INTRODUCTION
Many of the plants used today were known to the people of ancient
cultures throughout the world and they were valued their preservative
and medicinal powers (Kivanc, 1997). The antimicrobial properties of plants
have been investigated by a number of researchers world wide. The broad
bean (Vicia faba L.), is a member of the Legominosea family. It
contains toxic substances such as saponins, lathrogens, cyanogenic and
other glycosides, protease and amylase inhibitors and hemagglutinins (Rubatzky
and Yamaguchi, 1997).
By a comparison of the compound Aglycon of Deoxy-Niazimicine (N-benzyl,
S-ethyl thioformate) isolated from Moringa oleifera Lam. with crude
chloroform extract showed a strong antibacterial activity against gram
positive Straphylococcus aureus and gram negative Shigella dysenteriae,
Shigella boydii, Salmonella typhi and Pseudomonas aeruginosa
and produced zone of inhibition between 9 to 13 mm while the crud extract
showed compasratively lower activity against gram positive bacteria Straphylococcus
aureus and gram negative bacteria Shigella dysenteriae, Shigella
boydii and produced zone of inhibition between 9 to 11 mm (Nikkon
et al., 2003). The effect of three different extracts (hexanic,
ethyl acetate, methanol) obtained from various Brazilian Drosera species
against different bacteria such as Staphylococcus aureus, Escherichia
coli, Klebsiella peneumoniua, Salmonella choleraesuis,
Enterococcus faecium, Pseudomonas aeruginosa and Candida
albicans were evaluated. The best results were obtained with ethyl
acetate extracts from D. Montana var. montana and D.
communis (Ferreira et al., 2004). Nascimento et al.
(2000) also found antibacterial activity of Achillea millifolium,
Caryophyllus aromaticus, Melissa officinalis, Ocimun basilucum,
Psidium guajava, Punica granatum, Rosmarinus officinalis,
Salvia officinalis, Zyzygyum joabolanum and Thymus vulgaris.
The phytochemicals benzoic acid, cinnamic acid, eugenol and farnesol were
also utilized. The highest antibacterial potentials were observed for
the extracts of Caryophyllus aromaticus and, Zyzygyum joabolanum,
which inhibited 64.2 and 57.1% of the tested microorganisms, respectively,
with higher activity against antibiotic-resistant bacteria (83.3%). Salvia
officinalis, Achillea millifolium, extracts did not
present any antimicrobial activity.
As part of our investigation on the antibacterial activities of different
parts of broad bean plant, a microbiological study of four different extracts
was undertaken.
MATERIALS AND METHODS
Plant material: Fresh plant parts (flowers, leaves, seeds and seed hulls) of broad
bean were collected from the research field of Agricultural Faculty,
The Guilan University in Rasht, Iran in 2005 and dried in the sun for
5 days and finally in an oven below 50°C. The dried plant parts were
ground in to fine powders.
Preparation of the extracts: Each parts of plant material were extracted with the flowing solvents:
14 gram dried seed powder + 100 cc ethanol, methanol and sterile distilled
water,
14 gram flower powder + 100 cc ethanol, sterile distilled water and 2-Methylbutan-1-01,
14 gram seed hull powder + 100 cc ethanol, sterile distilled water and
2-Methylbutan-1-01,
14 gram leaves powder + 100 cc ethanol, sterile distilled water and 2-Methylbutan-1-01.
The extracts were concentrated to a dark syrupy residue. This syrupy
was dissolved once again in 10 cc in the above solvents. This liquid was
used for testing inhibitory activity.
Antimicrobial screening: In vitro antimicrobial screening is generally performed by
disc diffusion method (Vander and Vlietnck, 1999). Disc diffusion method
is highly effective for rapidly growing microorganisms and the activities
of the test drugs are expressed by measuring the diameter of the zone
of inhibition (Nikkon et al., 2003). The microorganisms in this research were collected from the Faculty of Science,
Microbiological Department, the Guilan University in Rasht, Iran. For antibacterial
activity seven pathogenic bacteria (four gram negative and three gram positive)
were selected. The plant extracts were dissolved separately in the solvents
to get a concentration of 100, 200 and 300 μL. Discs were dried in an oven
below 37°C for 8 days and then applied against seven pure cultured microorganisms.
RESULTS
The antimicrobial activity of the plant parts of broad bean (flowers,
leaves, seeds and seed hulls) are shown in Table 1 and 2.
While seed extract did not showed any antibacterial activity against
gram positive and gram negative bacteria, leaves extract in sterile distilled
water showed higher antibacterial activity against gram positive Bacillus
sabtilis (Fig. 1), Staphylococcus aureus (Fig.
2), Micrococcus pyogenes (Fig. 3) and gram
negative Escherichia coli (Fig. 4), Shigella
sp. (Fig. 5), Serratia marcescens (Fig.
6). The leaves extract on Klebsiella pneumonia did not showed
any antibacterial activity. The different parts of broad bean extracts
in sterile distilled water produced against all tested organisms zone
of inhibition between 13 to 32 mm by a concentration of 100 to 300 μL.
| Table 1: |
In vitro antibacterial activity of sterile distilled
water extract Zone of inhibition (Diameter in mm) |
 |
|
| Fig. 1: |
Leaves extract in sterile distilled water by Bacillus
sabtilis |
|
| Fig. 2: |
Leaves extract in sterile distilled water by Staphylococcus
aureus |
|
| Fig. 3: |
Leaves extract in sterile distilled water by Micrococcus
pyogenes |
|
| Fig. 4: |
Leaves extract in sterile distilled water by Escherichia
coli |
|
| Fig. 5: |
Leaves extract in sterile distilled water by Shigella sp. |
|
| Fig. 6: |
Leaves extract in sterile distilled water by Serratia marcescens |
|
| Fig. 7: |
Flowers extract in sterile distilled water by Sraphylococcus
aureus |
|
| Fig. 8: |
Flowers extract in sterile distilled water by Escherichia
coli |
|
| Fig. 9: |
Flowers extract in sterile distilled water by Serratia
marcescens |
|
| Fig.10: |
Flowers extract in sterile distilled water by Shigella
sp. |
|
| Fig. 11: |
Leaves extract in sterile distilled water by Escherichia
coli |
Flower extract in sterile distilled water affected also Staphylococcus
aureus between 13 to 22 mm (Fig. 7), Escherichia
coli between 25 to 29 mm (Fig. 8) and Serratia
marcescens Fig. 9), between 0 to 17 mm by a concentration
of 100 to 300 μL, respectively. Shigella sp. (Fig.
10) produced by 200 and 300 mm zone of inhibition 25 and 27 mm, respectively.
Leaves extract in ethanol produced against Escherichia coli zone
of inhibition between 25 to 34 mm by a concentration of 100 to 300 μL
(Fig. 11). Flower and Seed hull extracts in ethanol
showed in the tested concentration lower zone of inhibition.
Different parts of broad bean extracts in Methanol and 2-Methylbutan-1-01
couldn`t affect against all organisms tested in our research.
DISCUSSION
Our investigation showed that different parts of broad bean extracts
could produce relative highly antimicrobial activity against various bacteria.
Best results were obtained with sterile distilled water and ethanol extracts
from leaves and flowers as shown in Table 1 and 2.
Seed and seed hull of broad bean did not produced valuable zone of inhibition.
With the exception of Klebsiella pneumoni (resistant), the other gram-negative
and gram-positive bacteria were sensitive to all extracts (Bacillus
sabtilis, Staphylococcus aureus, Micrococcus
pyogenes, Escherichia coli, Shigella sp.,
Serratia marcescens). We consider these as preliminary results
and a larger number of bacterial isolated must be tested, with different
concentrations of broad bean extracts in order to establish their real
antimicrobial activity. Our investigation determined the relation between
extract concentration and bactericidal effect. The isolation of essential
oil of mature leaves of Cestrum diurnum (Bhattacharjee et al.,
2005), the root of Moringa pterygosperma (Kurup and Rao, 1952) and different
species of Drosera (Ferreira et al., 2004), are agreement with
the tests accomplished in this study.
CONCLUSIONS
Data from our results reveal the great potential of leaves and flower
of broad bean for therapeutic treatment, in spite of the fact that they
have not been completely investigated. Therefore, more studies need to
be conducted to search for new compounds. In conclusion, this investigation
reports that flower and leaves of broad bean extracts in sterile distilled
water possesses antibacterial activity and could be used in medicine after
toxicity test.
ACKNOWLEDGMENTS
We thank Dr. Fazel Najafi and Mr. Gh. Iranyar for helpful discussions
and assistance with laboratory.
