Evaluation of Antibacterial and Antioxidant potential
of Melia. Azedarach linn. and Psidium guajava linn. Leaf
extracts
Gade Kalyani1*,
Azka Fathima2, Guddeti Venkateswarlu3, D. Ravi Sankar
Reddy4,
Shaik Harun Rasheed5
1Associate Professor, Department of Pharmacology,
Bharat Institute of Technology,
Mangalpally, Ibrahimpatnam - 501510, Telangana, India.
2Assistant Professor, Department of Pharmaceutical Analysis,
Bharat Institute of Technology, Mangalpally,
Ibrahimpatnam - 501510, India.
3Assistant Professor, Department of Pharmacology,
School of Pharmacy,
Guru Nanak Campus, Ibrahimpatnam - 501506, Telangana,
India.
4Professor, Department of Pharmaceutical Chemistry, University College
of Pharmaceutical Sciences,
Acharya Nagarjuna
University, Nagarjuna Nagar, Guntur, Andhra Pradesh, India - 522510.
5Professor, Department of Pharmacy, School of Pharmacy,
Guru Nanak Campus,
Ibrahimpatnam - 501506, Telangana, India.
*Corresponding Author E-mail:
gadekalyani1989@gmail.com
ABSTRACT:
The present study is intended to evaluate and analyze in-vitro
antibacterial and antioxidative activities of leaf extracts of Melia
azedarach Linn (M.
azedarach L.) and Psidium guajava Linn (P. guajava L.). In-vitro antibacterial activity was measured using agar well
diffusion assay against Pseudomonas
aeruginosa and Staphylococcus
aureus strains.
In-vitro antioxidant activity was estimated using
2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging method. Among both plants high minimum inhibitory concentration values are observed with P. guajava L. ethanol extract at 47.6µg/ml for S. aureus and 43.7µg/ml
for P. aeruginosa, respectively. The results revealed that 20µg/ml
ethanol extracts of P. guajava L. exhibited the highest radical %
scavenging activity (76.24%) succeeded by methanol extracts (72.78%),
respectively indicating they are effective solvents to extract phenolic
compounds. We demonstrated
that for both bacterial strains, ethanol followed by methanol extracts showed
significant antibacterial activity in P. guajava L.in dose dependent
manner indicating the presence of high flavonoids, tannins, and steroids. Our results revealed that both plants are vital reservoirs of phytochemicals
with both antibacterial and antioxidant capacities.
KEYWORDS: Melia azedarach L., Psidium guajava L.,
Antioxidant activity, Antibacterial activity, In-vitro.
INTRODUCTION:
Inflammation is characterized by edema due to fluid
accumulation, heat, and which leads to hyperalgesia and involved in production
of fever during these infections. Current medicine, however, necessitates the
separation and purification of the active compounds.
Natural products have been and remain to be
exceptional sources of medicinal agents, which are templates for synthetic
agents with the potential for treatments of various diseases and to develop new
anti-inflammatory agents which could also be used to treat fever1. Algesia, pyrexia and Inflammatory
diseases being common with increase in prevalence, still have no proper
treatment till today. Medicinal
plants constitute one or more components for curing the life threating
infections caused by pathogenic microorganisms.
An imbalance between antioxidants and reactive oxygen
species results in oxidative stress induced cellular damage. It is linked to
most of the diseases such as cancer, diabetes, ageing, atherosclerosis,
ischemia injury, rheumatoid arthritis, inflammation, neurodegenerative, and
pulmonary disorders2. Epidemiological studies reinforce that
the prevalence of oxidative stress-associated conditions is mitigated by the
consumption of fruits and vegetables3.
Taking this into consideration our present study is aimed to assess
antibacterial and antioxidant activities of various solvent extracts (ethanol
(E.E), methanol (M.E), pet. ether (P.E.E), aqueous (A.E)) extracts obtained
from Melia. Azedarach Linn (M. azedarach L.) and Psidium
guajava Linn (P. guajava L.) leaves, as well as evaluating their total phenol
and flavonoid contents.
MATERIALS AND METHODS:
Collection and preparation of the plant extracts: Leaves of M. azedarach and P.
guajava were collected from various locations of Andhra Pradesh, India and authenticated by taxonomist P.
Satyanarayana Raju at the Department of Botany and Microbiology, ANU, India. The dried powdered leaves are extracted
continuously at 55oC with 500ml of 50% (v/v) of different solvents
petroleum ether (Pet.ether), ethanol, methanol, and water by soxhlet extraction4. The extracts obtained were filtered, evaporated
using rotary evaporator at various temperatures depending on the solvent. The
concentrated solutions obtained were freeze-dried, and then stored in sterile
glass desiccators until further analysis. Dimethyl sulfoxide (DMSO)is used to
prepare dose extracts that can be orally administered.
Chemicals
and reagents:
Pet. ether, Ethanol, Methanol, Chloroform, DMSO, Ampicillin were purchased from Anka chem, Telangana, India. 2, 2- Diphenyl-2-picryl hydrazyl (DPPH),
rutin, Gallic acid and Ascorbic acid, Nutrient agar, Luria
broth, Pseudomonas aeruginosa
(P. aeruginosa) and Staphylococcus aureus (S. aureus)
(MTCC1144, and 6388) are obtained from (HI media Lab., India).
In-vitro antibacterial and antioxidant activities of M. azedarach L. and P. guajava L. leaves:
Antibacterial activity using Agar well diffusion
method:
Agar well-diffusion method was followed to determine
the antibacterial activity5. Solidified nutrient agar plates were
cleansed using sterile cotton swabs with 8 hours old broth culture of
respective bacteria. Wells (2cm apart and 10mm diameter) were made in each of these
plates using sterile cork-borer. The control for experiment was made of DMSO
solution. Stock solution of both extracts were prepared at a concentration of 1
mg/ml in different solvents viz. petroleum ether, ethanol, methanol, water.
About 100µl concentrations of M. azedarach L. and P. guajava L.
solvent extracts were added individually using sterile syringe into the wells
of agar medium containing petri dish and done twice in a row and allowed to
diffuse at room temperature for about 2 hours [Figure 1].
Figure 1: Schematic representation of antibacterial
activity of both plant extracts using agar well diffusion and microdilution
method
Pure sub-cultures of P. aeruginosa and S.
aureus were used in the study and were obtained from college microbiology lab
that were initially procured from the Microbial type of culture collection and
gene bank (MTCC1144, 6388). Control experiments comprising only inoculums
without plant extracts were set up. The plates were further incubated at 37°C
for 18-24h for growth of pathogenic bacteria. For the antibacterial activity,
the inhibition zone diameter (IZ) (mm) was measured, and the activity index
(AI) was also calculated. Triplicates were maintained, the average values were
recorded6. Also, the activity index is calculated for both plant extracts using
the formula, AI- Activity
index = IZ of test sample/IZ of standard.
Microdilution method:
The Minimal inhibitory concentration (MIC) and Minimum
bacterial concentration (MBC) were performed by a serial dilution technique
employing 96-well microtiter plates. The serial dilution of the extracts was
made with Luria broth for bacterial culture with respective inoculum. The microplates
were incubated for 72hours at 28oC, respectively. The MICs were
determined by serial sub-cultivation of 2µl into microtiter plates containing
100µl of broth per well and further incubation for 72hours. The optical density
of each well was measured at a wavelength of 655nm by Microplate reader (Bio
Rad, iMark-1.02.01) and compared with the standard ampicillin for bacteria as
the positive control. All experiments were performed in triplicate [Figure 1]
Phytochemical Analysis:
The preliminary qualitative phytochemical screening of
various extracts was done to determine the presence of bioactive components.
The presence of phenolics (ferric chloride test), alkaloids (dragendroff test)7, flavonoids (ammonium test), tannins (lead acetate),
triterpenes/ steroids (liebermann burchardt test), saponins (foam test) and
glycosides was determined (legals test)8.
These are further confirmed using the quantitative tests.
Determination of total phenolics:
The total phenolics content in extracts were
determined with slight modifications using Folin- Ciocalteu’s phenol reagentmethod9. Stock solutions of extracts and gallic acid were
prepared by solvating 1mg of M. azedarach L. and P. guajava L. aliquots
separately andgallic acid in 1
ml of extract. A total of 50μl of different concentrations of extracts
ranging from 10μg to 100μg were taken into series of test tubes. To
each tube 250μl of 50% Folin- Ciocalteu’s reagent was added and properly
mixedand allowed to stand for 10 minutes. Then add 500μl 20% sodium
carbonate (Na2CO3)
to the above mixture. The mixtures were then vortexed to which autoclaved
distilled water was added to make up the final volume up to 5ml10.
A set of standard solutions of gallic acid (10, 20,
40, 60, 80 and 100μg/ml)
in distilled water were prepared. The absorbance of the blue colored complex was
measured for test and standard solutions against the reagent blank at 750nm
after 30min incubation at room temperature. The results were expressed as
gallic acid equivalent (GAE) (µg/mg of extracted compound) using gallic acid
calibration curve11.
Determination of flavonoids:
The total flavonoids content
(TFC) in extracts were measured through Aluminum chloride
colorimetric assay as described by Moneim12.
1mg/ml of stock solutions were prepared in distilled water for different
dilutions of rutin standard and M. azedarach L and P. guajava Lextracts (10-100μg/ml) concentrations separately in a series of test tubes. The total volume was made
up to 5ml using distilled water. Around 0.3ml of 5% NaNO2 was added
to the above mixture. Then after 5 minutes 0.3ml of 10% aluminum chloride (AlCl3)
was incorporated to the mixture resulting in the appearance of a yellow color13. The resultant mixture was incubated for 6 minutes at
room temperature. After the incubation period 2ml of 1M NaOH was incorporated
into the mixture. The total volume of about 10ml was made up with distilled
water. The absorbance was measured against a reagent blank devoid of the
extract at 510nm wavelength in the spectrophotometer after the solution was
mixed well. The results were expressed in terms of rutin equivalent (µg/mg of
extracted compound) calibration curve.
In-vitro antioxidative activity using DPPH assay:
The antioxidant activity of the extracts was
determined by free radical scavenging ability in correspondence to stable 2, 2-
diphenyl-2-picrylhydrazyl (DPPH) method14.
The scavenging activity was in correspondence to hydrogen donating ability and
was determined by the method described by Brand-Williams et al. (1995) with
minor modification15. The DPPH radical discolorizes in presence
of antioxidants (catechol moieties) indicating the scavenging potential towards
the free DPPH* radical by in-vitromethod. A
linear correlation was founded between the total phenol content estimation
(Folin-Ciocalteu assay) and the free radical scavenging potential16.
The extracts were prepared i.e., 20µg/ml, 40µg/ml,
60µmg/ml, 80µmg/ml, and 100µg/ml. 5ml of each prepared concentration was mixed
with 0.5ml of 1mM DPPH solution in DMSO withethanol and methanol, pet. ether
and aqueous solutions, respectively. Experiment was done in triplicate. The test
tubes were incubated for 30 min at room temperature with intermittent shaking
and then, absorbance measured at 517nm using UV- Visible spectrophotometer,
lower the absorbance of the reaction mixture indicates higher free radical
scavenging activity. Vitamin C (0.1mg/ml) was used as a standard and the same
concentrations were prepared as the test solutions [Figure 2]. The difference
in initial absorbance between the test extracts and the control (DPPH in
solvent) was calculated and expressed as % scavenging activity of DPPH radical.
The capability to scavenge the DPPH radical was calculated by using the
following equation. Scavenging effect (%) = (1-AS/AC) ×100, AS is the
absorbance of the sample at t =0 min. AC is the absorbance of the control at t
=30 min, which varied with different concentrations.
Figure 2: Schematic representation of antioxidant
activity of both plant extracts using DPPH method.
Statistical Analysis:
All experimental results were expressed as mean ±
standard deviation (SD) or standard error of mean (SEM), n=3. Statistical
analysis of the data was performed by one-way Analysis of Variance (ANOVA) and
mean comparison using Student's t-test. Graphical representation is performed
using software GraphPad prism software 9.0.0.121 version. P<*0.05 was
considered statistically significant.
Table 1: Antibacterial activity
(zone of inhibition (IZ), mm, and activity index (AI)) of M. azedarach
L. and P. guajava L. plant extracts.
|
Plant
|
Bacterial strain
|
Activity
|
E. E
|
M.E
|
P.E. E
|
A. E
|
Standard (Ampicillin)
|
|
M. azedarach L.
|
S. aureus
|
IZ
|
20.2±0.25
|
17.3±0.31*
|
15.0±0.35
|
8.9±0.15*
|
20.13
|
|
AI
|
1.003
|
0.859
|
0.745
|
0.442
|
|
M. azedarach L.
|
P. aeruginosa
|
IZ
|
23.4±0.14*
|
18.6±0.25*
|
14.0±0.13*
|
11.7±0.52
|
23.25
|
|
AI
|
1.006
|
0.886
|
0.602
|
0.503
|
|
P. guajava L.
|
S. aureus
|
IZ
|
24.6±0.15*
|
20.56±0.34
|
22.4±0.25*
|
16.3±0.13*
|
23.13
|
|
AI
|
1.222
|
1.021
|
1.11
|
0.809
|
|
P. guajava L.
|
P. aeruginosa
|
IZ
|
20.7±0.65
|
20.3±0.86
|
14.5±0.52
|
14.8±0.41
|
26.25
|
|
AI
|
0.890
|
0.787
|
0.623
|
0.636
|
Values are mean of triplicate readings (mean ± S.D),
IZ- includes the diameter of disc (6 mm); Standard - Ampicillin (1.0 mg/disc).
Readings were found to be significant with P value 
0.05.
RESULTS:
Phytochemical screening:
The % (w/w) yields of pet. ether,ethanol, methanol,
and aqueous extracts were 11.74gm (2.34%), 13.39gm (2.67%), 23.50gm (4.7%), and
25.37gm (5.07%), respectively while for P. guajava L.leaves the crude extracts weights were 10.16gm
(2.02%), 14.95gm (2.98%), 23.86gm (4.77%) and 27.43gm (5.48%) correspondingly.
The variation in yields obtained is due to the presence of higher percent of
polar content in the leaf extracts. Most of the polar contents are extracted
when most polar aqueous solvent is used followed by methanol, while pet. etherand
ethanol showed minimum yield. The quantitative analysis of phytochemical
constituents of crude extracts of both plants data shows the
presence of flavonoids,phenols,
tannins, glycosides in higher amount
while other phytoconstituents saponins, steroids were present in trace amounts
andanthraquinones are absent17. M. azedarach L. showed presence of higher concentration of
flavonoids while P. guajava
L. indicated higher
concentration of phenolics. The results obtained are in correspondence with
existing data18.
Antibacterial activity:
The antibacterial activity of M. azedarach L. (M. A. 1
and 2) and P. guajava L. P. G 1 and 2) leaf extracts was determined in-vitrousing
agar well diffusion method19 and micro dilution methods20. Results were evaluated according to their zone of
inhibition (IZ) against sample-1(S. aureus) and
sample-2 (P. aeruginosa) pathogens (Figure 3).
Sample 1: S. aureus
Sample 2: P. aeruginosa
Figure 3: Antibacterial effect of various extracts of leaves of both plants against bacterial sample 1 and 2 strains by
indicating zone of inhibition.
The obtained IZ, AI values were compared with that of standard,
viz., Ampicillin (1.0mg/disc) as summarized in (Table 1). For both the tested
bacterial cultures, ethanol extract showed maximum IZ value in P. guajavaL. (24.6mm for
S. aureus) which is followed by M. azedarach L
ethanol extract (23.4 for
P. aeruginosa).
The standard ampicillin IZ values for the two
bacterial strains closely resemble the IZ values of ethanol extract of
respective plants (23.25mm for P. aeruginosa and
20.13mm for S. aureus). The minimum IZ valuesof 8.9 and 11.7mm
are observed with aqueous extract of M. azedarach L. with respect
to S. aureus and P. aeruginosapathogens (Table 1). More specifically, aqueous extract represented
higher susceptibility to all bacterial strains. Overall, ethanol extract showed higher AI value with P. guajava L of
1.222 for S. aureus and for M. azedarach L. of
1.006 for P. aeruginosa. The minimum AI values are observed with M.
azedarach L. aqueous (0.503 for P. aeruginosa and 0.442 for S. aureus) (Table
1). Further, maximum MIC values are observed with P. guajava L. ethanol extract at 47.6 µg/ml for S. aureus and 43.7µg/mlfor
P. aeruginosa, respectively. Aqueous extract of P. guajava L. showed least MIC values of 37.0µg/ml and 35.9µg/ml
against S. aureus and P. aeruginosa, respectively. For M.
azedarach L. maximum MIC values of 12.7µg/ml for S. aureus and 17.8µg/ml
for P. aeruginosa are observed for ethanol extract, while minimum values
are observed with aqueous extract at 7.0µg/ml for S.
aureus and 9.6 µg/ml for P. aeruginosa, individually (Table 2).
Table 2: MIC (µg/ml), MBC performance of different
extracts of M. azedarach L. and P. guajava. L. against
pathogenic organisms
|
Bacterial strain
|
Plant Extract
|
Activity
|
E.E (µg/ml)
|
M.E (µg/ml)
|
P.E.E (µg/ml)
|
A.E (µg/ml)
|
|
S. aureus
|
P. guajava L.
|
MIC
|
47.6
|
45.6
|
40.7
|
37.0
|
|
MBC
|
94.4
|
83.3
|
82.5
|
73.1
|
|
P. aeruginosa
|
P. guajava L.
|
MIC
|
43.7
|
41.7
|
39.8
|
35.9
|
|
MBC
|
96.4
|
94.5
|
89.7
|
71.9
|
|
S. aureus
|
M. azedarach L.
|
MIC
|
22.7
|
21.27
|
19.7
|
17.0
|
|
MBC
|
48.8
|
42.5
|
38.7
|
34.8
|
|
P. aeruginosa
|
M. azedarach L.
|
MIC
|
27.8
|
24.5
|
22.7
|
19.6
|
|
MBC
|
54.6
|
48.3
|
44.5
|
38.3
|
In connection with MIC values the MBC values were
estimated accordingly. Ethanol extract of P. guajava L. showed comparatively
efficient MBC value of 94.4 µg/ml (S. aureus) and 96.4µg/ml (P.
aeruginosa) while aqueous extract has minimum value with 73.1µg/ml (S.
aureus) and 71.9µg/ml (P. aeruginosa) (Table 2). Whilst ethanol
extract of M. azedarach L. indicated maximum MBC of 24.8µg/ml and 34.6µg/ml
against S. aureus and P. aeruginosa correspondingly and aqueous
extract showed minimum MBC of 14.8µg/ml and 18.3µg/ml against S. aureus and
P. aeruginosa congruently. These results are in line with the findings of earlier reported activities
of these plants and the high antibacterial activity of P. guajava L. extracts could be due to presence of high content of flavanols, isoflavones and
flavones21.
The results all together revealed that ethanol followed by methanol extract of M.
azedarach L. and P.
guajava L. plants have potent antibacterial activity against the
two bacteria studied showing high degree of inhibition followed bypet. ether and aqueous extracts. The inhibition zone diameters
differ for the plant extracts which might be due to diffusion capacity and amphiphilic
character of substances used and their antibacterial action. The major
components in studied extracts including quercetin among phenolics and low
doses of gallic acid, oleic acid inhibit the growth of S. aureus and P.
aeruginosa bacteria in accordance with study conducted by Dilika et al.,22. From the results of study, it was understandable
that P. guajava L.
shows better antibacterial action than M. azedarach L. extracts and
could be used as a drug of choice in the treatment of bacterial co-infections
against dengue.
Antioxidant activity:
The total phenolics (TPC) and flavonoids (TFC) content
were determined using folin-
ciocalteu’s and aluminum
chloride colorimetric methods, respectively. Phenolic compounds in plants are
responsible for their antioxidant activity due to presence of hydroxyl groups23. The results were reported as equivalents of GAE
using gallic acid calibration curve (µg/mg) for TPC and equivalents of rutin
(RE) (µg/mg) using rutin calibration curve for TFC. The TPC values differed
significantly between the various extracts as shown in Table 3. The highest
concentration of TPC 469µg/mg was seen with ethanolic, followed by methanol
(397µg/mg), pet. ether (286 µg/mg) and least with aqueous
(189µg/mg) extracts for P.
guajava L. While the order
for M. azedarach L. follows 431, 359, 248 and 148µg/mg for
ethanol, methanol, pet. ether and water extracts, respectively. However, the
values of both extracts are comparatively lesser than the standard ascorbic
acid. Totally, P. guajava L. extracts showed better TPC values
indicating the presence of more phenolic compounds than M. azedarach L. extracts.
Flavonoids also serve as secondary antioxidant defense
system in plants when exposed to variable stresses23.
The TFC varied between the M.
azedarach L. andP. guajava L. as shown in Table 3. The highest concentration of
total TFC was seen in P.
guajava L. (496µg/mg
-ethanol), followed by methanolic (481 µg/mg), pet. ether, and aqueous extract
465, 431µg/mg, respectively. TFC values P. guajava L. are higher than the standard values. While in M. azedarach L. the values of 194, 184, 177 and 120µg/mg were
observed for ethanol, methanol, pet. ether and aqueous extracts, respectively.
These results align with TPC values indicating the presence of more flavonoids
along with phenolic compounds in P. guajavaL.
extracts.Several assays are
used to assess antioxidant activity but used methods are those that
includeproduction of free radical species which are formerly neutralized by antioxidant
compounds24. Table4shows the results of the free
radical activity (DPPH) in terms of % scavenging action for M. azedarach L. and P. guajava L. leaves extractin
comparison to standard vitamin C.
Table 3: Total phenolic contents
(TPC) and Total flavonoid contents (TFC) of P. guajava L.andM. azedarach L. extracts in
comparison with standard.
|
SI. no
|
Solvent
extracts used
|
TPC (µg/mg) of
Standard (gallic acid)
|
TPC (µg/mg) of
P. guajava L. leaves
|
TPC (µg/mg) of
M. azedarach L. leaves
|
TFC (µg/mg) of
standard (rutin)
|
TFC (µg/mg) of
P. guajava L. leaves
|
TFC (µg/mg) of
M. azedarach L. leaves
|
|
1
|
Ethanol (E.E)
|
518± 0.67
|
469± 1.23
|
431± 1.42
|
340± 1.03
|
496± 0.79
|
194± 0.89
|
|
2
|
Methanol (M.E)
|
496± 1.21
|
397± 1.13
|
359± 0.98
|
321± 1.11
|
481± 0.84
|
184± 0.73
|
|
3
|
Petroleum Ether (P.E.E)
|
374± 0.72
|
286± 0.18
|
248± 1.03
|
313± 0.67
|
465± 1.16
|
177± 1.19
|
|
4
|
Aqueous (A.E)
|
297± 0.67
|
189± 0.21
|
148± 1.35
|
299± 1.17
|
431± 1.17
|
120± 1.21
|
All data values were expressed as mean ± standard
deviation (SD) (n=3).
Table 4: DPPH radical scavenging activity (%) of in various solvent extracts
of M. azedarach L.,P. guajava L. leaves and standard.
|
Si. no
|
Conc (µg/
ml)
|
Scavenging effect (%) of Ascorbic acid (standard)
|
Scavenging effect (%) of M. azedarach L.
|
Scavenging effect (%) of P. guajava L.
|
|
E. E
|
M.E
|
P.E. E
|
A. E
|
E. E
|
M.E
|
P.E. E
|
A. E
|
E. E
|
M.E
|
P.E. E
|
A. E
|
|
1
|
20
|
84.76±
0.02
|
81.30±
0.05
|
77.19±
0.07
|
74.31±
0.06
|
74.79±
0.08
|
70.46±
0.04
|
67.15±
0.03
|
63.26±
0.07
|
76.24±
0.08
|
72.78±
0.04
|
68.67±
0.03
|
65.78±0.04
|
|
2
|
40
|
82.13±
0.09
|
77.25±
0.10
|
74.75±
0.03
|
71.86±
0.07
|
72.66±
0.06
|
68.33±
0.06
|
65.02±
0.07
|
62.13±
0.05
|
74.11±
0.06
|
69.23±
0.04
|
66.73±
0.07
|
63.84±0.09
|
|
3
|
60
|
80.57±
0.03
|
73.30±
0.07
|
71.95±
0.04
|
69.06±
0.04
|
71.11±
0.07
|
66.77±
0.08
|
63.46±
0.09
|
60.57±
0.03
|
73.83±
0.07
|
66.56±
0.08
|
65.21±
0.09
|
62.32±0.06
|
|
4
|
80
|
77.31±
0.12
|
66.29±
0.08
|
68.13±
0.07
|
65.24±
0.03
|
67.84±
0.04
|
63.51±
0.07
|
60.20±
0.06
|
57.31±
0.09
|
71.56±
0.04
|
60.54±
0.07
|
62.38±
0.06
|
59.49±0.07
|
|
5
|
100
|
71.04±
0.08
|
61.49±
0.04
|
60.28±
0.06
|
57.19±
0.07
|
61.57±
0.05
|
57.24±
0.10
|
53.93±
0.05
|
51.04±
0.03
|
69.89±
0.05
|
60.34±
0.10
|
59.13±
0.05
|
56.24±0.03
|
Each value is expressed as the mean ± SD (n = 3). P value is significant <0.05
(A) (B)
(C) (D) (E)
Figure 4: Discoloration of DPPH solution under the
influence of M. azedarach L.(M), P. guajava L. (P) and standard (S) using
ethanol (E), methanol (M), pet. ether (P) and aqueous (A) extracts. (A=M.S,
P.S), (B=M.E, P.E), (C=M.P, P.P), (D=M.M, P.M), (E=M.A, P.A)
The discoloration of the samples in extract solvents
was observed in accordance with addition of DPPH reagent (Figure 4). The
results showed that the decrease in scavenging activity with respect to increase
in absorbance of the DPPH radical, was due to its reduction by various
antioxidant concentrations of extracts relating to the standard.
DISCUSSION:
The results revealed that 20 µg/ml ethanol extract
fraction of P. guajavaL. exhibited the highest radical %
scavenging activity (76.24%) succeeded by methanol, Pet. ether and aqueous
extracts with 72.78%, 68.67%,and 65.78%, respectively. While lowest %
scavenging activity of 74.79%, 70.46%, 67.15%, and 63.26% demonstrated in M.
azedarachL. 100 µg/ml ethanol, methanol, pet. ether, and aqueous extracts,
respectively.
Finally, in the present study, both ethanolic extracts
showed the highest % scavenging activity followed by methanolic extracts
indicating they are effective solvents to extract phenolic compounds25. Ethanol is favored for the extraction of antioxidant
compounds mainly because of its low toxicity. Thus, it was apparent that DPPH
free radical scavenging activity is related to the presence of bioactive compounds
such as phenolic compounds in extracts26.
Also, the antioxidant capability of these plants revealed that scavenging
effect DPPH radical was proportional to phenolic content along with flavonoids
contribution indicating that higher the phenolic content in plants then higher
will be the radical scavenging action.
In conclusion, the findings of this study suggest the
ethanol and methanol solvent extracts of M. azedarach L. and P.
guajava L. as antibacterial agents to treat various bacterial co-infections
and function as a source of natural antioxidants by treating the oxidative
stress associated with dengue fever27.
The present investigation concludes P. guajava L. extracts
contain potential antibacterial and antioxidant components over M. azedarach
L. due to higher amounts of hydroxyl (-OH) groups in the phenolic compounds.
These phytochemical constituents may be of significant use for the development
of pharmaceutical drugs against dengue associated bacterial co-infections and
to improve endogenous antioxidant system by inhibiting decomposition of
hydroperoxides into free radicals28. Here we
demonstrated the ethanolic, methanolic, petroleum ether, and aqueous extracts
of these plants possess significant inhibitory effect against gram-positive S.
aureus and P. aeruginosa pathogens and function as promising
antioxidant agents. We further recommend prediction of active lead molecules
and conduct experimentation to validate the claims of their use in dengue
therapy management.
ACKNOWLEDGMENTS:
We, the authors would like to thank the university college
of pharmaceutical sciences, Acharya Nagarjuna University for providing support
during this study. Authors would also like to thank Dr. Rajasekhar Reddy, Johns
Hopkins University, USA for his help in editing the final draft of the
manuscript.
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