Pak. J. Bot., 47(4): 1587-1592, 2015.

ANTIMICROBIAL ACTIVITY OF NERIUM OLEANDER L. AND

NICOTIANA TABACUM L.: A COMPARATIVE STUDY
ROHMA MALIK1, TASVEER ZAHRA BOKHARI1, MUHAMMAD FAHEEM SIDDIQUI2,
UZMA YOUNIS1, MUHAMMAD ISHTIAQ HUSSAIN3 AND IMRAN AHMED KHAN4
1
Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan, Pakistan
2
Department of Botany, University of Karachi. Karachi, 75270
3
Centre for Plant Conservation, University of Karachi, Karachi 75270, Pakistan
4
Department of Geography, University of Karachi, Karachi 75270, Pakistan.
Corresponding author’s e-mail: mfsiddiqui2011@yahoo.com: mfsiddiqui@uok.edu.pk
Abstract

The antimicrobial activity of aqueous ethanolic extract of two plant species i.e. Nerium oleander L. and Nicotiana tabacum
L. from Multan district were checked, against three pathogenic bacteria viz: Staphylococcus aureus (gram positive),
Escherichia coli and Pseudomonas aeruginosa; (gram negative). Disc diffusion technique was used to check the antimicrobial
activity. The bacterial strains under question were found susceptible to plant extracts. Nerium oleander and Nicotiana tabacum
exhibited strong antibacterial action against tested bacteria. The ethanolic extract of Nerium oleander leaves showed highest
antibacterial action against Pseudomonas aeruginosa at 900mg/ml concentration, whereas, Nicotiana tabacum showed
maximum zones of inhibition against Staphylococcus aureus at 900mg/ml concentration. The results showed that the leaves
extracts of these plants have great power as anti-microbial agents against these common bacterial isolates. However, for future
pharmacological implication of these plants more research is needed for the isolation of active ingredient of these plants. The
results obtained could be attributed to the differential environmental conditions on the ecology of these plants.

Key words: Antimicrobial effect, Nerium oleander, Nicotiana tabacum, Pseudomonas aeruginosa, Staphylococcus
aureus and Escherichia coli.

Introduction of the plants that retard the growth of micro-organisms

have been investigated in different laboratories around the
Environmental circumstances play a vital role in world since 1926 (Bakht et al., 2012).
defining the function and floristic composition of plants. The plants are selected for study due to their
From the beginning of human civilization plant and plant following contrasting characteristics (Table 1).
products are usually used to treat different diseases (Joshi et
al., 2009). As plants have substances of medicinal values, Materials and Methods
therefore, they are used to treat number of diseases since
long time. Use of plants had minimal or less side effect on The study was carried out during October 2012-2013.
human beings (Doughari, 2006). With the passage of time In this study leaves of two different plants Nerium oleander
the usage of plants is increasing in pharmaceutical L. (Apocynaceae), Nicotiana tabacum L. (Solanaceae)
industries, suggested that if a plant is used as remedy of were used. The disc diffusion technique (Anon., 2002) was
disease then it would have some important ingredients used to evaluate the antibacterial activity. For antibacterial
(Nostro et al., 2000). Researchers have great interest in sensitivity extracts of these leaves were taken and
those substances which are derived from plants because processed. Experimental work was categorized as
they are versatile in their applications. Various following: 1) Collection of plant material, 2) Preparation of
phytochemicals can be obtained from plants which are very aqueous ethanolic leaves extracts and 3) Determination of
beneficial for mankind and medicinal plants have become Zone of Inhibition (Fig. 1). The leaves of both plants
(Nicotiana tabacum L. and Nerium oleander L.) were
the richest biological resource of such chemicals which are
collected from local field of Multan, Pakistan. The leaves
used in manufacturing of traditional drugs as well as in
were thoroughly washed and air dried under shade for
modern nutraceuticals, food supplements, medicines, folk about 15-20 days. The dried leaves were then grinded in a
medicines, raw material and pharmaceutical intermediates herbal grinder into a coarse form. Before extraction the
for synthetic drugs (Tumwine, 2011). powder was stored at room temperature in air tight
Antimicrobial compounds are a group of chemical polythene bags. The extract was prepared and crude extract
compounds which are synthetically or biosynthetically was stored in dried and air tight Petri dishes (Pyrex),
produced which either destroy or usefully suppress the labeled and stored in refrigerator for further study (Lavanya
growth and metabolism of variety of microorganisms & Brahmaprakash, 2011), then incubated for overnight at
(Lavanya & Brahmaprakash, 2011). Pharmacologically 37oC.The bioassay used was the standard disk diffusion
important plants present a large source of antimicrobial assay adapted from (Parekh & Sumitra, 2007). By dipping
agents and serve as a drug in many countries (Mahesh & and saturating commercially sterilized discs in plant extract
Satish, 2008). This antimicrobial activity of plant is due to test discs were prepared. Same sized sterilized disks
their constituents of oils and extracts which are used in absorbed the same volume of the extract. Aqueous ethanol
pharmaceutical and others (Hammer et al., 1999). Globally, paper discs were used for control, which prepared by
researchers are using extracts of plants for their antiviral, dipping the disks into aqueous ethanol solution
antibacterial, and antifungal activities. The characteristics (Sermakkani & Thangapandian, 2010).
1588 ROHMA MALIK ET AL.,

Collection

Drying

Grinding Soaking

Filtering
Fig. 1. Pictures showing methodology of experiment.
ANTIMICROBIAL ACTIVITY OF NERIUM OLEANDER & NICOTIANA TABACUM 1589

Table 1. Characteristics of selected plants species (Nerium oleander &Nicotiana tabacum) for antimicrobial assay.
Sr. Part Chemical
Plants Family Medicine Uses References
No. used Constituents

Folk medicine, Used in suicide Haynes et al., 1985; Edward et al.,

Nerium Leaves Cardiac glycosides, 1993; Nostro et al., 2000; Doughari,
Apocynaceae An oral anticancer drug equipment’
1. oleander extract Neriin & Oleandrin 2006; Joshi et al., 2009 and
known as Anvirzel insecticidal properties Lavanya & Brahmaprakash, 2011.

Raw material for

Nicotiana Leaves Insecticide, Denduangboripant et al., 2005
Solanaceae Nicotine cigarette industry,
2. tabacum extract pesticide and Jude, 2013
Smoking

Observation of results: On the basis of absence or minimum diameter (18mm) for zone of inhibition was
presence of zone of inhibition results were measured. observed. A slight difference in antimicrobial activity
Zone reader was used to measure zone of inhibition in 24mm, 23mm and 22mm was observed at 800-
mm (Ahmad & Arina, 2011). 600mg/ml dilutions respectively.

Antimicrobial assay of plant extract: Three species of Antibacterial effect of Nicotiana tabacum: Fig. 3(D);
bacteria (Escherichia coli, Staphylococcus aureus & indicated that Staphylococcus aureus had highest
Pseudomonas aeruginosa) were used for antimicrobial diameter for zone of inhibition (26mm) at highest plant
assay. For further tests nutrient agar broth, nutrient agar extract (900mg/ml) concentration while at lowest extract
suspension and Hi-sensitivity test agar were prepared by concentration (500mg/ml) the minimum diameter (15mm)
following manufacture’s instruction. The commercially for zone of inhibition was observed. A gradual decline in
sterilized discs (HIMEDIA, Mumbai) were used by diameter of zone of inhibition i.e. 23, 20 and 17mm was
incorporating 20µl of plant extract using micropipette and observed at 800, 700 and 600mg/ml dilutions
then discs were kept into sterile petri plates (Pyrex) and respectively. Fig. 3(E); data indicated that Escherichia
coli had highest diameter for zone of inhibition (20mm) at
incubated for overnight for further use after checking
highest 900mg/ml concentration while at lowest level of
sterility. A swab dipped in standard inoculum was used
extract 500mg/ml concentration the minimum diameter
for the transformation of bacteria of petri dish with
13mm for zone of inhibition was observed. A slight
nutrient agar. These were
difference in antimicrobial activity 19mm was observed at
Results 800mg/ml. A gradual decline in diameter of zone of
inhibition 17mm and 14mm was observed at 700-
Antibacterial effect of Nerium oleander: The plant 600mg/ml dilutions respectively. Fig. 3(F); data indicated
extracts of both the species are applied on three tested that Pseudomonas aeruginosa had highest diameter for
bacteria, different zone of inhibitions are exposed in zone of inhibition (21mm) at highest 900mg/ml
(Fig. 2). Escherichia coli had highest diameter for zone concentration. While at lowest level of extract 500mg/ml
of inhibition (24mm) at 900mg/ml concentration of concentration the minimum diameter (11mm) for zone of
Nerium oleander extract while at 500mg/ml inhibition was observed. A gradual decline in diameter of
concentration showed the minimum diameter (15mm) of zone of inhibition 18mm, 16mm and 13mm was observed
zone of inhibition (Fig. 3(A). A decline in diameter of at 800-600mg/ml dilutions respectively.
zone of inhibition 22mm was observed at 800mg/ml, Plants contain many biologically active compounds
which have potential as medicinal agents (El-Mahmood
then zone of inhibition showed gradual decline 20mm
et al., 2010). The main theme of the present research
and 17mm with decreasing dilution of the extract from
work was to analyze anti-bacterial actions of Nerium
700-600mg/ml concentrations. The mean values of oleander and Nicotiana tabacum. The bacterial species
antibacterial activity (Zones of inhibition in mm) of had shown different susceptibilities for different
Staphylococcus aureus under different concentration concentration i.e. (900, 800, 700, 600 and 500mg/ml) of
(900mg/ml, 800mg/ml, 700mg/ml, 600mg/ml and ethanolic extracts of leaves of these two species as also
500mg/ml) of Nerium oleander leaf extract is observed by Garima et al. (2011). The results showed
represented in Fig. 3(B). The data indicated that that both plants are affected against all tested
Staphylococcus aureus had highest diameter for zone of organisms/bacteria. The effects against gram-negative
inhibition (22mm) at highest 900mg/ml concentration bacteria were more dominant than those gram-positive
while at lowest level of extract 500mg/ml concentration but N. tabacum showed maximum results against
the minimum diameter (13mm) for zone of inhibition Staphylococcus aureus at maximum concentration
was observed. A slight difference in antimicrobial (900mg/ml) and zone of inhibition was 26mm.
activity 19mm and 18mm was observed at 800- In most of the cases the leaves of the plants have
700mg/ml, then zone of inhibition showed gradual reported to contain more phytoconstituents than any other
decline (15mm) with decreasing dilution of the extract part of the plant. The occurrence of metabolites in these
600mg/ml concentration. Fig. 3(C); indicated that plants is more concentrated that’s why this indicated that
Pseudomonas aeruginosa had highest diameter for zone they contain more active metabolites. So this was the
of inhibition (28mm) at 900mg/ml concentration while strong reason to choose the leaves of these species
at lowest level of extract 500mg/ml concentration the (Kawalekar, 2012).
1590 ROHMA MALIK ET AL.,

Fig. 2. Nerium oleander and Nicotiana tabacum plates showing zones of inhibition againt various bactreial strains.

Discussion lowest concentration (500mg/ml) of extract showed the

minimum diameter (18mm) for zone of inhibition was
The disc-diffusion method was mainly used for the observed against Pseudomonas aeruginosa. While at the
determination of antibacterial action. Among the different concentration of (900, 500mg/ml) N. oleander showed
methods, disk diffusion has been used more frequently to different zones of inhibition which were 22mm and 13mm
examine the antimicrobial activity of natural respectively against Staphylococcus aureus as also
antimicrobials (Kim & Kim, 2007; Mayachiew et al., observed by Wong et al. (2013).
2010). The antibacterial activities monitored by different In case of N. tabacum, the highest diameter for zone
concentration of extracts to investigate that which of inhibition was found at (26mm) at highest 900mg/ml
concentration showed maximum antimicrobial activity. concentration, while at lowest level of extract 500mg/ml
Simultaneously increasing the concentrations of extracts concentration the minimum diameter (15mm) for zone of
and notifies the trend in activity. The antibacterial inhibition was observed against Staphylococcus aureus.
activities of both the plants were checked on all three At the concentration of 900mg/ml and 500mg/ml N.
bacterial strains. The two plant species showed the tabacum showed different zones of inhibition which were
highest values for zone of inhibition against Pseudomonas 21mm and 11mm respectively against Pseudomonas
aeruginosa and Staphylococcus aureus. Among these, the aeruginosa. Ethanolic extract of Nerium oleander showed
least activity was shown by N. tabacum against the maximum diameters for zone of inhibition against
Escherichia coli. The results showed that Nerium three bacterial species. The highest activity of this plant
oleander had biggest diameter for zone of inhibition might be due the presence of terpenoids, saponins and
(28mm) at highest (900mg/ml) concentration, while at amino acids.
ANTIMICROBIAL ACTIVITY OF NERIUM OLEANDER & NICOTIANA TABACUM 1591

A D

B E

C F

Fig. 3. A, B, C, D, E & F showing different zones of inhibition against Escherichia coli, Staphylococcus aureus and Pseudomonas
aeruginosa for both plants (Nicotiana tabacum L. and Nerium oleander L.)

Ethanolic extract of N. tabacum showed the According to the Jude (2013) Nerium oleander,
maximum diameters for zone of inhibition (26mm) Lippia nodiflora, Wattakaka volubilis and Weinmannia
against Staphylococcus aureus while there was a less tinctoria were possessed the highest inhibitiory zone
difference against Escherichia coli and Pseudomonas against E. coli, K. pneumoniae, S. typhi, P. vulgaris and
aeruginosa which were 20mm and 21mm respectively. P. mirabilis. Methanol extract of Nerium oleander
exhibited the highest activity (28 mm) against S. typhi
The phytoconstituents of this specie were mainly
than standard antibiotics around 22 mm. According to the
saponins, flavoids and anthraquinones. While amino report of Jeyachandran et al. (2010) the methanolic
acids were absent in N. tabacum. extract of Nerium oleander had maximum zone of
1592 ROHMA MALIK ET AL.,

inhibition (28mm) against S. typhi. Results in the present Jeyachandran, R., W. Baskaran and L. Cindrella. 2010.
investigation showed the similar zone of inhibition of Screening of phytochemical and antibacterial potential of
ethanolic extract N. oleander against Pseudomonas four Indian medicinal plants. Libyan Agriculture Research
aeruginosa. Center Journal International. 1(5): 301-306.
Joshi, B., L. Sunil and S. Anuja. 2009. Antibacterial property of
different medicinal plants: Ocimum sanctum, Cinnamomum
Conclusion zeylanicum, Xanthoxylum armatum and Origanum
majorana. Kathmandu University Journal of Science,
1. These plant species have antimicrobial potential Engineering and Technology, 5(1): 143-150.
against common pathogenic bacterial strains. Jude, C.A. 2013. Extraction, Characterization and industrial
2. The maximum concentration 900mg/ml is found to applications of Tobacco Seed Oil (Nicotiana tabacum).
be most efficient against all bacterial strains. Chemistry and Materials Research, 3(2): 19-21.
3. So, we can use this level of plant extracts as a source Kawalekar, J.S., P. Varsha and N. Vijayalakshmi. 2012.
of natural medicine against these bacteria. Preliminary phytochemical investigations on Roots of
Nerium Oleander, Linn. Inter. J. Pharmacognosy and
Phytochemical Res., 4(3): 134-138.
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(Received for publication 5 May 2014)