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Journal of Pharmacy and Alternative Medicine www.iiste.org
ISSN 2222-5668 (Paper) ISSN 2222-4807 (Online)
Vol. 3, No. 3, 2014
36
Research Article
Assessment of antibacterial potential of Saccharum spontaneum Linn. (family:
Poaceae), against different pathogenic microbes- an in vitro study.
Musaddique Hussain* 1, 2, Muhammad Razi Ullah Khan1, Shahid Masood Raza1, Abdul Aziz2, Hazoor
Bakhsh2, Abdul Majeed2, Faiza Mumtaz3
1School of Pharmacy, The University of Faisalabad, Faisalabad, Pakistan.
2Faculty of Pharmacy, Bahauddin Zakariya University, Multan, Pakistan.
3Department of Physiology and Pharmacology, University of Agriculture, Faisalabad, Pakistan
*E-mail of the corresponding author: musaddique.ph@gmail.com
Accepted Date: 3 July 2014
n this study, Saccharum spontaneum (Family: Poaceae), was evaluated for its antibacterial potential against
human pathogenic bacterial strains. In-vitro antibacterial tests were performed by disc diffusion method
on nutrient agar, in order to analyze the percentage zone of inhibition. Whole plant’s extract showed the
significant zone of inhibition (mm), against Staphylococcus aureus (17.00), Streptococcus pneumoniae (16.50),
Bacillus cereus (15.90), Bacillus pumilus (15.45), Escherichia coli (18.00), Klebsiella pneumoniae (17.10),
Pseudomonas aeruginosa (15.20) and Citrobacter freundii (14.00), with relative percentages of inhibition of
76.90, 71.60, 57.40, 56.85, 70.40, 69.90, 61.05 and 54.30 respectively. Modified agar well diffusion method
was used to measure the minimum inhibitory concentration (MIC) and MIC values lies within the range of 75
to 300μg /ml for the G+ve strains while 75 to 600μg /ml for G-ve. Due to presence of tannins and flavonoids,
it inhibits the growth of bacteria on most regulatory levels such as peptidoglycan, DNA, RNA and protein
synthesis.
Keywords: Saccharum spontaneum, Methanolic crude extract, Antibacterial assay, Nutrient agar.
1. INTRODUCTION
Traditional use of medicinal plants and its products
have a long history that began with folk medicine
and through the years has been incorporated into
allopathic medicine (Dubey et al., 2011). Since
antiquity, many plants species reported to have
pharmacological properties as they are known to
possess various secondary metabolites like
flavonoids, glycosides, alkaloids, saponins, steroids,
tannins, tirpenes which is therefore, should be
utilized to combat the disease causing pathogens
(Kamali and Amir, 2010). Side effects and the
resistance against antibiotics, recently much
attention has been paid to extracts and biologically
active compounds isolated from plant species used in
herbal medicine (Essawi and Srour, 2000).
Plant-based antimicrobials represent a vast
untapped source of medicines and further
exploration of plant antimicrobials need to occur.
Antimicrobials of plants origin have enormous
therapeutic potential (Hussain et al., 2011). They are
effective in the treatment of infectious diseases while
simultaneously mitigating many of the side effects
that are often associated with synthetic
antimicrobials.
Due to the increase of resistance to antibiotics, there
is an urgent need to develop new antimicrobial
agents. Among the potential sources of new agents,
plants have long been investigated. Because, they
contain many bioactive compounds that can be of
interest in therapeutic. Because of their low toxicity,
there is a long tradition of using dietary plants in the
treatment of infectious disease in Pakistani folk
medicine.
Saccharum spontaneum Linn (Family: Poaceae), is
referred by multiple Synonyms, i.e., Saccharum
aegyptiacum Wild, Saccharum biflorum Forssk,
Saccharum punctatum Schumach, and is known by
vernacular name of wild sugar cane, false sugar cane,
thatch grass (English), kans, kansi, kans grass (Urdu
and Hindi). It is distributed throughout Asia
(Kiritikar and Basu, 2005). It is a perennial grass,
growing up to three meters in height, with
spreading rhizomatous roots. Leaves are harsh and
I
37
linear, 0.5 to 1 meter long; 6 to 15 mm wide.
Pannicles are white and erect, measuring 15-30 cm
long, with slender and whorled branches, the joints
covered with soft white hair. Spikelet’s are about 3.5
mm long, much shorter than the copious, long, white
hairs at the base (Khare, 2007; Vardhana, 2008).
Phytochemical investigations revealed presence of
quinones, terpenes, alkaloids, flavonoids, saponins,
tannins, carbohydrates, protein, lignin, starch,
polyphenolic compounds, amino acids, coumarin,
phenol, steroids and glycosides (Ghanni, 2003;
Suresh Kumar et al., 2009; Suresh Kumar et al.,
2010).
Aerial parts possess laxative and aphrodisiac
properties, and are useful in burning sensations,
strangury, phthisis, vesical calculi, blood diseases,
biliousness and haemorrhagic diathesis (Chopra et
al., 1956). Roots are sweet, astringent, emollient,
refrigerant, diuretic, lithotriptic, purgative, tonic,
aphrodisiac and useful in treatment of dyspepsia,
burning sensation, piles, sexual weakness,
gynecological troubles, respiratory troubles (Trease
and Evans, 2002; Kiritikar and Basu, 2005). Leaves
are employed for broom (cathartic and diuretics)
(Yoganarashimhan, 2002). The stems (clums) are
useful in dyspepsia, menorrhagia, and general
debility (Yoganarashimhan, 2002; Khare, 2007).
Whole plant is used to treat vomiting, anemia, mental
diseases, abdominal disorders and obesity
(Anonymous, 1996; Kiritikar and Basu, 2005).
Present study was focused to evaluate the
antibacterial activity (In vitro) of methanolic crude
extract of Saccharum spontaneum (Ss.Cr) against
G+ve strains, i.e., Bacillus cereus, Bacillus pumilus,
Staphylococcus aureus, Streptococcus pneumoniae,
G-ve strains, i.e., Pseudomonas aeruginosa,
Escherichia coli, Citrobacter freundii, Klebsiella
pneumoniae.
2. MATERIAL AND METHODS
2.1 Plant collection and extraction:
Fresh plants of Saccharum spontaneum (Linn.) were
collected from surrounding areas of T. P. Link Canal,
near Kot Addu (Pakistan) during the month of
May-June, 2014. The identity of the plant was
confirmed by using all official monograph (Kiritikar
and Basu, 2005; Khare, 2007). Plant material was
dried under shade for 20 days and grinded into
coarse powdered material (# 40) by an electrical
grinder.
Triple maceration process was adopted for
extraction by macerating coarse powdered material
with 70% aqueous-methanol in air tight amber glass
bottles at 27oC, with occasional shaking thrice a day
for one week (Harborne, 1973). After maceration,
the soaked coarse powdered material was passed
through muslin cloth (double layered), in order to
remove vegetative debris and the obtained filtrate
was subsequently filtered through a Whatman-1
filter paper. The filtrate was stored in amber glass
air-tight container. The previously mentioned
extraction procedure was subsequently repeated
twice after each two days and filtrates of these three
macerations were combined. Rotary evaporator
(Rotavapor, BUCHI labrotechnik AG, Model 9230,
Switzerland) attached with a vacuum pump and a
recirculation chiller was used for evaporation of the
filtrate, under reduced pressure at 37°C to a thick,
semi-solid paste. The dark green crude extract was
lyophilized to remove moisture contents. The dried
extract was transferred to amber glass jar and stored
at −4oC in a refrigerator.
2.2 Test organisms and standard drugs (discs) used
All standard drug discs (gentamicin, flucloxacillin,
vancomycin, ciprofloxacin, ceftriaxone, levofloxacin)
having drug conc. of 20μg/disc (Oxobid Ltd.
Basingstoke, Hampshire, England) were purchased
from Al-Mujhaid Scientific shop, Faisalabad, Pakistan.
Whereas, all the test organisms (Bacillus cereus,
Bacillus pumilus, Staphylococcus aureus,
Streptococcus pneumoniae, Pseudomonas
aeruginosa, Escherichia coli, Citrobacter freundii,
Klebsiella pneumoniae) were collected from the
pathology lab of Faisalabad Institute of Cardiology
(FIC), Faisalabad, Pakistan. All microbes were
cultured overnight in a nutrient agar (pH 5)
containing peptone (0.5%), agar (1.2%), yeast
(0.3%), and NaCl (0.8%) (Cruiskshank et al., 1975).
2.3 In-vitro antimicrobial potential assessment:
In-vitro antimicrobial assay was performed by
adopting the standard disc diffusion method (Taylor
et al., 1995). Three different types of discs were used,
i.e., standard discs (gentamicin; inhibiting the
bacterial protein synthesis, flucloxacillin, ceftriaxone
and vancomycin; inhibiting the bacterial cell wall
biosynthesis; levofloxacin and ciprofloxacin,
inhibiting the DNA synthesis) as positive control,
crude extract discs (sample discs), and discs
containing the DMSO (negative control). All the discs
have diameter of 6 mm. Glass wares and prepared
nutrient agar media, were sterilized in autoclave at
121oC for 25 minutes. Agar plates were prepared
with thickness of gels layer ranging between 2-3 mm.
The petri-dishes were incubated overnight at 37oC
38
and those showing no growth were selected for
further work. Streak plate method was adopted for
the inoculation of bacterial culture on agar plates.
Bacterial cultures were incubated at 37oC in
incubator for 12-14 hours. Experiments were
carried out in triplicate. At the end of the incubation
period, zone of growth inhibition around the discs
(mean value n=3) was measured in comparison with
the positive and negative control (Khyade and Vaikos,
2011). Modified agar well diffusion method was
used to determine the MIC of the Ss.Cr by using
concentration range of 75, 150, 300, 600, 1200,
5000 and 10000μg/ml (Tagg et al., 1976; Ajay et al.,
2002).
2.3.1 Relative percentage inhibition
The relative percentage inhibition of the crude
extract with respect to positive control was
calculated by using the following formula (Ajay et al.,
2002),
Relative percentage inhibition= 100 x (a – b)
(c - b)
Where a, b and c, represent the total area of
inhibition of the test extract, solvent and standard
drugs respectively.
The total area of the inhibition was calculated by
using
Area of inhibitory zone = πr2
Where r is radius of zone of inhibition
2.4 Statistical analysis
The results were expressed as mean ± SEM of
triplicate samples. Statistically significant differences
between groups were measured using one-way
analysis of variance (ANOVA). Results were analyzed
statically by using “Graph pad Prism” version 6,
(Graph Pad Software, San Diego, CA, USA).
3. RESULTS
Methanolic crude extract of Saccharum spontaneum
(Ss.Cr) at the dose range of 150mg/ml showed the
zone of growth inhibition (mm), (including 6 mm
disc) of 17.00 against S. aureus, 16.50 against S.
pneumoniae, 15.90 against B. cereus, 15.45 against B.
pumilus, 18.00 against E. coli, 17.10 against K.
pneumoniae, 15.20 against P. aeruginosa and 14.00
against C. freundii, as compared with standard drugs
(mm) flucloxacillin (20.00), ceftriaxone (19.50),
ciprofloxacin (21.00), vancomycin (20.50),
ceftriaxone (21.45), levofloxacin (20.50), gentamicin
(19.45) and ciprofloxacin (19.00) with relative
percentages of inhibition 76.90, 71.60, 57.40, 56.85,
70.40, 69.90, 61.05 and 54.30 respectively and MIC
values of Ss.Cr are depicted in table 2. After statistical
analysis, P value was determined which was
significant, i.e., less than 0.05 (P < 0.05).
Table 1: Zone of inhibition (mm) of sample (Ss.Cr), positive control (standard drug discs) and negative control
(DMSO) against different bacterial species (mean ± SEM., n = 3).
Bacterial Strains
Zone of Inhibition (mm/sensitive strain)
*Sample
*Positive Control
Negative Control
S. aureus
17.00
Flucloxacillin
20.00
NR
S. pneumoniae
16.50
Ceftriaxone
19.50
NR
B. cereus
15.90
Ciprofloxacin
21.00
NR
B. pumilus
15.45
Vancomycin
20.50
NR
E. coli
18.00
Ceftriaxone
21.45
NR
K. pneumoniae
17.10
Levofloxacin
20.50
NR
P. aeruginosa
15.20
Gentamicin
19.45
NR
C. freundii
14.00
Ciprofloxacin
19.00
NR
* = diameter of the zone of inhibition including diameter of 6mm disc, Sample = Ss.Cr; Positive control =
Standard drugs; -ve control = DMSO; NR = No response
39
Table 2: Relative percentage inhibition and MIC of Ss.Cr against different bacterial species (values are
expressed as mean ± SEM., n = 3).
Test Bacteria
RPI (%)
MIC(μg/ml)
S. aureus
76.90
75
S. pneumoniae
71.60
150
B. cereus
57.40
300
B. pumilus
56.85
300
E. coli
70.40
75
K. pneumoniae
69.90
150
P. aeruginosa
61.05
300
C. freundii
54.30
600
Figure 1: Zone of inhibition of the crude extract of
Saccharum spontaneum (Ss.Cr) in diameter (mm)
against different bacterial species (values are
expressed as mean ± SEM., n = 3).
Figure 2: Relative percentage inhibition of crude
extracts of Saccharum spontaneum (Ss.Cr) against
different bacterial species.
4. DISCUSSION
The researchers are trying their best to develop new
natural products from medicinal plants against
multidrug resistant microbial strains because multi
drug resistance is the major hurdle of this era, which
is leading toward mortality and morbidity (Braga et
al., 2005). Medicinal plants are the major source of
the secondary metabolites which have been
reported to possess the antimicrobial property
(Hussain et al., 2013). In vitro evaluation of the
plants for the antimicrobial property is the first step
toward achieving the goal for developing
eco-friendly management of the infectious diseases
(Nushad, 2012).
Considering these, Saccharum spontaneum (Ss.Cr)
was screened in vitro for its antibacterial activity
against human pathogenic bacteria. On the basis of
the results of the present study it may be revealed
that extract of Saccharum spontaneum (Ss.Cr)
possess activity against Gram+ve and Gram –ve
bacteria. In general Gram+ve bacteria are
considered more sensitive than Gram –ve bacteria
toward different antimicrobial compounds because
of the difference of cell wall structure of both
(Veeramuthu et al., 2006; Khan et al., 2010) but
methanolic crude extract of Saccharum spontaneum
(Ss.Cr) showed the higher inhibition against S.
aureus (76.90%), S. pneumoniae (71.60%), and E.
coli (70.40%), supporting the view, that medicinal
40
plants might be useful in the development of novel
antibacterial agents (Heinrich and Simon, 2001).
In-vitro results of this plant appear as interesting and
promising and may be effective as potential source
of novel antibacterial drug.
5. Conclusion
Saccharum spontaneum is believed to possess the
antibacterial activity due to presence of tannin,
alkaloids saponins and flavonoids, which have been
studied (Ghanni, 2003; Suresh Kumar et al., 2009;
Suresh Kumar et al., 2010). Tannin and flavonoids
are the potent antioxidant and free radical scavenger
which prevent oxidative cell damage and also have
strong antimicrobial activities (Trease and Evans,
1983; Okwu, 2004; Nushad, 2012). Hence these
compounds may be responsible for the antimicrobial
activity of the plant. Further research is necessary
to determine the identity of the therapeutic
compound within this plant and also to determine
their full spectrum of efficacy. However, the present
study may serve as the primary platform for the
further in-vivo studies.
Conflict of Interests
Authors declared no competitive interests for the
presented work.
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