In Vitro Antibacterial Activity of Leaf
Extracts of Hibiscus Syriacus (L)
Rakesh Punasiya1* and Sujit Pillai2
1Department of Pharmaceutical
Biotechnology, GRY Institute of Pharmacy, Borawan,
Khargone-451228, India
2Department of Pharmaceutical Chemistry,
GRY Institute of Pharmacy, Borawan, Khargone, MP
*Corresponding Author E-mail: rakeshpunasiya@yahoo.com
ABSTRACT:
The
antimicrobial activity of different crude extract like petroleum ether,
benzene, chloroform, methanol, and aqueous of Hibiscus syriacus L. plants used in
traditional Indian medicine was tested by well diffusion method against four
bacterial pathogens: Bacillus
cereus
(MTCC-430), Staphylococcus epidermis (MTCC-435), Klebsiella
pneumonia
(MTCC-432), and
Bacillus subtilis
(MTCC 121) bacteria. The minimum inhibitory concentration (MIC) was determined
for evaluating the potential leaves extract. The antibacterial activity of
methanol extracts (0.5 g/ml) of Hibiscus syriacus L. showed maximum zone of inhibition (19.1 mm,
19.6 mm, 22.9 mm and 25.4 mm, respectively) against Bacillus cereus (MTCC-430), Staphylococcus epidermis (MTCC-435), Klebsiella
pneumonia
(MTCC-432), and
Bacillus subtilis
(MTCC 121) bacteria. MIC was tested at various concentrations from 0.625 mg/ml
to 100 mg/ml for all the leaves extracts. The Klebsiella
spp was the most resistant strain and various
concentrations of all the leaves extract benzene extract showed less activity
against the tested microorganism. The present screening result demonstrated
that the methanol leaves extract of plants Hibiscus
syriacus L. has potent antibacterial activity and
the studied plants may be new source for novel antibacterial.
KEYWORDS: Antibacterial activity, Well diffusion method, Zone of
inhibition, Pathogens
INTRODUCTION:
In the recent times, focus on the research
has all over the world and a large body of evidence has been collected to show
the immense potential of medicinal plants used in traditional system. Various
medicinal plants have been studied using modern scientific approaches, and the
results have revealed the potential of medicinal plants. [1] According
to World Health Organization, medicinal plants would be the best source to
obtain a variety of drugs. Therefore, such plants should be investigated to
better understand their properties, safety and efficacy. [2]
Now a
day, there have been increases in antibiotic resistant strains of clinically
important pathogens, which have led to the emergence of new bacterial strains
that are multi-resistant. The non-availability and high cost of new generation
antibiotics with limited effective span have resulted in increase in morbidity
and mortality. Therefore, there is a need to look for substances from other
sources with proven antimicrobial activity. [3] Plant compounds are of interest
as a source of safer or more effective substitutes than synthetically produced
antimicrobial agents. Phytochemical progress has been aided enormously by the
development of rapid and accurate methods of screening plants for particular chemicals.
[4] The treatment of infectious diseases is facing a major problem at present
with many microbes developing resistance to widely used antibiotics and
antiviral therapies. [5] One way to prevent antibiotic resistance of pathogenic
species is by using new compounds that are not based on existing synthetic
antimicrobial agents. [6] Within the last few decades, there has been the emergence of about
30 threatening infectious diseases with the majority capable of affecting
humans. [7]
The
genus Hibiscus is widely distributed
over Korea, china, India, and Siberia. The Hibiscus
syriacus L. (Malveceae)
are used as a fork medicine in the orient [8] for the cure of hematochezia, dysentery, obstruction due to wind phlegm,
and vomiting of food. [9] Hibiscus syriacus L. (Rose-of-Sharon) is valued for large
flowers produced in summer when few other shrubs bloom. It is useful as a
garden accent due to its strict, upright habit. The open, loose branches and
light green leaves make Rose-of-Sharon ideally suited to formal or informal
plantings, and with a little pruning makes an attractive, small specimen tree.
The plant grows in sun or partial shade and in any soil. Rose-of-Sharon
grows 8 to
Hibiscus rosasinensis Linn. is commonly known as
China rose belongs to family Malvaceae. The flowers
are considered emollient and demulcent, soacked in
coconut water are given to induce labor. Decoction of flower is given in
bronchial catarrh. Leaves are emollient, aperient,
anodyne and laxative. Roots are demulcent and used for coughs. Decoction of
roots is used for venereal diseases and as antipyretic. [13] Hibiscus anthocyanins, groups of phenolic
natural pigments present in the dried flower of Hibiscus sabdariffa
and Hibiscus rosasinensis have been found to have cardioprotective, hypocholesterolemic,
antioxidative and heaptoprotective
effects in animals. [14] The antibacterial activity in vitro as well as
the wound-healing activity of the ethanol extracts of H. rosa-sinensis
flowers in vivo. Flowers contain anthocyanins,
which may be responsible for its antioxidant effects. Bacterial infection play
important role in many of the pathological conditions where extracts of H. rosa-sinensis extracts have been used as traditional
medication. [15]
MATERIALS AND
METHODS:
Selection of plant
Drug discovery from medicinal plants has evolved to include numerous
fields of inquiry and various methods of analysis. The process typically begins
with a botanist ethanopharmacologist who identifies
the plant of interest. Collection may involve species with known biological activity.On the basis of intensive literature survey Hibiscus
Syriacus were
selecting for present study.
Collection of plant materials
The
leaves of Hibiscus syriacus L. was collected from the month of
august-September from the Garden of Jawaherlal
Institute of Technology and G. R. Y. Institute of Pharmacy vidya
vihar Borawan district Khargone western nimar region of
Madhya Pradesh, India.
Authentication of plant
The
plant Hibiscus syriacus L. was identified and authenticated by
Dr S.K. Mahajan, (Retd)
Botanist from Government
College, Khargone Madhaya
Pradesh. The
herbarium of the plant specimens were prepared and deposited in the Department
of Pharmacognosy, G. R. Y. Institute of Pharmacy vidya
vihar Borawan district Khargone madhaya Pradesh, India,
under voucher no. G.R.Y.I.P. 43.
Preparation of extract
Extraction of organic component
The leaves
were initially separated from the main plants body and rinsed with distilled
water and shade dried and then homogenized into fine powder and stored in air
tight bottles. It was then passed through the 40 mesh sieve Dried and powered
plant defatted firstly to remove fatty material for this purpose 1000 gram of
weighed powered leaves of Hibiscus syriacus L. was packed in Soxhlet and extracted with
petroleum ether at 60-80°c for 36 hours. The marc was removed and dried then it
was subjected to continuous hot extraction with organic solvent like benzene,
chloroform, and methanol respectively extracts in soxhlet
for 36 hours according to their polarity index. After complete extraction the
solvent was evaporated and concentrated to dry residue. The petroleum ether,
benzene, chloroform, and methanolic extract of Hibiscus
syriacus L. leaves yielded greenish brown and deep blue semi solid
residue then it were filtered with the help
of muslin cloth. The supernatant was collected and the solvent was evaporated
by distillation and the extract was concentrate under reduce pressure.
Extraction of aqueous component
Dried
leaf material was extracted with 2000 ml of water by boiling the mixture. The
sample was then filtered through Whatman No. 1 filter paper and concentrated to
dryness under reduced pressure. Crude extracts were then stored in the dark at
10oC until use. The percentage yield for each extract was
determined. [16]
Preliminary phytochemical
screening
The
methods described by Harborne (1978) with slight
modifications were used to test for the presence of the active ingredients in
the test sample. The plant materials were screened for the presence of
different classes of secondary metabolites including alkaloids, flavonoids, phenols, saponins,
tannins, anthocyanins, anthraquinones,
sterols, and triterpenes using previously
described methods. [17]
Tested
microorganisms
Two
Gram-positive bacteria (Bacillus
cereus
(MTCC-430), Bacillus subtilis (MTCC 121), two Gram-Negative organisms (Staphylococcus epidermis
(MTCC-435), Klebsiella pneumoniae (MTCC-432) were included. The
microorganisms were kindly provided by the Microbial type culture collection Chandigard India. The microorganisms were cultivated on
nutrient agar medium and stored at 4oC.
Antimicrobial activity by agar
well-diffusion method
500
mg of each extract of Hibiscus syriacus L was
dissolved in 1ml of each solvent. Suspension of micro-organisms was made in
sterile normal saline and adjusted. Culture media was prepared using nutrient
agar and were autoclaved at 121o, 15 psi for 15 minutes. A volume of 20 ml agar
was transfer on Petridis and allowed to solidify. Each labelled
medium plate was uniformly inoculated with a test organism by using a sterile
cotton swab rolled in the suspension to streak the plate surface in a form that
lawn growth can be observed. Each Petridis were divided into four sectors and
in each sector 4 mm bore was made using sterile borer. 0.1ml of the various
extract concentration were dropped into each, appropriate labeled well. Other
solvents used for extraction apart from water were tested neat for each
organism. The inoculated plates were kept in the refrigerator for 1 hour to
allow the extracts to diffuse into the agar. [18] The Nutrient Agar plates were
incubated at 37OC for 24 hours. Antimicrobial activity was
determined by measuring the diameter of zones of inhibition (mm) produced after
incubation. O.O5% of chloramphenicol was used as control. [19]
Minimum inhibitory concentration
Determination
of MIC and MBC
The
minimum inhibitory concentration (MIC) of the crude extracts was also
determined by comparing the various concentrations of plant extracts, which
have different inhibitory effect, and selecting the lowest concentration of
extract showing inhibition. After incubating at 24 h at 37°C, the MIC of each
sample was determined by measuring the optical density in the spectrophotometer
(620 nm), and comparing the result with those of the non inoculated nutrient
agar. All the samples were prepared in
triplicates. [20]
Data Analysis
Each
experiment of the antimicrobial activity was assessed with six serial dilutions
for each compound and then replicated three times. Results were expressed as
mean ±SE and statistically analyzed using an analysis of variance (ANOVA).
Differences are considered significant when P0.05.
RESULT:
Preliminary
phytochemical screening
Our preliminary phytochemical
screening of H.syriacus L. leaves (table 1) revealed the
presence of carbohydrate, glycosides, steroids, triterpenes,
flavonoids, alkaloid, tannins, and saponins.
Table
1 Phytochemical screening of leaves extract of Hibiscus syriacus L.
S.No. |
Phytochemical |
Pet.
Ether |
Benzene |
Chloroform |
Methanol |
Water |
1 |
Carbohydrates |
- |
+ |
+ |
+ |
+ |
2 |
glycosides |
- |
- |
- |
+ |
+ |
3 |
Steroids |
+ |
+ |
+ |
- |
- |
4 |
Proteins |
- |
- |
+ |
+ |
- |
5 |
Flavonoids |
- |
- |
- |
+ |
+ |
6 |
Tannins |
+ |
+ |
+ |
+ |
+ |
7 |
Alkaloids |
- |
- |
- |
- |
+ |
Table2. Antibacterial
activity of different leaves extract by disc diffusion method.
Bacterial sp. |
Zone of Inhibition in mm of leaves extract of Hibiscus syriacus |
|||||
Petroleum ether extract |
Benzene extract |
Chloroform extract |
Methanol extract |
Water extract |
Chloramphenicol (std Drug) |
|
Bacillus cereus (MTCC-430) |
08.1 |
07.2 |
10.1 |
19.6 |
17.1 |
27.0 |
Bacillus subtilis (MTCC-121) |
10.3 |
9.8 |
10.6 |
25.4 |
21.8 |
35.2 |
Staphylococcus epidermis (MTCC-435) |
09.3 |
8.6 |
10.4 |
22.9 |
20.9 |
31.5 |
Klebsiella pneumonia (MTCC-432) |
08.0 |
7.4 |
9.2 |
19.1 |
16.4 |
22.4 |
Antibacterial Activity of the Plant Extracts
The
antibacterial activity of the plant extracts are depicted in Table 2. The
results indicated that the plants extracts showed antibacterial activities at
variable degrees against Bacillus
cereus
(MTCC-430), Staphylococcus epidermis (MTCC-435), Klebsiella
pneumonia
(MTCC-432) and Bacillus subtilis (MTCC 121) bacteria. Methanolic
leaves extract of Hibiscus syriacus displayed the
most important spectrum of activity, its inhibitory effects being observed
against all the bacterial strains, followed by the all other leaves extract of
Hibiscus syriacus. The methanolic
leaves extract of Hibiscus syriacus showed the
highest activity against Bacillus subtilis (MTCC-121). Antibacterial activity of
leaves extract of Hibiscus syriacus L. was evaluated
in vitro against four bacterial species. Among the five extract (petroleum
ether, benzene, chloroform, methanol, and aqueous) used in the study. Methanol extract displayed maximum
antibacterial activity against all the bacterial species tested. Among four
bacterial species screened Bacillus
cereus
(MTCC-430), Staphylococcus epidermis (MTCC-435), Klebsiella
pneumonia
(MTCC-432) and Bacillus subtilis (MTCC 121) bacteria were most susceptible
to the methanol extract of leaves of Hibiscus syriacus
L. (19.1 mm, 19.6 mm, 22.9mm, 25.4mm) respectively. Whereasv
the water extract of Hibiscus syriacus L. exhibited
significant antibacterial activity (16.5 mm, 17.1 mm, 20.9 mm, and 21.8 mm)
against Bacillus cereus (MTCC-430), Staphylococcus epidermis (MTCC-435), Klebsiella
pneumonia
(MTCC-432) and Bacillus subtilis (MTCC 121) bacteria respectively.
Petroleum ether(8.1 mm, 8.0 mm, 9.0 mm, 10.3 mm), benzene (7.2 mm, 7.4 mm, 8.6
mm, 9.8 mm) and chloroform (9.2 mm, 10.1 mm, 10.4 mm, 10.6 mm) leaves extract
of Hibiscus syriacus L. fails to elict
significant antibacterial activity against tested bacterial species.
All
The leaves extract of Hibiscus syriacus show
antibacterial activity against the majority of the bacteria, their inhibitory
effect being noted against Bacillus cereus (MTCC-430), Staphylococcus epidermis
(MTCC-435), Klebsiella pneumoniae (MTCC-432), and
Bacillus subtilis (MTCC 121) bacterial strains tested
respectively. Chloramphenicol used as reference antibiotic showed variable
inhibitory activity on different strains of bacteria. Result obtained revealed
potential application of leaves extract of Hibiscus
syriacus L. for the treatment of bacterial
infection.
The
MIC values (table 2) ranged 0.001 and 0.625 mg/ml. The lowest minimal value of
0.078 mg/ml was recorded with the methanol leaves extract of Hibiscus syriacus L against Bacillus subtilis (MTCC-121).
Table3. Minimum inhibitory
concentration of different leaves extract.
Bacterial sp. |
Minimum inhibitory concentration (mg/ml) |
|||||
Petroleum ether extract |
Benzene extract |
Chloroform extract |
Methanol extract |
Water extract |
Chloramphenicol (std Drug) |
|
Bacillus cereus (MTCC-430) |
0.312 |
0.625 |
0.625 |
0.312 |
0.312 |
0.002 |
Bacillus subtilis (MTCC-121) |
0.312 |
0.312 |
0.625 |
0.078 |
0.156 |
0.001 |
Staphylococcus epidermis (MTCC-435) |
0.312 |
0.312 |
0.625 |
0.156 |
0.156 |
0.001 |
Klebsiella pneumonia (MTCC-432) |
0.625 |
0.625 |
0.625 |
0.312 |
0.312 |
0.004 |
DISCUSSION:
The
Medicinal plants have been main source for drugs over many countries in both
developed and developing world. Traditional medicines products are not
officially recognized in many countries, and the European Union presently developing regulatory laws for quality traditional medicines. [21]
Plants are important source of potentially useful structures for the
development of new chemotherapeutic agents. The first step towards this goal is
the in vitro antibacterial activity assay. [22]
Hibiscus rosasinensis was screened
for antibacterial activity against human pathogenic bacterial strains. Most of
the extracts have shown antibacterial activity against these pathogens. E.
coli are common member of the normal flora of large intestine. It
is predominant facultative organism in the gastrointestinal tract and colonizes
the tract within hours or few days. It is responsible for causing diarrhea,
which is characterized by rapid onset of watery non-bloody
fluid. [23]
Each
of the extract tested in the present study displayed antibacterial activity on Bacillus cereus (MTCC-430), Staphylococcus epidermis (MTCC-435), Klebsiella
pneumonia
(MTCC-432), Bacillus subtilis (MTCC 121) bacterial strains tested.
However, differences were observed between antibacterial activities of the
extracts. Therefore, the inhibitory
activity found herein against Gram-positive as well as Gram-negative bacteria.
Phytochemical screening results of leaves extract of Hibiscus syriacus was in accordance with the results previously
obtained. This latter suggested that the presence of flavonoids
which interfere with cell division) in Hibiscus syriacus
could account for its antimicrobial activity. They demonstrated that methanol
extract of Hibiscus syriacus possess inhibitory
activities against Bacillus
cereus
(MTCC-430), Staphylococcus epidermis (MTCC-435), Klebsiella
pneumonia
(MTCC-432), Bacillus subtilis (MTCC 121). In this report, the
antibacterial activity was observed against Bacillus cereus (MTCC-430), Staphylococcus epidermis (MTCC-435), Klebsiella
pneumonia
(MTCC-432), Bacillus subtilis (MTCC 121). The results of the present
work also bring additional data on the antibacterial activity of Hibiscus syriacus, since we report for the first time its activity
against Bacillus cereus (MTCC-430),
Klebsiella Staphylococcus
epidermis
(MTCC-435), Klebsiella pneumonia (MTCC-432), and Bacillus
subtilis (MTCC
121). The different phytochemicals found here should
then explain its antibacterial activity against different bacterial strains
tested. The plants of the Hibiscus family were already found to possess flavonoids, terpenoids and
glycosides that could explain their antibacterial activity. [24-25]
The emergence of multiple drug resistant bacteria has become a major
cause of failure of the treatment of infectious diseases. [26] The
antibacterial activity of the methanol and aqueous extracts were additional
information of the antibacterial activities of this plant Hibiscus syriacus. Chloramphenicol
confirmed that an active efflux system expressed by tested bacteria is
responsible for their resistance to chloramphenicol. The wide substrate
specificity of these pumps could allow them to provoke extrusion of various
active antibacterial compounds, preventing their inhibitory effects. Therefore,
the low antibacterial activities of these plants shown in the present work
should thus be due to the resistance of bacteria strains tested. The contrast
between high numbers of secondary metabolite classes found in these extracts
reinforces the idea that the detection of the classes of phytochemicals
in plants is not a guarantee for a good antibacterial property. [27] This shows
that the studied extract mostly exhibited bacteriostatic
effects.
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Received
on 21.11.2014 Accepted on 20.12.2014
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