore, Manasagangotri, Mysore (animal
as a potential anticancer agent and provide insights into sanction order UOM/IAEC/05/2017).

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Cancer Plus Bioefficacy of Melia azedarach leaf extract

Consent for publication soxhlet ethanol extraction to produce and test plant material
(essential oils) for their antimicrobial properties. J Microbiol
Not appliable. Biol Educ. 2014;15(1):45-46.

Availability of data doi: 10.1128/jmbe.v15i1.656

The data of this study are available from the corresponding 11. Harborne AJ. Phytochemical Methods a Guide to Modern
Techniques of Plant Analysis. Berlin: Springer Science and
author upon reasonable request.
Business Media; 1998.
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