Antibacterial and Antifungal Studies of the Crude Extract and Solvent Fractions
of Onosma khyberianum
Murad Ali Khan,
2013, 4(9), 525-528.
Background: The most commonly used plant namely Onosma khyberianum
was brought under antimicrobial study after observing their medicinal values.
The present study aim to assess antimicrobial activity of ethanolic extracts
and subsequent solvent soluble fractions of Onosma khyberianum against
five bacterial strains Salmonella typhi, Shigella dysenteriae,
Escherichia coli, Staphylococcus aureus, Vibrio cholerae
and three fungal strains Aspergilus flavus, Alternaria alternate
and Fusarium oxysporum. Materials and Methods: Briefly the stock
solution of crude extract and other fractions were prepared in DMSO (3 mg mL-1
for antibacterial essay, 4 mg 1 mL-1 for antifungal essay). The antibacterial
activity was evaluated by agar diffusion method while for antifungal assay the
disc diffusion method was used. Results: The ethanolic and chloroform
fractions showed excellent activity against all the selected bacterial strains.
The same fractions also showed best activity against all selected fungal strains.
Conclusion: The present study suggests Onosma khyberianum to
be a source for isolation of antimicrobial compounds for human health care and
use as preservatives in food processing industries.
Sher, 2009). The genus Onosma (Boraginaceae)
consists of 85 species, occurring mainly in Iran and westwards to Syria, Turkey
and Europe. Onosma is a genus of plants that grow biennially or perennially.
It is represented in Pakistan by 08 species, namly O. khyberianum,
O. limitaneum, O. chitralicum, O. hispida, O. dichroantha,
O. hypoleucum, O. griffithii and O. thomsonii (Ali
and Nasir, 1989). The plants of the genus has cooling, laxative, anthelminitic,
alexipharmic effects and are effective in the diseases of the eye, derangements
of blood, bronchitis, abdominal pain, stangury, thirst, itch, leucoderma, fever,
wound, pile and in urinary calculi (Kirtikar, 1994).
Most of the ailments like diarrhea, diabetes, malaria, skin diseases and microbial
diseases are cured by plants in different ways like intake of fruits and vegetables,
in form of drugs and extracts. The world health organization (WHO) reported
that 80% of people favor to use medicinal plants to cure these diseases (Sahito
et al., 2003). Since herbal drugs have compounds having antimicrobial
properties so they act as antimicrobial agents (Prince
and Prabakaran, 2011). Antimicrobial agents or antimicrobials are substances
that kill or restrain the growth of other microorganisms such as bacteria, fungi
or protozones. In early 20th century, antibiotics were considered only those
drugs that were only formed by one microorganism that demolish or inhibit the
growth of another microorganism but in current days antibiotics are referred
to all those drugs that are used to cure microbial diseases. It means not only
antibiotics but all synthetic compounds also come under the class of antimicrobial
agents (Levy, 1994). Fungal diseases are not common
as bacterial diseases but if occurred they are complicated to cure especially
in those having weak immune system (Kivcak et al.,
2009). After the discovery of antibiotics it was considered that infectious
diseases would be entirely treated but with the enlarged use of antibiotics,
the causal agents of the diseases i.e., bacteria and fungi modified their physiology
and in this new form these are resistant to the pre-existing antibiotics. Moreover
most of the antibiotics are toxic. That is why the search of a secure source
of new antibiotics or antimicrobial agents are required to overcome this global
therapeutic problem (Sibanda and Okoh, 2007).
Botanist, ethnopharmacologist, natural products chemist and microbiologist
are trying to uprooting new antimicrobial drugs from medicinal plants in order
to cover the demand for natural and non costly drugs to cure infectious diseases
without any side effects (Cowan, 1999). The unnecessary
use of antibiotics for crop production and resistance of microorganism against
these antibiotics has made various health problems to human being. Medicinal
plants have compounds to control microorganisms, so it is significant to isolate
those compounds and to know those strategies by which they operate and kill
pathogenic bacteria (Kuntal et al., 2010). The
process of evaluating antimicrobial compounds is therefore a continuing process.
Plant extracts have great potential as antimicrobial compounds against microorganisms.
Thus, they can be used in the treatment of infectious diseases caused by resistant
microbes. Surprisingly no antimicrobial studies have been made so far on Onosma
khyberianum. The objective of this research was to evaluate the potential
of Onosma khyberianum extract and solvents partitions on microbial strains.
MATERIALS AND METHOD
Collection of plant material: The Onosma khyberianium plant was
collected from Khyber Agency which was used for the treatment of infection diseases
by tribal peoples and was authenticated by plant taxonomist at Department of
Botany (voucher No. 156), KUST, Kohat, Khyber Pakhtunkhwa, Pakistan.
Preparation of plant extract: The whole plant was shade dried and then
chopped into fine powder. The fine powder of plant was soaked in ethanol for
15 days, extracted three times at room temperature in the same solvent and filtered.
The filtrates were then processed through rotary evaporator to get crude extract
and dried. The dried extract was further suspended in water and was partitioned
successively with n-hexane, ethyl acetate, chloroform and n-butanol to obtain
n-hexane, chloroform, ethyl acetate, n-butanol and aqueous soluble fractions,
respectively. The crude extract and its solvent soluble fractions were tightly
packed and stored in refrigerator at 4°C.
Antibacterial activity: The crude extract and their respective solvents
soluble fractions were subjected to antibacterial evaluation against five bacterial
stains i.e., Salmonella typhi, shigella dysenteriae, Escherichia
coli, Staphylococcus aureus and Vibrio cholerae. Solutions
of crude extract and various fractions at concentration of 3 mg mL-1
were prepared in DMSO.
The Nutrient agar media 14 g was prepared in 500 mL conical flask and it was
sterilized along with Petri dishes, cork borer and pipette in autoclave for
15 min at 121°C at high pressure. The nutrient agar media was poured into
Petri dishes under laminar flow hood to evade bacteria from environment. Wells
of 7 mm were punched in the agar media by using sterile metallic borer. The
modified agar diffusion method as reported by Khan and
Tewari (2011), was followed. Nutrient agar media was inoculated with a given
bacterial culture corresponding to 106 CFU mL-1. Bacterial
strains was spread on the solidified agar media. The prepared stock solutions
of extract and fractions were poured to wells. The petri dishes were incubated
at 37°C for 24 h and control wells containing antibiotic Chloramphenicol,
which is a positive control, was also run side by side in the same petri dishes
and DMSO was used as negative control. After 24 h antibacterial activities were
measured by measuring the diameter of the zones of inhibition and were compared
these values of zone of inhibitions with the zone of inhibition of standard
drug levoflaxcine. The amount of growth in each well was measured (Khan
and Tewari, 2011).
Antifungal assay: Three fungal strains i.e., Aspergilus flavus,
Alternaria alternate and Fusarium oxysporum were used for the
antifungal activity. For Antifungal assay disc diffusion method was used (Perez
et al., 1990). Briefly the stock solution of crude and other fractions
were prepared in DMSO i.e., 4 mg mL-1. Nutrient broth was used for
culturing fungal strains. The media was prepared according to manufacture specification
and was transferred to plates at 50-60°C under laminar flow hood. These
plates were inoculated with the test fungi and incubated at 27°C for growth.
For the antifungal assays nutrient agar was prepared by dissolving and autoclaving
the specified quantity of dry powder of Nutrient agar in a given quantity of
distilled water and was transferred to plates. One milliliter solution of the
test sample was added and inoculated with different fungal species. Plates were
incubated at 27°C for 5 to 8 days and inhibition of fungal growth was measured
(Perez et al., 1990).
RESULTS AND DISCUSSION
Among most critical health issues of the globe in 21st century, bacterial
infection is considered to be one of them (Morris and
Masterton, 2002). Bacterial defiance to antibiotics is the key health issue
and thus, it is essential to overcome this problem by the development of new
drugs with innovative mechanism of action (Wang et
al., 2003). Plants herbal mixtures contributed a lot to human welfare
and health, thus it provides a foundation for distinctive drug compounds. The
utilization of plants various extracts with well known antimicrobial potential
has significant value for therapeutic remedies.
In our present exploration, the antibacterial and antifungal activity of Onasma
khyberianum various extract/fractions in comparison with Chloramphenicol
and Terbinafine standards were determined against five bacterial Salmonella
typhi, Shigella dysenteriae, Escherichia coli, Staphylococcus
aureus, Vibrio cholera and three fungal strains Alternaria alterrnata,
Aspergillus flavus and Fusarium oxysporum, respectively.
The antibacterial activities of Onasma khyberianum have been shown by
Table 1. None of the fractions were completely inactive against
any bacterial strain. Results of antibacterial attempt revealed that chloroform
and methanol fractions of plant displayed more potent activity against various
strains as compared to n-hexane, n-butanol and aqueous fractions. Promising
activity 28 mm was shown by chloroform fraction against Salmonella typhi
followed by same fraction against Shigella dysenteriae and Vibrio
cholera with inhibition zone of 26 mm for each. Ethanol fraction showed
best activity against Shigella dysenteriae (21 mm) and Vibrio cholera
(20 mm). n-hexane showed activity against Shigella dysenteriae (8
mm) and Vibrio cholera (12 mm) but completely ineffective against Salmonella
and E. coli. All fractions showed excellent activity against
shigella, Stap. aureus and Vibrio cholera.
The antifungal activities of Onasma khyberianum against three fungal
strains Alternaria alterrnata, Aspergillus flavus and Fusarium oxysporum
have been presented in Table 2 from which it is clear
that ethanol and chloroform fractions of plant were most active against all
the selected fungal strains as compared to n-hexane, ethyl acetate, n-butanol
and water. The activity of ethanol fraction against F. oxysporum was
18 mm, A. alternata 13 mm and A. flavus was 7 mm whereas the activities
of chloroform fraction against F. oxysporum, A. alternata and A.
flavus were 17, 11 and 9 mm, respectively. The activity of n-hexane recorded
against A. alternata was 7 mm but it was entirely inactive against F.
oxysporum and A. flavus. Similarly ethyl acetate and n-butanol fractions
were only active against F. oxysporum and inactive for all other fungal
strains while water fraction showed no activity against none of the fungal strain.
Ozgen et al. (2003) studied the antibacterial
activity of two species of genus onosma. The ethyl acetate fraction was
found to be effective on Staphyloccoccus aureus, Bacillus subtilis
and Escherichia coli. The chloroform fractions was the most effective
on S. aureus and E. coli, respectively. These two species did
not have any antifungal activity against any fungal strain. Here ethyl acetate
fraction donot show any promising activity against any of the bacterial strains.
The chloroform fraction show excellent activity against S. aureus, S.
dysenteriae and V.cholerae (Ozgen et al.,
2003). In antifungal study crude ethanolic extract show good results
against Fusarium oxysporum and Alternaria alternate. In fractions
chloroform fraction show good activity against Fusarium oxysporum (Ozgen
et al., 2003).
Naz et al. (2006) reported antibacterial activity
of Onosma hispidum for the first time in this species. In addition to
these compounds, the crude ethanolic extract and methanol fraction exhibited
substantial bioactivity against species of corynebacteria, enterococci, staphylococci
and streptococci (Naz et al., 2006).
These findings suggest that Onosma khyberianum has good antibacterial
and antifungal properties that can be used for infection control and treatment
and could also be as new source for antibiotics discovery and infection treatment.
Further studies are necessary to isolate and characterize the active components
of the extracts/fractions and also to elucidate their antibacterial mechanisms
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