In Vitro Antibacterial and Antifungal Assay of Tectona grandis-A
Screening Study
K. G. Purushotham1*, P.
Arun1, J. Johnsy Jayarani1, R.
Vasanthakumari1, and D. Chamundeeswari2
1Dr. M.G.R. Educational
and Research Institute, Dr. M. G. R
University, Maduravoyal. Chennai-600 095, Tamil
2Sri
ABSTRACT:
Medicinal plants are
potential of antimicrobial compounds. The present study deals with the antibacterial
and antifungal activity of methanolic extracts of Tectona grandis (Verabinaceae) leaves were collected from the
KEYWORDS: Antibacterial activity, Antifungal activity,
Disc diffusion method, MIC, MBC, MFC, Tectona grandis,
1.
INTRODUCTION:
Infectious diseases
are the second leading cause of death World wide1. Bacterial
infections are the most serious global health issues in 21st century2.
Antimicrobial resistance settings have failed to address this essential aspect
of drug usage3. There are a number of clinically efficacious
antibiotics becoming less effective due to the development of resistance. The
emergence of bacterial resistance to antibiotic is a major health problem and
therefore, it is critical to develop new antibiotic with novel mechanism of
action to overcome there problems4. Plants and plants products have
been used extensively throughout history to treat medicinal problems. Numerous
studies have been carried out to extract various natural products for screening
antimicrobial activity5, 6.
Tectona grandis is a large deciduous tree, 10-12 meter tall; branchlets 4-angled, density clothed with yellowish grey tomentum. Leaves opposite, elliptic or obviate, 30-50 x
15-20 cm, cuneate at base, entire or crenulate, acute or acuminate, rough and glabrous above, stellate, grey to tawny tomentose
beneath7. It commonly known as Indian teak and it belongs to family Verabinaceae. Lapachal, a napthoquinone isolated from T. grandis is reported to have anti ulcer8
and nitric oxide scavenging activity9. Tectona grandis has been reported to contain
1-hydroxy2methylanthraquinone. tectoquinone,
pachybasin, dehydrotectol, tectol, lapachol,
2-methylquinizarin, deoxy lapachol,
β-sitoserol, squalence
and betulinic acid10-12. The present
investigation was made to evaluate the antibacterial and anti fungal activity
of T. grandis
leaves.
2. MATERIAL
AND METHODS:
2.1 Collection of
plant material:
Tectona grandis leaves were collected from the
2.2 Solvents
and Chemicals:
Methanol was used for the extraction purpose.
The commercial grade solvent was purchased (Ranbaxy Fine Chemicals Ltd., New
Delhi, India) which was distilled and this purified form was used for the
extraction procedure in this study.
2.3 Preliminary
phytochemical screening:
Preliminary phytochemical
screening of T. grandis
was carried out from the various phytoconstituents
using standard procedures13. The methanolic
extract was found to contain more flavonoids. The
preliminary phytochemical screening of methanolic extract revealed the presence of alkaloids, flavonoids, and tannins.
2.4 Preparation
of crude extract:
Weighed quantities of coarsely powdered
leaves of T. grandis
were placed in maceration flask and added with sufficient quantity of methanol.
Complete maceration took place for about 72 hours, with occasional shaking
during first 6 hours14. After 72 hours, the men strum was collected
and evaporated to obtain the dried extract.
2.5 Preparation
of test solution and disc:
Test solution was prepared with known weight
of crude extracts, dissolved in 5% dimethyl sulphoxide (DMSO). Whatman no.1 sterile filter paper discs
(6mm) were impregnated with 20µl of this extract (corresponding to 500 µl/ml to 1000 µl/ml of crude extract) and allowed to
dry at room temperature.
2.6 Microorganisms
used:
Six different bacterial strains used for
study were Klebsiella pneumoniae
(MTCC 432), Psedomonas aeruginosa
(MTCC 1688), Proteus mirabilis (MTCC
425), Escherichia coli, (MTCC 729), Salmonella typhimurium
(MTCC 98), and Streptococcus species
(MTCC 389). These standard stains were obtained from Microbial Type Culture
Collection and gene bank (MTTC); Institute of Microbial Technology,
In vitro antibacterial
activity was determined by using Muller Hinton Agar and Muller Hinton Broth. In vitro antifungal activity was
determined by using Sabouraud Dextrose Agar, yeast
Nitrogen base (for yeast) and Sabouraud Dextorse Broth (for mycelia fungi) and they were obtained
from Himedia Ltd., Mumbai15.
2.7 Preparation
of inoculam:
Twenty-four hours old culture of selected
bacterial/yeast was mixed with physiological saline and the turbidity was
corrected by adding sterile physiological saline until a Mac Farland turbidity standard of 0.5 [106colony
forming units(CFU) per ml].
The isolates were sub cultured on Sabouraud Dextrose Agar and incubated at 35°C for 7-14
days. The growth was scraped aseptically, crushed and macerated thoroughly in
sterile distilled water and fungal suspension was standardized spectrophotometrically to an absorbance of 0.600 at 450 nm.
2.8 Antibacterial
and antifungal assay:
The agar diffusion method was followed for
antibacterial and antifungal susceptibility test. Petri plates were prepared by pouring 15-20
ml of Muller Hinton Agar for bacteria and Sabouraud
Dextrose Agar for fungi respectively and which were allowed to solidify. Plates
were dried and 0.1 ml of standardized inoculum
suspension was poured and uniformly spread. The excess inoculum
was allowed to dry for 5 minutes in incubator. The discs were then applied and
the plates were incubated in incubator at 37°C for 24 hours (bacteria), 28°C
for 48 hours (yeast) and 28°C for 72-96 hours (mycelia fungi). The inhibition
zones were measured from the edges of the disc to the inner margin of the surrounding
pathogens. Each assay in this experiment was repeated thrice.
2.9 Minimum
Inhibitory Concentration (MIC):
Minimum Inhibitory Concentration of the plant
extract was tested as previously by the two-fold serial dilution method. The
test extract was dissolved in 5% DMSO to obtain 1000 µg/ml stock solution. 0.5
ml of stock solution was incorporated into 0.5 ml of Muller Hinton broth for
bacteria, Yeast Nitrogen Base for yeasts and Sabouraud
Dextrose Broth for mycelia fungi to get a concentration of
500 µg/ml and serially diluted by double dilution method to achieve 250,
125, 62.5 and 31.25 µg/ml, respectively. 50 µl of standardized suspension of
the test organisms were transferred on to each tube. The control tube contained
only organisms and not the plant extract. The culture tubes were incubated the
in incubator at 37°C for 24hours (bacteria), 28°C for 48 hours (yeasts),
and 28°C for 72-96 hours (mycelial fungi). The lowest concentrations, which did not
show any growth of tested organism after macroscopic evaluation was determined
as MIC.
2.10 Minimum
Bactericidal Concentration (MBC) and Minimum Fungicidal Concentration (MFC):
All the tubes used in
the MIC study which did not show any growth of the bacterial and fungal after
the incubation period were first diluted (1:4) in fresh Muller Hinton Broth for
bacteria and Yeast Nitrogen Base for
yeast and Sabouraud Dextrose Broth for mycelial fungi and then sub cultured on to the surface of
the freshly prepared Muller Hinton agar (for bacteria) and Sabouraud
Dextrose Agar (for fungi) plates and incubated in incubator at 37°C for 24
hours (bacteria), 28°C for 48 hours (for yeast) and 28°C for 72-96 hours (mycelial fungi). The MBC and MFC were recorded as the
lowest concentration of the extract that did not permit any visible bacteria
and fungal colony growth on the appropriate agar plates after the period of
incubation.
|
Table:
- 1 Antibacterial activity of Tectona grandis leaves Antibacterial activity of Tectona grandis |
|||||
|
S. No |
Bacteria |
Methanol |
|||
|
Mean Zone of Inhibition (mm) |
MIC(µg/ml) |
MBC(mg/ml) |
|||
|
500 µg/ml |
1000µg/ml |
||||
|
1 |
Streptococcus
species
(MTCC 389). |
8 ± 0.3 |
14 ± 0.37 |
62.5 |
125 |
|
2 |
Proteus
mirabilis
(MTCC 425) |
6 ± 0.21 |
10 ± 0.32 |
125 |
250 |
|
3 |
Salmonella
typhimurium(MTCC 98) |
5 ± o.24 |
9 ± 0.41 |
125 |
500 |
|
4 |
Psedomonas aeruginosa (MTCC 1688) |
5 ± 0.18 |
10 ± 0.34 |
250 |
500 |
|
5 |
Klebsiella
pneumonia
(MTCC 432) |
4 ± 014 |
8 ± 029 |
250 |
500 |
|
6 |
Escherichia
coli,
(MTCC 729) |
4 ± 0.11 |
7 ± 0.28 |
250 |
500 |
±: Standard deviation
Table: -
2 Antifungal activity of Tectona grandis
leaves
|
Antifungal activity of Tectona
grandis |
|||||
|
S. No |
Fungi |
Methanol |
|||
|
Mean Zone of Inhibition (mm) |
MIC (µg/ml) |
MFC (mg/ml) |
|||
|
500 µg/ml |
1000 µg/ml |
||||
|
1 |
Candida
albicans |
13 ± 0.45 |
20 ± 0.52 |
31.25 |
62.5 |
|
2 |
Cryptococcus
neoformans |
6 ± 0.21 |
10 ± 0.44 |
250 |
500 |
|
3 |
Aspergillus flavus |
12 ± 0.37 |
15 ± 0.49 |
62.5 |
125 |
|
4 |
Aspergillus
fumigates |
12 ± 0.40 |
15 ± 0.56 |
62.5 |
125 |
|
5 |
Aspergillus |
11 ± 0.30 |
14 ± 0.32 |
62.5 |
125 |
|
6 |
Rhizopus sp |
10 ± 0.48 |
13 ± 0.35 |
62.5 |
125 |
|
7 |
Trichophyton rubrum, |
9 ± 0.45 |
13 ± 0.49 |
125 |
125 |
|
8 |
Trichophyton mentagrophytes |
7 ± 0.24 |
7 ± 0.24 |
250 |
500 |
|
9 |
Trichophyton gypseum |
7 ± 0.28 |
7 ± 0.28 |
250 |
500 |
±: standard deviation
3.
RESULTS:
Our results (Tables 1 and 2) showed that the
methanol extract of Tectona grandis
possessed antibacterial and antifungal activities against the microorganisms
tested. A total of 15 microorganisms which consisted of six bacteria, two
yeasts and seven fungi (including three dermatophytic
fungi) were tested. When the methanol extract was assayed against the test
organisms by agar diffusion assay (table 1 and 2), the mean zones of inhibition
obtained were between 2 and 20 mm. The blind control (dimethyl
sulphoxide) did not inhibit any of the microorganisms
tested.
MIC values of 62.5-250 µg/ml were obtained
against bacterial pathogens. On other hand, the values obtained in the range of
31.25-250 µg/ml were recorded against the fungal isolates. The results of MBC
of the extract (Table 1) showed that with the exception of the antibacterial
assays against Streptococcus species
(MTCC 389) and Proteus mirabilis
(MTCC 425), the methanolic extract exhibited a MBC at
a concentration of 500 µg/ml. MFC of the extract (Table 2) showed that with the
exception of the antifungal assays against Candida
albicans, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Rhizopus sp. And Trichophyton rubrum the methanolic extract exhibited a MFC at a concentration of 500 µg/ml.
4.
DISCUSSION:
The inhibitory effect of (Tables 1 and 2) methanolic extract of Tectona grandis gave favourable
results against all the tested microorganisms with MIC values between 31.25 and
500 µg/ml. The present study revealed that the leaves extract of T. grandis was
very effective against Candida albicans, Aspergillus flavus, Aspergillus fumigates, Apergillus
The higher zone of inhibition was recorded at
1000 µg/ml concentration than 500 µg/ml concentration of the extract. As the disc dosage level
increases, the inhibitory effect also increased. The values obtained in the MBC
and MFC studies were higher than those of the MIC studies (Tables 1and 2). Similar
observations were made by (Chattopadhyay D) 19
while studying the antimicrobial activities of Alstonia macrophyla and Mollotus peltatus leaves.
The fact that the methanolic
extract of Tectona grandis
leaves exhibited inhibitory activity against some of the microorganisms
implicated in the pathogenesis of the skin diseases (yeasts, such Candida albicans
and dermatophytes such as Trichophyton rubrum, Trichophyton
mentagrophytes and Microsporum gypseum). T. grandis leaves contain tannin, which
are used as anti-inflammatory agents and also used topically for treatment of
burns20.
Further study on the fractions of active
components in Tectona grandis
leaves and the maximal species may provide better understanding of the
antimicrobial activity. In since Tectona grandis appear to be most promising, bioassay–guided
fractionation which is currently underway with a goal of elucidating their
active antimicrobial compound
5.
ACKNOWLEDGEMENT:
The authors are thankful to staff members
from ACS medical college and Hospital Velappanchavadi,
Chennai 600 077, and sincerely acknowledges to Dr. K.Balakrishnan
Research Officer, central Research institute for Ayurvda
and Siddha (Central Council for Ayurveda
and Siddha), Arumbakkam, and Chennai. For identification of plant material
6.
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Received on 17.11.2009
Accepted on 20.12.2009
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Research Journal of Pharmacognosy and Phytochemistry. 2(1): Jan.-Feb. 2010, 57-60