Antimicrobial Activity and Phytochemical Screening of Aloe vera (Aloe barbadensis Miller)

Abstract

The present study was conducted to assess the antimicrobial potential and phytochemical analysis of Aloe vera (Aloe barbadensis Miller) leaves extracts.The extracts were prepared by the sequential cold maceration method by using hexane, ethyl acetate, methanol and distilled water as a solvent. Antimicrobial activity of four extracts was performed by agar well diffusion method against different bacteria and fungi. Determination of Minimum Inhibitory Concentration (MIC) of different extracts, Thin Layer Chromatography (TLC), TLC bioautography and qualitative phytochemical analysis were also performed. The antimicrobial activity of A. barbadensis leaves extracts was found maximum against S. marcescens with a Zone of Inhibition (ZOI) of 13.67±0.57mm by hexane extract. The MIC of different extracts ranged between 6.25 and 50.00 mg/ml. Among all the fungi used in the study, all the three Aspergillus species were slightly inhibited by the specific extracts. The finding of TLC bioautography showed that compounds eluted at Rf 0.65 demonstrated strong antimicrobial activity whereas compounds eluted at Rf 0.41 and Rf 0.82 exhibited moderate antimicrobial activity against S. marcescens. Phytochemical analysis indicated the presence of phytochemicals present in various extracts. The results of the investigation clearly indicate that A. barbadensis leaves extract have a potential antimicrobial activity against various microorganisms due to the presence of various phytochemicals.

Full text

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Original Research Article https://doi.org/10.20546/ijcmas.2017.603.246
Antimicrobial Activity and Phytochemical Screening of
Aloe vera (Aloe barbadensis Miller)
Darshan Dharajiya1*, Nalin Pagi2, Hitesh Jasani3 and Payal Patel4
1Department of Plant Molecular Biology and Biotechnology, C. P. College of Agriculture,
Sardarkrushinagar Dantiwada Agricultural University, Sardarkrushinagar-385506, Gujarat, India
2Department of Genetics and Plant Breeding, CPCA, SDAU, Sardarkrushinagar-385506,
Gujarat, India
3Department of Microbiology, College of Computer, Science and Information Technology
(CCSIT), Junagadh-362015, Gujarat, India
4Department of Biotechnology, ARIBAS, New V.V.Nagar-388121, Gujarat, India
*Corresponding author
A B S T R A C T
Introduction
The resistance of microorganisms against
antimicrobial drugs is a major problem of
recent times, which is increasing day by day
(Cohen, 2000; Kumar et al., 2013). As
synthetic antimicrobials or antibiotics have
considerable side effects over natural
antimicrobial agents it is compulsory need to
search for drugs which are effective against a
wide range of microorganisms with minimal
or no side-effects (Shrikanth et al., 2015). To
tackle this problem, medicinal plants with
ethnobotanical importance can be act as a
source for the identification of the new drugs.
Medicinal plants are considered as the
International Journal of Current Microbiology and Applied Sciences
ISSN: 2319-7706 Volume 6 Number 3 (2017) pp. 2152-2162
Journal homepage: http://www.ijcmas.com
The present study was conducted to assess the antimicrobial potential and phytochemical
analysis of Aloe vera (Aloe barbadensis Miller) leaves extracts.The extracts were prepared
by the sequential cold maceration method by using hexane, ethyl acetate, methanol and
distilled water as a solvent. Antimicrobial activity of four extracts was performed by agar
well diffusion method against different bacteria and fungi. Determination of Minimum
Inhibitory Concentration (MIC) of different extracts, Thin Layer Chromatography (TLC),
TLC bioautography and qualitative phytochemical analysis were also performed.The
antimicrobial activity of A. barbadensis leaves extracts was found maximum against S.
marcescens with a Zone of Inhibition (ZOI) of 13.67±0.57mm by hexane extract.The MIC
of different extracts ranged between 6.25 and 50.00 mg/ml. Among all the fungi used in
the study, all the three Aspergillus species were slightly inhibited by the specific extracts.
The finding of TLC bioautography showed that compounds eluted at Rf 0.65 demonstrated
strong antimicrobial activity whereas compounds eluted at Rf0.41 and Rf 0.82 exhibited
moderate antimicrobial activity against S. marcescens. Phytochemical analysis indicated
the presence of phytochemicals present in various extracts. The results of the investigation
clearly indicate that A. barbadensis leaves extract have a potential antimicrobial activity
against various microorganisms due to the presence of various phytochemicals.
Ke ywords
Antimicrobial
activity, Aloe vera,
A. barbadensis
Miller, TLC
Bioautography,
Phytochemical
analysis.
Accepted:
20 February 2017
Available Online:
10 March 2017
Article Info

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2153
greatest pharmaceutical stores existing on the
earth as they can produce eternal secondary
phytochemicals having bioactive properties.
These phytochemicals work efficiently to cure
various diseases and illnesses since ancient
times (Abdallah, 2011).
Aloe barbadensis Miller (Aloe vera L.) is an
herb found all over the world. It is revealed
that it has conspicuous pharmacological
activities such as antibacterial (Subramanian
et al., 2006; Arunkumar and Muthuselvam,
2009; Saritha et al., 2010; Fani and Kohanteb,
2012; Nejatzadeh-Barandozi, 2013),
antifungal (Bajwa et al., 2007; Rosca-Casian
et al., 2007; Khaing, 2011; Sitara et al.,
2011), antiviral (Zandi et al., 2007),
antioxidant (Baradaran et al., 2013; Ray et al.,
2013; Kang et al., 2014), cytotoxic (Jose et
al., 2014; Shalabi et al., 2015), antidiabetic
(Tanaka et al., 2006; Choudhary et al., 2014;
Suleyman et al., 2014), anti-inflammatory
(Vijayalakshmi et al., 2012; Bhattacharjee et
al., 2014), antitumor (El-Shemy et al., 2010;
Srihari et al., 2015), nephroprotective
(Iftikhar et al., 2015; Virani et al., 2016),
antiulcer (Borra et al., 2011) and anti-aging
effects which can be used as a moisturizing
agent to cure cardiovascular diseases as well
as to enhance the immune system (Chatterjee
et al., 2013). It is used as an herbal medicine
since long time which contains more than 100
bioactive constituents. Aloe plant is a rich
source of many natural phytochemicals
possessing health-promoting effects like,
anthraquinones, vitamins, minerals,
polysaccharides, sterols, amino acids,
saponins, salicylic acids and may more
(Surjushe et al., 2008; Chatterjee et al., 2013).
This might be the first report of the evaluation
of antimicrobial activity of A. barbadensis
leaves extracts against two bacteria viz.,
Serratia marcescens and Bacillus cereus as
well as four fungi used in the present study.
Thus, the aim of the present investigation was
to evaluate the inhibitory effects of A.
barbadensis leaves extracts against
pathogenic bacteria and fungus in addition to
elucidate the possible class of phytochemicals
responsible for their antimicrobial activity.
Materials and Methods
Plant material used
Fresh leaves of A. barbadensis were collected
from the botanical garden of G. J. Patel
Institute of Ayurvedic Studies and Research,
New Vallabh Vidhyanagar, Gujarat, India.
The taxonomical identification was done by
the taxonomist. The fresh leaves were washed
with distilled water and air dried. After
drying, leaves were powdered and stored at
4°C in airtight bottles for further study.
Preparation of plant extracts
Four solvents viz., hexane, ethyl acetate,
methanol and distilled water were used in the
sequential cold maceration method (Dharajiya
et al., 2014) as described in flow chart given
in Figure 1. At the end of extraction process
four different extracts were prepared and
further used for antimicrobial study. Test
samples of 100 mg of extract/ml of dimethyl
sulphoxide (DMSO) were prepared to
perform antimicrobial assay.
Test microorganisms
All the microorganisms used in the present
study were collected from the Department of
Microbiology, ARIBAS, Gujarat, India. Total
four bacteria were used in the study, of which
three were Gram negative bacteria viz.,
Escherichia coli (MTCC No. 448),
Pseudomonas aeruginosa (MTCC No. 7436)
and Serratia marcescens (MTCC No. 3124)
while one was Gram positive bacterium
namely, Bacillus cereus (MTCC No. 135).
Total five fungal strains were used viz.,

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Aspergillus niger, Aspergillus flavus,
Aspergillus oryzae, Penicillium chrysogenum
and Trichoderma viridae. The bacterial
cultures were maintained on nutrient agar
medium and the fungal strains were
maintained on Potato Dextrose Agar (PDA)
medium at 4°C.
Antimicrobial activity
The antibacterial and antifungal activities of
the extracts were carried out by agar well
diffusion method as described by Dharajiya et
al., 2014 and Dharajiya et al., 2015a.The
positive control wells were filled with
Gentamicin (10 µg/ml) and Fluconazole (10
mcg/disc) against bacteria and fungi,
respectively. The negative control wells were
filled with DMSO.
Determination of Minimum Inhibitory
Concentration (MIC)
The determination of MIC of different
extracts with respect to different bacteria and
fungi was determined by using the broth
dilution method as explained by Dharajiya et
al., 2014.
Analytical Thin Layer Chromatography
(TLC)
Analytical TLC was performed to identify an
appropriate solvent system to generate the
chromatogram. Various solvent systems were
applied on the pre-coated TLC plates (Merck,
silica gel 60 F254 plate, 0.25 mm) for the
development of the chromatogram.
Among all the solvent systems, chloroform:
methanol: distilled water (50:40:10) was
found best and used for the TLC analysis as
well as TLC bioautography analysis. The
TLC plates were visualized under visible light
for compounds separated followed by the
calculation of Rf values.
TLC Bioautography
The hexane extract of A. barbadensis leaves
was separated on TLC plate and the same
plate was used for the TLC bioautography
against S. marcescens. The TLC plate was
developed using chloroform: methanol:
distilled water (50:40:10) solvent, which
separated components. The same TLC plate
was dried at room temperature for the
complete removal of solvents and placed in
the petri plate followed by over laying of
nutrient agar seeded with an overnight culture
of S. marcescens. The petri plate was
incubated at 37°C for 24 h. After incubation,
an aqueous solution of 5 mg/ml of
methylthiazoletetrazolium (Sigma-Aldrich)
was sprayed on the plate. The clear zone of
inhibition was observed against pink/purple
background and their Rf values were
compared with the reference TLC plate
(Dharajiya et al., 2016).
Qualitative phytochemical analysis
The extracts were tested for the presence of
alkaloids, tannins, saponins, cardiac
glycosides, steroids, phenols and flavonoids
according to the standard protocols for
detecting the presence of different
phytochemicals in the plant extracts as
described by Dharajiya et al., 2012 and
Dharajiya et al., 2015b.
Results and Discussion
The problem of microbial resistance towards
antimicrobial drugs is becoming a major
problem for humankind as it leads to the
death of millions of people (Cohen, 2000).
Most of the world’s population relies on plant
derived traditional medicines for the need of
their primary health care (Duraipandian et al.,
2006). Plants can be a very important source
of newer drugs or antimicrobial compounds
as they exhibit a vast range of

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phytochemicals. Various Aloe species are
found all over the world which are used in
cosmetics, medicine/pharma and food
industry (Park and Jo, 2006). Aloe leaves
contain various chemicals from different
classes which have antimicrobial activity
(Arunkumar and Muthuselvam, 2009). Hence,
the present study was carried out to evaluate
the efficiency of different four extracts as an
antimicrobial agent as well as to access the
presence of phytochemicals in each extract.
Antimicrobial activity
Antimicrobial activity (in terms of the zone of
inhibition) of the extracts was evaluated
against selected pathogenic bacterial and
fungal strains by agar well diffusion method.
In the present investigation, total four extracts
viz., hexane, ethyl acetate, methanol and
aqueous extracts of A. barbadensis leaves
with a concentration of 100 mg/ml were tried.
All the extracts except ethyl acetate showed
antimicrobial activity against different test
microorganisms. The maximum antibacterial
effect of A. barbadensis leaves extracts was
found against S. marcescens [Zone of
inhibition (ZOI) = 13.67±0.57mm] by hexane
extract followed by inhibition of B. cereus
(ZOI = 12.33±0.57 mm) by the methanol
extract. The methanol extract showed
inhibitory effect against all the tested bacterial
strains while ethyl acetate extract failed to
inhibit the growth of any of the bacterial
strains evaluated in the present study. In case
of antifungal activity, the maximum
inhibitory activity was found by aqueous
extract against A. niger with 09.6±0.57mm
zone of inhibition. Out of the four extracts
tested, two extracts viz., hexane and ethyl
acetate failed to express antifungal activity
against any of the fungal strains use in the
study. The methanol extract exhibited slight
inhibitory action against A. oryzae. Out of all
the microorganisms, P. chrysogenum and T.
viridae were found to be resistant to all the
four extracts of A. barbadensis leaves. The
complete findings regarding antimicrobial
activity are represented in Table 1.
The inhibitory activities of A. barbadensis or
Aloe vera leaves against some bacteria viz.,
Aeromonas hydrophius, Aggregatibacter
actinomycetemcomitans, Bacillus sphaericus,
Bacteroides fragilis, Enterococcus faecalis,
Escherichia coli, Klebsiella pneumoniae,
Listeria monocytogenes, Micrococcus luteus,
Morganella morganii, Mycobacterium
smegmatis, Porphyromonas gingivalis,
Proteus mirabilis, Proteus vulgaris,
Pseudomonas aeruginosa, Shigella boydii,
Staphylococcus aureus, Streptococcus
mutans, Streptococcus pyogenes and Vibrio
parahaemolyticus have been evaluated
(Alemdar and Agaoglu, 2009; Arunkumar and
Muthuselvam, 2009; Pandey and Mishra,
2010; Saritha et al., 2010; Fani and Kohanteb,
2012; Nejatzadeh-Barandozi, 2013) in the
recent past.
There are very few reports on antifungal
activity of Aloe sp. which included the
antifungal activity against some fungi viz.,
Alternaria alternata, Aspergillus flavus,
Aspergillus niger, Botrytis gladiolorum,
Candida albicans, Colletotrichum coccodes,
Crytococcus neoformans, Drechslera
hawaiensis, Fusarium oxysporum,
Heterosporium pruneti, Microsporium canis,
Penicillium gladioli, Penicillium maneffei,
Penicillum digitatum, Phythium sp.,
Rhizoctonia solani, Trichophyton
mentagraphytes and Trichophyton schoenleini
(Agarry and Olaleye, 2005; De Rodrıguez et
al., 2005; Rosca-Casian et al., 2007; Alemdar
and Agaoglu, 2009; Khaing, 2011; Sitara et
al., 2011).
Hence, possibly it is the first study showing
antimicrobial activity of A. barbadensis
leaves extracts against two bacteria viz., S.
marcescens and B. cereus as well as four

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fungal strains viz., Aspergillus flavus,
Aspergillus oryzae, Penicillium chrysogenum
and Trichoderma viridae.
Determination of MIC
The MIC values of various extracts with
respect to specific microorganism were
resolute using the broth dilution method as
given in Table 2. All the extracts exhibiting
antimicrobial activity in the agar well
diffusion method were advanced to determine
MIC values. As per the MIC results found in
the present study, the range of MIC of various
extracts was 6.25 to 50.00 mg/ml. In the
present investigation, the lowest MIC value
recorded was 6.25 mg/ml for the hexane
extract against S. marcescens which indicated
maximum power to inhibit the growth of the
specific bacterial strain. The highest MIC
value was 50 mg/ml for methanol and
aqueous extracts against A. oryzae and A.
flavus, respectively.
There are few reports of determination of
MIC of various extracts of Aloe sp. against
different bacterial strains. One of the previous
study indicated that the range of MIC of A.
vera gel was 12.5-50.0 µg/ml against some
periodontopathic and cariogenic bacterial
isolates (Fani and Kohanteb, 2012). Another
report revealed that the range of MIC of A.
barbadensis extract against various
pathogenic bacteria was 0.10-10.0 mg/ml
(Pandey and Mishra, 2010). Ultimately, there
are very few reports of MIC determination for
A. barbadensis leaf extracts against the strains
used in the present study. Hence, present
study can be utilized as a base for the
development of the antimicrobial drugs from
A. barbadensis leaf against some bacteria.
TLC and TLC Bioautography
Total five components from hexane extract of
A. barbadensis leaves were separated by TLC
and their Rf values are given in Table 3. The
same plate was used for the TLC
bioautography against S. marcescens. It
allowed determining the active components of
the hexane extract having antimicrobial
activity against S. marcescens.
Table.1 Antimicrobial activity (Zone of Inhibition) of A. barbadensis leaves extracts
Microorganisms
Name of extract
(Concentration = 100 mg/ml)
Positive control
Negative
control
Hexane
Ethyl
Acetate
Methanol
Aqueous
Gentamicin
(10 µg/ml)
Fluconazole
(10 mcg/disc)
DMSO
Bacteria
S. marcescens
13.67±0.57
-
11.00±1.00
11.67±1.15
19.00±1.00
NA
-
B. cereus
-
-
12.33±0.57
10.83±0.76
15.17±0.76
NA
-
P. aeruginosa
-
-
08.83±0.76
-
15.00±1.00
NA
-
E. coli
-
-
10.33±0.57
09.5±0.50
14.67±1.04
NA
-
Fungi
A. niger
-
-
-
09.6±0.57
NA
16.16±1.04
-
A. flavus
-
-
-
08.1±0.28
NA
21.33±1.15
-
A. oryzae
-
-
08.6±0.57
-
NA
16.00±1.00
-
P. chrysogenum
-
-
-
-
NA
18.66±1.52
-
T. viridae
-
-
-
-
NA
22.33±0.57
-
(-): No zone of inhibition, NA: Not Assessed, DMSO: Dimethyl sulphoxide,The test was done in triplicate,
Diameter of the zone of inhibitions is given here as mean±standard deviation

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Table.2 Minimum Inhibitory Concentration (MIC) values of A. barbadensis leaves extracts
Microorganisms
Name of extract
Hexane
Ethyl Acetate
Methanol
Aqueous
Bacteria
S. marcescens
06.25
NA
12.50
12.50
B. cereus
NA
NA
12.50
25.00
P. aeruginosa
NA
NA
25.00
NA
E. coli
NA
NA
25.00
25.00
Fungi
A. niger
NA
NA
NA
25.00
A. flavus
NA
NA
NA
50.00
A. oryzae
NA
NA
50.00
NA
P. chrysogenum
NA
NA
NA
NA
T. viridae
NA
NA
NA
NA
MIC: Minimum Inhibitory Concentration (mg/ml),
NA: Not Assessed
Table.3 Thin Layer Chromatography (TLC) of hexane extract of A. barbadensis leaves
No. of Compound
Rf value
Band colour in visible light
1
0.35
Dark brown
2
0.41
Brown
3
0.65
Light yellow
4
0.82
Brown
5
0.90
Brown
Table.4 Qualitative phytochemical analysis of A. barbadensis leaves extracts
Name of test
Name of extract
Hexane
Ethyl Acetate
Methanol
Aqueous
Alkaloids
+
-
+
+
Saponins
+
+
+
-
Tannins
-
-
-
+
Sterols
+
+
+
+
Cardiac glycoside
-
-
-
-
Flavanoids
-
-
+
+
Phenol
+
+
+
+
(+): Present, (-): Absence

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Fig.1 Sequential cold maceration method for preparation of plant extracts
Fig.2 TLC bioautography of hexane extract of A. barbadensis leaves against S. marcescens

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2159
The result of TLC bioautography represented
that components separated at Rf 0.65
possessed strong antimicrobial activity,
whereas components with 0.41 and 0.82 Rf
values exhibited moderate antimicrobial
activity against S. marcescens which is
represented as a clear zone of inhibition in
Figure 2. Hence, the components with
specific Rf values and having antimicrobial
activity can be detected and purified for
further specific analysis. The ethanol, acetone
and methanol extracts of A. vera gel were
used for the separation of the active
components possessing antimicrobial activity
(Lawrence et al., 2009). Another study
revealed that the component with 0.8 Rf value
exhibited antimicrobial activity and identified
as aloe-emodin (Nidiry et al., 2011). In the
present investigation, the component with
0.82 Rf value possessed antimicrobial activity
which is indicative of the extraction of aloe-
emodin in the hexane and other extracts
showing antimicrobial activity.
Qualitative phytochemical analysis
The preliminary phytochemical analysis gives
valuable information regarding the presence
of important classes of phytochemicals
present in the extracts. The outcomes of the
qualitative phytochemical analysis of various
extracts of A. barbadensis leaves are given in
Table 4. The results point out to the presence
of some phytochemicals in methanol, aqueous
and hexane extracts as compared to ethyl
acetate extract. It might be the reason behind
no antimicrobial activity of ethyl acetate
extract against the selected microorganisms.
Similar investigations were carried out by
other researchers for the determination of the
class of phytochemicals present in various
extracts of Aloe species (Arunkumar and
Muthuselvam, 2009; Raphael, 2012).
In Conclusion, the current study revealed that
the methanol extract of A. barbadensis leaves
possessed overall more antimicrobial activity
followed by aqueous and hexane extracts,
however hexane extract showed antimicrobial
activity only against S. marcescens but with
maximum zone of inhibition. Various
phytochemicals may play role as
antimicrobial agent which were extracted in
different solvents. These phytochemicals
having antimicrobial activity should be
identified and purified from the crude extracts
by various analytical techniques and can be
implicated in the development of
antimicrobial drugs against various
pathogenic microorganisms.
Acknowledgement
We (authors) express our genuine
appreciations to ARIBAS, New V.V. Nagar
for the financial assistance and providing
facilities to carry out the present investigation.
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How to cite this article:
Darshan Dharajiya, Nalin Pagi, Hitesh Jasani, Payal Patel. 2017. Antimicrobial Activity and
Phytochemical Screening of Aloe vera (Aloe barbadensis Miller). Int.J.Curr.Microbiol.App.Sci.
6(3): 2152-2162. doi: https://doi.org/10.20546/ijcmas.2017.603.246