b e n i - s u e f u n i v e r s i t y j o u r n a l o f b a s i c a n d a p p l i e d s c i e n c e s 3 ( 2 0 1 4 ) 1 4 0 e1 4 8

Available online at www.sciencedirect.com

ScienceDirect
journal homepage: www.elsevier.com/locate/bjbas

Antioxidant, antimicrobial activity and in silico

PASS prediction of Annona reticulata Linn. root
extract
Prasad G. Jamkhande a,*, Amruta S. Wattamwar a, Sanjay S. Pekamwar a,
Prakash G. Chandak b
a

Department of Pharmacology, School of Pharmacy, S.R.T.M. University, Nanded 431606, Maharashtra, India
Insitute of Nephrology and Hypertension, University Hospital and University of Bern, Inselspital 3010, Bern,
Switzerland

article info

abstract

Article history:

Microbial infections and diseases are frequently associated with several pathogenic strains

Received 29 November 2013

of bacteria and fungi. Plants of the reticulata genus are a notable source of new therapeutic

Accepted 9 May 2014

agents including antioxidant and antimicrobial. This study reports the antioxidant and

Available online 20 May 2014

antimicrobial activities of methanolic root extract of Annona reticulata Linn. The antioxidant property of extract was evaluated using DPPH free radical scavenging and hydrogen

Keywords:

peroxide assay. Antibacterial tests were performed using the agar cup method whereas

Annona reticulata

Poison plate method was used to assess sensitivity of fungal strains. The biological po-

Antioxidant activity

tential of major phytoconstituents as antimicrobial agent was screened by new software

PASS prediction

based tool, PASS. The dose dependent scavenging was observed at concentrations 20, 40,

Antimicrobial activity

60, 80 and 100 mg/ml which were compared to ascorbic acid. The probable activity (Pa) of

DPPH radical scavenging assay

neoannonin using PASS was found to be 0.541. The extract was significantly active against

H2O2 assay

all strains of bacteria but the largest zone of inhibition was found against B. cereus. Predominant growth reduction was observed in fungi Tricoderma viride and Candida albicans.
The results indicate that the extract show potential as a source of new antimicrobial drug
and may impart health benefits by its antioxidant property.
Copyright 2014, Beni-Suef University. Production and hosting by Elsevier B.V. All rights reserved.

1.

Introduction

Beginning from the history, there has been a continual battle

between humans and the multitude of microorganisms which

are basis of infectious disease. Different infectious diseases

like Bubonic plague, tuberculosis, malaria and more recently,
the human immunodeficiency virus/acquired immunodeficiency syndrome, have affected significant portions of the

* Corresponding author.
E-mail addresses: pjamkhande@gmail.com, pjamkhande@rediffmail.com (P.G. Jamkhande).
Peer review under the responsibility of Beni-Suef University

Production and hosting by Elsevier

http://dx.doi.org/10.1016/j.bjbas.2014.05.008
2314-8535/Copyright 2014, Beni-Suef University. Production and hosting by Elsevier B.V. All rights reserved.

b e n i - s u e f u n i v e r s i t y j o u r n a l o f b a s i c a n d a p p l i e d s c i e n c e s 3 ( 2 0 1 4 ) 1 4 0 e1 4 8

human population, causing significant morbidity and mortality (Tenover, 2006). The crucial fact about the treatment of
bacterial infections is the ability of bacteria to develop resistance to antimicrobial agents which is variable in different
geographic areas and it has been correlated with the consumption of antibiotics in the general population (Francesco
et al., 2010; Sengupta and Chattapadhyay, 2012). The multiple drug resistance not only increases morbidity and mortality
but also increased expenditure on patient management and
implementation of infection control measures (Woodford and
Livemore, 2009). Antimicrobial agents like penicillin induce
allergic responses while broad spectrum antibiotics kill the
normal flora of the body thereby impairing normal body
functions (Ahmad et al., 2012). Antimicrobial agents principally act by interference with cell wall synthesis, inhibition of
protein synthesis, interference with nucleic acid synthesis,
inhibition of a metabolic pathway and disruption of bacterial
membrane structure (Tripathi, 2008). Environmental stress
like microbial infection may increases generation of highly
reactive oxygen species (ROS) which causes significant damage to cell structures (Dubovskiy et al., 2008). Phytoconstituents of phenolic structure like flavonoids, catechins,
carotenoids, beta carotene and lycopene produce antioxidant
effect by stabilizing the free radicals and prevent cellular
damage (Arulselvan et al., 2012).
Medicinal plants represent a rich source of antimicrobial
agents (Mahesh and Satish, 2008). The plant Annona reticulata
belongs to genus Annona and family Annoanaceae (Pinto et al.,
2005; Saad et al., 1991). The plant also known as Ramphal,
Bullock's heart and Custard apple (Pinto et al., 2005; Nirmal
et al., 2010). It is a small tree with glabrous branches which
is found in tropical region (Pinto et al., 2005). Various plant
parts like leaves, bark, seed and root are medicinally useful
and they show many therapeutic activities as anticancer, CNS
depressant, analgesic, antihyperglycemic, anti-inflammatory,
antiproliferative, wound healing and antiulcer activity (Bhalke
and Chavan, 2011; Rahman et al., 2011). The root contains
aporphine alkaloids like liriodenine, norushinsunine, reticuline and one acetogenin neoannonin (Suresh et al., 2012).
Other species of this plant have been reported to have antibacterial and antioxidant property (Aamir et al., 2013; Pandey
and Brave, 2011). So the current study was directed for
exploration of these phytochemicals from A. reticulata for
antioxidant and antibacterial potency.

2.

Material and methods

2.1.

Plant material collection and authentication

The roots of A. reticulata were collected in the month of

January from Beed district and Authenticated by Dr. B. D.
Gachande, Associate Professor of Botany department, N. E. S.
Science College, Nanded, Maharashtra, India.

2.2.

Preparation of extract

The roots were rinsed well with deionised water, made into
small pieces of 2e3 cm and shade dried for 10 days. Dried
plant material was powdered by using dry grinder and passed

141

through sieve no. 85 to obtain fine powder. Prepared root

powder was extracted with 500 ml methanol using Soxhlet
apparatus. The solvent was evaporated and concentrated
extract was obtained. The extract was stored in well closed
container (Mehta, 2007; Bart, 2011).

2.3.

Preliminary phytochemical screening

Freshly prepared extract were subjected to standard phytochemical analysis to ensure the presence of major chemical
constituents according to the standard procedure (Kokate
et al., 2008).

2.4.

Reagents, chemicals and microorganism

Methanol, dimethyl sulphoxide, sodium hydroxide and

hydrogen peroxide (H2O2) were purchased from Rankem (India).
2,2-diphenyl-1-picrylhydrazyl (DPPH), Nutrient agar and Potato
dextrose agar were obtained from Himedia. Ascorbic acid (Oxford Laboratories) and Potassium dihydrogen phosphate (S. D.
Fine, Mumbai) were purchased from respective vendors.
The gram negative bacteria used in this study were
Escherichia coli (E. coli) (NSBCN B06), Salmonella Typhi (S. Typhi)
(NSBCN B08) and Pseudomonas aeruginosa (P. aeruginosa) (NCIM
2053). The gram positive bacteria that were used in this study
were Staphylococcus aureus (S. aureus) (NSBCN B09), Bacillus
subtilis (B. subtilis) (NCIM 2063) and Bacillus cereus (B. cereus)
(NSBCN B01). The fungal strains selected for antifungal study
were Aspergillus niger (A. niger) (NCIM 596), Penicillium chrysogenum (P. chrysogenum) (NSBCN F01), Fusarium moneliforme (F.
moneliforme) (NCIM 1099), Aspergillus flavus (A. flavus) (NCIM
538), Tricoderma viride (T. viride) (NSBCN F03) and Candida albicans (C. albicans) (NCIM 3055).

2.5.

Antioxidant activity

2.5.1.

DPPH free radical scavenging assay

The scavenging activity of the extracts was estimated by using

DPPH as a free radical scavenging assay and a method adapted
from Madaan et al., 2011. Solution of DPPH (0.1 mM) in
methanol was prepared by dissolving 1.9 mg of DPPH in
methanol and volume was made up to 100 ml with methanol.
The solution was kept in darkness for 30 min to complete the
reaction.1 ml of DPPH solution was added to 1 ml of different
(20, 40, 60, 80 and 100 mg/ml) concentrations of extract and
allowed to stand at room temperature for 30 min. The mixture
was measured spectrophotometrically (UV-1800, UV-VIS
spectrophotometer, Shimadzu) at 517 nm. The free radical
scavenging activity was calculated as using following formula,

% inhibition (Ac  At)/Ac  100

where, Ac is the absorbance of control and At is the absorbance of test sample. A standard of ascorbic acid was run
using same concentrations as that of extract (Madaan et al.,
2011; Mulla et al., 2010). The antioxidant activity of the sample was expressed as IC50 value, was defined as concentration
(in mg/ml) of sample that inhibits the formation of DPPH radicals by 50% (Chew et al., 2012).

142

2.5.2.

b e n i - s u e f u n i v e r s i t y j o u r n a l o f b a s i c a n d a p p l i e d s c i e n c e s 3 ( 2 0 1 4 ) 1 4 0 e1 4 8

Hydroxyl (H2O2) radical scavenging activity assay

The ability of the extract sample to scavenge H2O2 was

determined according to the method of Singh et al. (2010).
Solution of 0.2 M potassium dihydrogen phosphate and 0.2 M
sodium hydroxide solutions were prepared as per the Indian
Pharmacopoeia 1996 standards. 50 ml potassium dihydrogen
phosphate solution was placed in a 200 ml volumetric flask
and 39.1 ml of 0.2 M sodium hydroxide solution was added and
finally volume was made up to 200 ml with distilled water to
prepare phosphate buffer (pH-7.4). 50 ml of phosphate buffer
solution was added to equal amount of hydrogen peroxide to
generate the free radicals and solution was kept aside at room
temperature for 5 min to complete the reaction. Extracts (1 ml)
in distilled water were added to 0.6 ml hydrogen peroxide
solution and the absorbance was measured at 230 nm in a
spectrophotometer (UV-1800, UV-VIS spectrophotometer,
Shimadzu) against a blank solution containing phosphate
buffer solution without hydrogen peroxide. Concentrations
selected for extract were ranging from 20 to 100 mg/ml. The
percentage of scavenging of H2O2 of extract was measured
using the following equation:
Percent scavengedH2 O2

Ao  A1
 100
Ao

where Ao is the absorbance of the control, and A1 is the

absorbance in the presence of the extract sample (Singh et al.,
2010; Gulcin et al., 2004).

2.6.

PASS computer program

Prediction of methanolic extract of A. reticulata root for antibacterial and antifungal activity was done with the help of
computer program, PASS (Prediction of activity spectra for
substances) (Khurana et al., 2011; Mittal et al., 2008). PASS is a
computer based program used for the prediction of different
types of pharmacological activities for different substances
(Khurana et al., 2011) including phytoconstituents. Prediction
of this spectrum by PASS is based on structural activity relationship (SAR) analysis of the training set containing more
than 205,000 compounds exhibiting more than 3750 kinds of
biological activities. The predicted activity spectrum of a
compound is estimates as probable activity (Pa) and probable
inactivity (Pi) (Goel et al., 2011). The compounds showing more
Pa value than Pi are the only constituents considered as
possible for a particular pharmacological activity (Khurana
et al., 2011; Goel et al., 2011).

2.7.

Assay for antibacterial activity by agar cup method

Susceptibility pattern of bacteria was obtained by agar cup

method (agar well method) according to the method of
Cruickshank et al. (1998) against methanolic extract of A.
reticulata root. Nutrient agar was prepared and sterilized at 15
psi for 15 min using autoclave. It was allowed to cool below
45  C and seeded with turbid suspension of test bacteria
separately, prepared from 24 h old slant cultures. The 3%
inocula were used every time. This seeded preparation was
then poured in sterile petri plate under aseptic condition and
allowed to solidify. The agar cups of 10 mm diameter were
made by punching the agar container with a sterile cork borer.

The 100 ml of extract solution prepared in dimethyl sulphoxide

(1%) was added in the cup under aseptic condition with the
help of micropipette. For blank (negative control), 100 ml of
DMSO was placed in one of the cup. A standard antibiotic disk
impregnated with 10 units of penicillin was placed on the
seeded nutrient agar surface as standard reference antibiotic
(positive control). The plates were incubated at 37  C for 24 h.
The inhibition zones which appeared around the agar cup
indicated the presence of antimicrobial activities that was
evaluated using zone scale or antibiotic scale (Cruickshank
et al., 1998).

2.8.
Assay for antifungal activity by poison plate
method
Antifungal activity was performed by poison plate method
according to the method of Cruickshank et al. (1998). Potato
dextrose agar was used as media to evaluate the activity. The
medium was prepared and sterilized at 10 psi for 15 min using
autoclave. The sample was added to the sterile medium in
aseptic condition so as to get final concentration as 1%. A plate
with DMSO was prepared as blank (negative control) and
similarly a plate with 1% griseofulvin was prepared as standard reference plate (positive control). The fungal suspension
spot was inoculated on the plates with the help of nichrome
wire loop. The plates were incubated for 48 h at room temperature and plates were observed for the growth of inoculated fungi. Results were recorded as growth of fungi (no
antifungal activity), reduced growth of fungi (moderate antifungal activity) and no growth of inoculated fungi (antifungal
activity) (Cruickshank et al., 1998).

2.9.

Statistical analysis

All the results are presented as mean standard error of the

mean (SEM) of three determinations. Obtained data were
statistical analysed by one way ANOVA and post hoc tests
using Graphpad Instat (Version 3, USA) software. The difference was considered to be statistically significant when
P < 0.05.

3.

Result

3.1.

Phytochemical prospective

Methanolic extract of A. reticulata root showed presence of

alkaloids, flavonoids, steroids, triterpenoids, glycosides, carbohydrates, proteins and tannins.

3.2.

Antioxidant activity

Extract were subjected to screening for their possible antioxidant activity using DPPH free radical scavenging assay and
H2O2 radical scavenging assay.

3.2.1.

DPPH free radical scavenging assay

DPPH is a stable free radical which shows maximum ultraviolet and visible (UVeVis) absorbance at 517 nm. It gets reduced
in presence of antioxidant present in the sample which is

143

b e n i - s u e f u n i v e r s i t y j o u r n a l o f b a s i c a n d a p p l i e d s c i e n c e s 3 ( 2 0 1 4 ) 1 4 0 e1 4 8

Table 1 e Dose-dependent DPPH free radical scavenging and H2O2 radical scavenging activity of methanolic extract of A.
reticulata.
Treatment

DPPH free radical

scavenging
(% inhibition)
H2O2 scavenging
activity assay
(% inhibition)

Concentration (mg/ml)

Absorbance
wavelength (nm)

IC50

20

40

60

80

100

Methanolic extract
Ascorbic acid

517

27.21 0.12
48.57 1.98

32.54 0.26
57.76 1.81

37.27 0.15
65.94 0.55

42.60 0.00
72.53 0.55

47.92 0.04
80.63 1.51

108.71
21.80

Methanolic extract
Ascorbic acid

230

27.38 0.57
50 0.57

39.28 0.04
61.90 0.57

41.66 0.30
72.61 0.57

51.19 0.68
84.52 0.00

55.95 0.08
91.66 0.00

80.08
18.85

All values are expressed as mean SEM for three determinations.

considered as a measure of their antioxidant activity. The

ability of samples to scavenge DPPH radical was measured on
the bases of their concentrations providing 50% inhibition
(IC50). The results of one way ANOVA test and post hoc test
indicates significant difference of mean percentage scavenging between different concentrations of tested extract.
The scavenging activity of extract was well pronounced at
higher concentrations of 80 mg/ml and 100 mg/ml with a mean
percentage of 42.60 0.00 and 47.92 0.04 respectively, which
was lower compared with standard antioxidant, ascorbic acid
but exhibits similar pattern of concentration dependent free
radical scavenging effect. The IC50 values of extract (108.71)
and ascorbic acid (21.80) were obtained using the linear
regression equation. The radical scavenging ability, IC50 of
extract and ascorbic acid were presented in Table 1.

3.2.2.

H2O2 radical scavenging assay

The scavenging ability of the extract on H2O2 is shown in Table

1. The extract demonstrated hydrogen peroxide radical scavenging activity in a dose dependent manner. The hydrogen
peroxide radical scavenging activity of extract was
(27.38 0.57)%, (39.28 0.04)%, (41.66 0.30)%, (51.19 0.68)%
and (55.95 0.08)% at the concentrations of 20, 40, 60, 80 and
100 respectively. Similar dose dependent scavenging was
observed for standard, ascorbic acid. The IC50 value of the
extract was 80.08 mg/ml whereas for ascorbic acid it was
18.85 mg/ml using linear regression equation.

3.3.
PASS predictions for antibacterial and antifungal
activities
The biological activity spectra of previously identified phytoconstituents were obtained by online PASS version. These
predictions were interpreted and used in a flexible manner.
PASS predicted probable activity (Pa) of acetogenin alkaloid,
neoannonin for antibacterial and antifungal activity was 0.541
and 0.528 respectively which is greater than 0.5. This type of
probable activity spectra was not shown by other phytochemicals from A. reticulata root extract.

3.4.

Antibacterial activity of root extract

Using the gar cup diffusion method, extract showed significant

antibacterial effects against all the strains of bacteria and their
potency were assessed qualitatively and quantitatively by the
presence and absence of zone of inhibition. Results of antibacterial activity of extract were listed in Table 2. The mean

zone of inhibition produced by extract on different bacterial

strain was between 20 0.57 mm to 29 0.57 mm and standard,
penicillin was between 19.66 0.33 mm to 40 0.57 mm
respectively which is shown in Table 3. The zone of inhibition
produced by penicillin was larger than those produced by
extract. The highest activity of extract was observed against B.
cereus (29 0.57) and least was against S. aureus (20 0.57) and S.
Typhi (20.33 0.33). Also extract exhibit moderate antibacterial
activity against B. subtilis (23.66 0.33), E. coli (25.66 0.33) and P.
aeruginosa (22 0.57). Extract exhibits prominent effects
against B. subtilis (23.66 0.33), E. coli (25.66 0.33) and S. Typhi
(20.33 0.33) as compared to Penicillin.

3.5.

Antifungal activity of root extract

The antifungal activity of root extract was evaluated using

poison plate method. Increase or decrease in fungal growth
after treatment was considered as a result for the assessment
of antifungal potency of extract, DMSO and standard, griseofulvin. Antifungal potency of extract was listed in Table 4. The
extract exhibited potent antifungal activity against T. viride
and C. albicans as that of griseofulvin whereas moderate
reduction in growth was observed in A. niger, P. chrysogenum, F.
moneliforme and A. flavus. No growth was observed in griseofulvin treated fungi whereas normal pattern of growth was
observed in DMSO treated fungi.

4.

Discussion

Lack of safe and effective antimicrobial agents leads to

diversion of research towards new strategies such as

Table 2 e Antibacterial activity (zone of inhibition) of

methanolic extract and penicillin.
Bacterial strain

Zone of inhibition (mm)

Extract

S. aureus
B. subtilis
B. cereus
E. coli
S. Typhi
P. aeruginosa

20
23.66
29
25.66
20.33
22

0.57***
0.33***
0.57***
0.33***
0.33
0.57***

Penicillin
33.66
19.66
50
12
19.66
40

0.33
0.33
0.57
0.57
0.33
0.57

All the values are expressed as mean SEM. *P < 0.05, **P < 0.01,
***P < 0.001.

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Table 3 e Showing zone of inhibition of produced by methanolic extract (E),

penicillin (S) and DMSO (C) against selected bacterial strains.

Bacillus subtilis (B. subtilis)

Pseudomonas aeruginosa (P. aeruginosa)

Staphylococcus aureus (S. aureus)

Bacillus cereus (B. cereus)

exploration of traditional ethnobotanical knowledge for the

development of efficient antimicrobial agents. Plants constitute an important source of numerous secondary metabolites
(Shanmugapriya et al., 2012). Root material of A. reticulata was
extracted with methanol as it dissolves several secondary

Salmonella Typhi (S. Typhi)

metabolites of plant (Cowan, 1999). Preliminary phytochemical investigation confirmed presence of alkaloid, flavonoids,
steroid, triterpenoids, glycosides, carbohydrates, proteins and
tannins which point out towards richness of plant in secondary metabolites (Suresh et al., 2012; Leitao et al., 2013).

b e n i - s u e f u n i v e r s i t y j o u r n a l o f b a s i c a n d a p p l i e d s c i e n c e s 3 ( 2 0 1 4 ) 1 4 0 e1 4 8

Table 4 e Antifungal potency of methanolic extract,

DMSO and griseofulvin.
Bacterial strain

A. niger
P. chrysogenum
F. moniliforme
A. flavus
T. viride
C. albicans

Antifungal activity (growth of fungi)

Extract

DMSO

Griseofulvin

RG
RG
RG
RG










, growth of fungi (no antifungal activity); , no growth of fungi

(antifungal activity); RG, reduced growth (moderate antifungal
activity).

Also the plants of these species abundantly supplied with

acetogenin (Kojima and Tanaka, 2009).
Oxygen is essential for survival but under certain abnormal
physiological conditions converts some of oxygen to ROS by
univalent reactions that makes them highly reactive and
damages nucleic acids, proteins, lipids and carbohydrates
components of cell (Prakash and Gupta, 2009). These free
radicals regulate critical aspects of cellular physiology like
signal transduction, gene transcription, and regulation of
soluble guanylate cyclase activity (Fang et al., 2002). The
excess production of free radicals results in abnormal physiological conditions like oxidative stress that impedes various
cellular and metabolic functions leading to neurodegenerative diseases, gastro-duodenal disorders, cancer,
cataracts, premature ageing, inflammation, cardiovascular
diseases (Bahorun et al., 2006), Chronic kidney disease (Small
et al., 2012) and metabolic dysfunction (Lopez-Alarcon and
Denicola, 2013). The antioxidant activity of natural and synthetic compounds attributed to various mechanisms such as
inhibition of chain initiation, binding of transition metal ion
catalysts, peroxides decomposition, prevention of continued
hydrogen abstraction and radical scavenging (Bahorun et al.,
2006; Gulcin et al., 2004) that hamper consequences of
oxidative damage. Phytochemicals like carotenoids, tocopherols, ascorbates, lipoic acids, flavonoids and related polyphenols (Devasagayam et al., 2004) are potential natural
antioxidants with strong antioxidant capacity that offers
protection against oxidative deterioration by the radicals
(Prakash and Gupta, 2009).
The cumulative capacity of root extract to scavenge free
radicals was determined using DPPH reaction. DPPH method
is a valid, accurate and sensitive method to evaluate scavenging ability of antioxidant compounds since the radical is
stable and need not to be generated as in other scavenging
assays (Singh and Singh, 2008). DPPH is a coloured stable free
radical which accepts an electron or hydrogen radical to
become a stable diamagnetic molecule (Lopez-Alarcon and
Denicola, 2013) and has UVevis absorption maximum at
515 nm (Huang et al., 2005). Reaction between antioxidants
and DPPH leads to decrease in absorbance of DPPH radical
which results in scavenging of the radical by hydrogen
donation and causes discolouration from purple to yellow
(Subramanian et al., 2013). Similar significant reduction was
observed when extract and ascorbic acid treated with DPPH
due to their scavenging ability.

145

H2O2 has wide physiological role that raises intracellular

Ca2 level, activate transcription factors, repress expression of
certain genes, promote or inhibit cell proliferation, activate or
suppress certain signal transduction pathways and promote
or suppress apoptosis (Prakash and Gupta, 2009). During
certain adverse physicochemical, environmental or pathological conditions the level of H2O2 increases that yields
potent species OH, highly reactive free radical in biological
system (Devasagayam et al., 2004; Singh and Singh, 2008).
These adversely alter lipids, proteins and DNA, especially
thiamine and guanosine and have been implicated in number
of pathologies like cardiovascular diseases, cytotoxicity,
ageing (Small et al., 2012; Badole et al., 2011). The scavenging
of H2O2 by the extract may be attributed to active secondary
metabolites, phenolics which neutralize H2O2 by donating
electrons thereby neutralizing it to water (Mathew and
Abraham, 2006). The scavenging capacity of root extract was
due to structural specificity of phytochemicals, which determine their electron donating abilities. Extract exhibited strong
H2O2 radical scavenging activity in a dose-dependent manner.
This scavenging was might be because of reacting directly
with H2O2, reacting with intermediates formed from enzyme
and H2O2 and inhibiting the horseradish peroxidase from
binding H2O2 (Huang et al., 2005).
Numbers of epidemiological studies have highlighted the
inverse correlation between antioxidants and occurrence of
disease and mortality due to these diseases (Devasagayam
et al., 2004). Phenolic compounds are products of secondary
metabolism and have strong redox properties which can play
an important role in chelating transitional metals, inhibiting
lipoxygenase and scavenging free radicals (Mohamed et al.,
2013). Preliminary phytochemical investigation revealed that
root part of A. reticulata is rich in secondary metabolites
(Suresh et al., 2011). The presence of phytochemicals, phenolic
and polyphenolic constituents might be responsible for the
free radical scavenging activities in the present experiment.
The diet rich in antioxidants, polyphenolic compounds may
have inhibitory effects on mutagenesis and carcinogenesis in
humans (Amir et al., 2012) that also involved in stabilizing
lipid peroxidation (Devasagayam et al., 2004).
Acetogenin alkaloids exhibit potent bioactivities through
diverse mechanisms like through depletion of ATP via inhibiting complex I of mitochondria and inhibiting the NADH oxidase of plasma membrane (Alali et al., 1999; Zeng et al., 1996).
A. reticulata is rich in acetogenin alkaloid (Suresh et al., 2012).
The possible biological role of phytochemicals has been
evaluated using PASS which shown effectiveness of neoannonin as antibacterial and antibacterial agent.
The possible antibacterial activity of extract was screened
against eight strains of bacteria using agar cup method.
Interestingly extract showed significant zone of inhibition
against all the strains but prominent was against B. cereus. The
B. cereus, gram-positive bacteria produces heat-labile enterotoxins in the small intestine and induce diarrhoea in human.
It also produces cereulide, a single heat-stable peptide toxin
that causes emesis (Guinebretiere et al., 2010). The significant
potency of extract against this bacterium indicates effectiveness for the management of B. cereus induced pathophysiological changes. Although E. coli, belonging to the normal flora
of humans, responsible for death in wide population annually

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particularly in children under the age of five. These serious life

threatening effects are because of chronic diarrhoea and food
poisoning (Fleckenstein et al., 2010). Although B. subtilis is a
benign organism to human but sometimes contaminate food
and causes food poisoning (Sundaram et al., 2011). P. aeruginosa causes a broad range of acute and chronic infections like
cystic fibrosis, keratitis, skin infections, urinary tract infections, infections respiratory tract and bloodstream infections (Lavoie et al., 2011). S. aureus is both commensal and
an opportunistic pathogen in humans and produces wide
range of infections (Colonna et al., 2013). The typhoid fever, a
major communicable disease is because of S. Typhi infection
(Walawalkar et al., 2013). The extract found to be effective
against E. coli, B. subtilis and P. aeruginosa however its effectiveness against S. aureus and S. Typhi is least. Root part of A.
reticulata abundantly contains aporphine alkaloids, acetogenins and sesquiterpenes (Bhalke and Chavan, 2011) of which
aporphine and acetogenins posses antibacterial activities
(Suresh et al., 2012; Alali et al., 1999).
The fungal infections in humans are either because of
allergic reactions to fungal protein and toxic reactions to
toxins present in certain fungi or infections (mycoses)
(Kathiravan et al., 2012). Infections increases rate of morbidity
and mortality in severely ill and immunocompromised patients like species of Candida is the most common cause of
opportunistic mycoses worldwide (Barros et al., 2013). A. niger
is economically important microorganisms (Schuster et al.,
2002) but it is associated with otomycosis, cutaneous infections and pulmonary disease (Kathiravan et al., 2012;
Person et al., 2010). Although Penicillium (P. chrysogenum)
species have low pathogenicity and infection, still these are
involved in direct infection, hypersensitivity reaction and
pulmonary fibrosis (Barcus et al., 2005). F. moniliforme are
molds that can cause local cutaneous infections (Nucci and
Anaissie, 2002) including onychomycosis and infections of
surgical and burn wounds (Bodey et al., 2002), keratitis and
occasionally peritonitis and cellulitis (Dignani and Anaissie,
2004). The second leading cause of invasive aspergillosis is
A. flavus and is a most common cause of superficial infection
in human. In immunocompromised patients it causes otitis,
keratitis, pulmonary and systemic infections (Hedayati et al.,
2007). T. viride infections contain nodular pulmonary infiltrates, peritonitis, cutaneous lesions and disseminated
infection that are predominantly observed in immunocompromised patients (Walsh et al., 2004). The extract found to be
moderately effective against A. niger, P. chrysogenum, F. moniliforme and A. flavus. The potent growth inhibiting activity was
observed against T. viride and Candida albicans. C. albicans is a
most common agent of superficial and invasive fungal infections that causes severe infections including candidaemia,
disseminated candidiasis, endocarditis and meningitis
(Norgaard et al., 2013). The plants is rich in various phytochemicals, one of them is tannin. Previously tannins have
been reported to exhibit antifungal activity because of their
ability to form complex with microbial enzymes, deprivation
of substrates and metal ions required for microbial growth
and alter metabolic pathways through inhibition of oxidative
phosphorylation (Liu et al., 2009). Considerable microbe killing
activity of plant based drugs is because of secondary metabolites (phenols, phenolic glycosides, flavonoids, anthocyanins

and aromatic amino acids) that concentrated in the different

plant parts and appear to function primarily in disease resistance and defence systems against predators, harmful microorganisms and pathogens (Lattanzio et al., 2006), resulting
in the high phytochemicals, antioxidant and antimicrobial
properties.

5.

Conclusion

Thus the findings of present investigation support the traditional ethanomedicinal claims of A. reticulata for the treatment of diverse infections. The broad spectrum antibacterial
and antifungal efficacy of the plant can be used for the management of disease causing pathogens. Further studies are
needed to uncover the therapeutic relevance of this plant
material as a effective antimicrobial agent. The antioxidant
activities of A. reticulata can be used as a natural antioxidant or
nutraceuticals and might be effective to diminish oxidative
stress associated with different pathophysiological conditions. Moreover, such screening of plants can provide a source
of new bioactive compounds with functional properties
beneficial to restore health.

Acknowledgements
We are very thankful to Director Prof. S. G. Gattani, School of
Pharmacy, Swami Ramanand Teerth Marathwada University,
Nanded, Maharashtra, India for providing laboratory facilities
for this research work.

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