JMRI 2005; 26 (1): 92 – 96
Journal of the Medical Research Institute
JMRI Vol.26 No.1; 2005
Journal of The
Medical
Research
Institute
Original Article
Antimicrobial Activity of Plectranthus Tenuiflorus Extracts
Saleh M. Al-Garni and Saleh A. Kabli
Department of Biological Sciences, Faculty of Science, King Abdulaziz University, P. O. Box
80203, Jeddah, 21589, Saudi Arabia.
Abstract:
The antimicrobial activity of different extracts of
Plectranthus tenuiflorus, on Gram +ve (Staphylococcus aureus) and Gram -ve (Escherichia coli and
Pseudomonas aeruginosa) bacteria, Candida
albicans, Aspergillus fumigatus, and A. niger
revealed that essential oil extract of the plant (85%
thymol ) showed higher antimicrobial activity on all
the tested organisms than pure thymol. Aqueous and
organic solvents extracts showed antimicrobial
activity on the tested organisms, but not comparable
to the high activity obtained by essential oil extract.
Aqueous extracts using cold and boiling water were
more efficient in inhibiting the growth of P.
aeruginosa and the two moulds, on the other hand,
organic solvents, polar and non polar (ethyl alcohol
and diethyl ether), extracts showed stronger
antimicrobial activity on E.coli, S. aureus and C.
albicans than aqueous extracts.
Key words: Plectranthus tenuiflorus, Antimicrobial
activity, Aqueous extraction, Organic solvents
extraction.
Introduction
Plectranthus tenuiflorus (Labiatae) plant is a small
downy, very leafy herb, with stems about 60 cm long
and small lilac-blue flowers. It is widely distributed in
Saudi Arabia [1]. The plant is commonly used in folk
medicine to treat different diseases, e.g. respiratory
system infections, inflammation of ear and throat and
abdominal disorders[2]. Several substances were
extracted and identified from Plectranthus species.
These materials were mainly thymol and different
types of diterpenoids [3-6].
The antimicrobial activity of Plectranthus species
extracts was studied against bacteria, fungi and
viruses[3-5,7-10]. On the other hand, some workers
revealed that oily extract of P. deloliatus leaves had
no antimicrobial activity against bacterial flora of
milk[11].
Therefore, the present work aimed to study the
antimicrobial activity of local P. tenuiflorus extracts
on some pathogenic microorganisms (Staphylococcus
aureus, Escherichia coli, Pseudomonas aeruginosa,
Candida albicans, Aspergillus fumigatus and A.
niger)[12]. Also, to elucidate the most proper method
for extraction of plant materials of high antimicrobial
activity, using broth medium technique.
Material and Methods
Plant material:
P. tenuiflorus (Labiatae) was collected from the
garden of King Abdulaziz University. Its identification was clarified by the staff member of
taxonomy at the Faculty of Science, Biological
Sciences Department, King-Abdulaziz University,
according to Collenette[1], after comparison with the
herbarium samples at the department.
Extraction of plant materials:
About 5kg of fresh leaves were collected at the
same time from one place, chopped into pieces and
homogenously mixed and used for extraction.
Different methods of extraction [13] were used, as
follows:
1-Extraction of essential oil:
Fresh leaves (3kg) of P. tenuiflorus were cut into
pieces and subjected to steam distillation. The
distillate was then extracted with petroleum ether
(40-60C). The resulting extract was dried on
anhydrous sodium sulphate. Petroleum ether was
removed using a rotary evaporator and the essential
oil was obtained [14]. Essential oil was dissolved in
petroleum ether to prepare the different
concentrations to test their antimicrobial activity.
2-Aqueous extraction:
a)Cold water
Fifty grams of chopped leaves were blended in a
blender with 100 ml distilled water for 15min and
centrifuged at 4000 rpm for 10 min. The filtrate was
completed to 100 ml using distilled water and
sterilized using bacterial filters.
b) Boiling water:
Fresh chopped leaves (50g) pieces were boiled in
a distillatory flask for 15 min in 100 ml distilled
water.
93
Al-Garni and Kabli
After cooling, the filtrate was separated by
centrifugation at 4000 rpm for 10 min, completed to
100 ml, and sterilized using bacterial filters.
3-Extraction using volatile organic solvents:
Extraction using polar (ethyl alcohol) and nonpolar (diethyl-ether) volatile organic solvents was
carried out.
a) Alcohol extraction:
Fifty grams of P. tenuiflorus leaves pieces were
extracted by boiling 96% ethyl alcohol (100ml) in
distillatory flask for 15 min. The alcoholic extract
was separated by filtration and completed to 100 ml
volume with 96% ethyl alcohol and sterilized through
bacterial filter.
b) Diethyl ether extraction:
Instead of 96% ethyl alcohol, diethyl – ether was
used for extraction by the same method. The extracts
were kept in a refrigerator at 10°C until used.
Microbial cultures growth conditions:
Test microorganisms included Gram +ve cocci:
Staphylococcus aureus (ATCC 118592), and Gram
-ve bacilli: Escherichia coli (ATCC 25922) and
Pseudomonas aeruginosa (ATCC 10145). The yeast
Candida albicans (ATCC 90028) and both
Aspergillus fumigatus and Aspergillus niger were
also tested.
Cultures of bacteria were grown in nutrient broth
(Difco) at 37C and maintained on slopes of nutrient
agar (Difco) at 4C. While, the yeast and mould fungi
were grown in Czapek's broth (Difco) at 28oC and
maintained on slopes of Czapek's agar (Difco) at 4oC.
Antimicrobial activity assay:
The different extracts of P. tenuiflorus were tested
for their antimicrobial activity using test tubes
method, each containing 8 ml of the sterilized broth
medium (nutrient for bacteria and Czapek for fungi).
Each test tube was inoculated with one ml containing
106 bacterial cells or 104 yeast cells/ml or 103 mould
spores / ml. Each tube accepted one ml of the tested
extract containing (0, 5, 10, 20 mg) prior to
microorganism inoculation. The tubes were vortexed
and incubated at 37oC for 24h for bacteria and at
28oC for 72h for fungi.
The inocula were freshly prepared; 24h old
cultures of bacteria in nutrient broth, 72h old yeast
hhhhhhhhhhhhhhhhhhhhhhhhhhhh
cultures in Czapek's broth, and 96h old fungal
cultures.
After incubation, one ml from each tube, after
vortexing, was mixed with cool (45C) sterilized
specific medium (nutrient agar or Czapek's agar) in
Petri-dish, incubated for 48h for bacteria at 37C and
at 28C for fungi for 4 days. Thereafter, CFU of the
tested organisms were estimated [15]. Three replicats
from each concentration were made and the recorded
data were the arithmetic mean.
Identification of major constituents of essential
oil:
The steam distilled from P.tenuiflorus leaves was
examined using a Carlo Erba 800 gas chromatograph
fitted with a Carlo Erba MD800 mass spectrometer
(GC.MS)[16,17]. .
Statistical Analysis:
With respect to observed antimicrobial activities,
the values of difference between two means that
would just achieve significance (least significance
differences) were calculated [18].
Results
Chemical composition of the essential oil:
The essential oil, steam distilled from leaves of
P.tenuiflorus, was analyzed by GC. MS. The main
component of the oil was identified as thymol (85%),
beside minor components of diterpenoids.
Antimicrobial activity of essential oil:
The antimicrobial activity of the steam distillate
(essential oil) from the leaves of P. tenuiflorus
(consists of 85% thymol) as compared with pure
thymol (SIGMA) on the test organisms (Table I)
revealed that the essential oil was more efficient in
inhibiting the growth of the different tested
organisms at concentrations of 5, 10mg/ 100ml. On
the other hand, 20mg / 100ml was lethal (no growth)
to the tested organisms. However, 10mg/100ml of
both thymol and essential oil were also lethal to the
tested mould fungi; A. fumigatus and A. niger as well
as to S. aureus bacterium, while only essential oil
was lethal to Candida albicans in concentration of
10mg/100ml.
Table (1). Antimicrobial activity of essential oil extract of Plectranthus tenuiflorus on colony forming unit (CFU) of the
test organisms, as compared with pure thymol
Test
material
None
(cntrol)*
Thymol
Essential
oil
extract
L.S.D
at 5%
Conc.
(mg/100ml)
E.coli
X 104
P.aeruginosa
X 104
CFU of tested organisms
S. aureus
C.albicans
X 104
X 103
A.fumigatus
X 102
A.niger
X 102
0.0
45.3 2.35
43.7 4.60
39.5 3.96
31.3 3.52
49.1 3.65
59.2 3.17
5
10
20
5
10
20
34.1 2.19
18.7 4.14
0.0
29.5 2.36
11.9 1.62
0.0
39.5 2.48
29.8 4.39
0.0
40.6 3.75
25.5 1.32
0.0
9.1 1.71
0.0
0.0
4.5 0.34
0.0
0.0
17.5 1.94
7.5 1.28
0.0
13.7 1.17
0.0
0.0
18.4 2.49
0.0
0.0
15.2 1.90
0.0
0.0
25.6 2.13
0.0
0.0
20.4 1.67
0.0
0.0
5.71
2.37
3.92
2.15
6.10
4.08
*One ml of petroleum-ether.
٩٤
JMRI 2005; 26 (1): 92 – 96
Antimicrobial activity of aqueous extracts:
The antimicrobial activity of aqueous extracts
(cold and boiling water) of P. tenuiflorus leaves
(Table II). Results were recorded at the different
tested extracts concentrations. Thus, boiling water
extract resulted in inhibition percentages of 82.6,
33.4, 67.1 and 79.6 for P.aeruginosa, C.albicans,
A.fumigatus and A.niger, respectively, as compared
to 36.9, 6.8, 11.3 and 39% inhibition for the same
organisms using cold water extract.
growth of P.aeruginosa and both mould fungi than
polar extracts. Thus, alcohol extracts resulted in
inhibition percentages of 48.2, 57.5, and 51 for
E.coli, S.aures and C.albicans, respectively. While,
ether extracts gave inhibition percentages of 33.3, 51
and 26.5 for the same organisms, respectively. Both
extracts gave comparable results for the inhibition
of A.fumigatus. The results (Tables II,III) generally
indicated that organic solvents extraction gave
substances (quantity and / or quality) more inhibitory
for the growth of E.coli, S. aureus and C. albicans
Antimicrobial activity of organic solvents extracts: than aqueous extracts (mean of inhibition percentages
Two different volatile organic solvents, one polar were 40.75, 54.25 and 38.75, respectively, as
(ethyl alcohol) and the other non – polar (diethyl- compared to 23.65, 38.55, and 20.1 for water
ether) were used to extract materials that may have extract), while aqueous treatments result in materials
antimicrobial activity against the tested organisms. (quantity and / or quality ) more efficient against the
The results (Table III) indicated that the polar solvent growth of P. aeruginosa and mould fungi than
(ethyl alcohol) extracts substances with higher organic solvents extracts (mean of inhibition
concentrations and / or not extracted by diethyl- ether percentages were 59.75, 39.2, and 59.3,respectively,
that have high antimicrobial activity against E.coli, for aqueous treatment as compared to 25, 35.7, and
S.aureus, and C.albicans , while non- polar (diethyl- 38.2 for organic solvents extraction for P.aeruginosa,
ether) extracts were more inhibitory against the A.fumigatus and A.niger, respectively) .
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Table (II): Antimicrobial activity of aqueous extracts (cold and boiling water) of Plectranthus tenuiflorus on colony
forming unit (CFU) of the tested organisms
Boiling water
Cold water
Extract
Conc. (mg/100ml)
0.0
5
10
20
L.S.D. at 5%
% of inhibition*
0.0
5
10
20
L.S.D. at 5%
% of inhibition*
Mean of inhibition**
E.coli
X 104
51.3 4.13
47.9 3.99
28.5 2.38
25.42.09
4.32
50.5
51.3 3.17
51.0 2.87
50.5 3.04
49.8 3.02
12.02
2.9
23.65
P.aeruginosa
X 104
49.3 4.05
47.53.79
38.2 3.56
31.1 2.31
5.33
36.9
49.3 2.92
41.7 2.67
23.4 2.28
8.60.57
19.35
82.6
59.75
CFU of test organisms
S. aureus
C.albicans
X 104
X103
45.4 3.46
51.5 2.63
39.02.37
51.22.39
31.22.03
49.02.58
24.61.28
48.0 2.38
3.09
4.50
45.8
6.8
45.4 3.18
51.5 3.38
44.7 2.98
49.8 3.51
40.2 2.71
41.7 2.39
31.2 1.75
34.32.59
18.80
11.17
31.3
33.4
38.55
20.1
* % of inhibition= (CFU at 0.0 conc. - CFU at 20 mg/100ml)
CFU at 0.0 conc.
** Mean of inhibition = (Sum of inhibition % of cold and boiling water)
2
A.fumigatus
X 102
57.7 3.28
56.44.26
52.93.31
51.2 3.02
6.54
11.3
57.73.89
42.9 3.23
30.9 2.44
19.0 2.11
2.28
67.1
39.2
A.niger
X 102
59.3 4.8
46.54.19
39.12.56
36.2 2.62
5.76
39
59.3 3.45
33.5 2.01
31.2 2.14
12.10.49
21.51
79.6
59.3
Table (III): Antimicrobial activity of volatile organic solvents (ethyl alcohol and diethyl ether) extracts of Plectranthus
tenuiflorus on colony forming unit (CFU) of the tested organisms
Diethyl ether
Ethyl alcohol
Extract
Conc. (mg/100ml)
0.0
5
10
20
L.S.D. at 5%
% of inhibition
0.0
5
10
20
L.S.D. at 5%
% of inhibition
Mean of inhibition
E.coli
X 104
49.6 4.07
42.4 2.95
34.1 1.63
25.71.28
15.26
48.2
44.4 2.97
41.5 2.74
37.2 2.05
29.6 1.57
3.51
33.3
40.75
P.aeruginosa
X 104
46.2 3.93
44.7 2.42
39.2 2.03
37.4 1.47
11.71
19
51.2 2.92
42.8 2.43
42.4 2.43
35.4 2.17
4.16
31
25
CFU of test organisms
S. aureus
C.albicans
X 104
X 103
41.6 2.88
23.5 1.51
35.4 2.28
22.6 1.92
21.3 1.35
19.4 1.16
17.7 1.13
11.5 0.84
17.96
9.34
57.5
51
42.7 2.46
26.4 1.71
40.4 2.69
26.3 2.12
35.1 1.97
22.5 1.18
20.9 1.41
19.4 0.74
4.60
2.52
51
26.5
54.25
38.75
A.fumigatus
X 102
45.9 2.79
44.2 2.68
34.8 1.83
30.2 1.27
10.45
34.2
49.7 3.15
48.3 3.10
42.6 2.24
31.2 1.82
3.12
37.2
35.7
A.niger
X 102
54.6 3.83
49.3 3.43
44.2 3.18
38.1 1.59
7.32
30.2
64.3 3.77
62.7 2.99
41.4 2.40
34.6 1.74
4.70
49
38.2
٩٥
Al-Garni and Kabli
Discussion
It was reported that thymol was the main
component of essential oils of P. tenuiflorus,
harvested in Saudi Arabia [6,17]. Thymol as a
phenolic alcohol inhibits the growth of several
microorganisms and therefore, used in several
antiseptics and pharmaceutical preparations[10].
The components of essential oil of P. tenuiflorus,
(in addition to its 85% thymol content) have more
lethal effect on the tested organisms than pure thymol
at the different tested concentrations. The
antimicrobial activity of essential oil extract of
Plectranthus species against different microorganisms was reported [10]. The plant species are
rich in essential oils and diterpenoids which are the
common secondary metabolites. Several diterpenoids
were identified from the plant species, beside a group
of long-chain alkylphenols and other miscellaneous
constituents [6]. These components may reflect the
highest antimicrobial activity of P. tenuiflorus
essential oil (85% thymol beside minor components
of diterpenoids) as compared with pure thymol.
Boiling water extraction was able to extract
substances with more inhibitory action for the growth
of P. aeruginosa, yeast and mould fungi than cold
water treatment, i.e. boiling leads to extraction of
more substances and / or higher amounts of the
antimicrobial substances. However, cold water
extract was more efficient in inhibiting E.coli and S.
aureus. This may be attributed to the denaturation of
substances due to boiling some materials that were
more inhibitory to these bacteria.
The aqueous extract of plants used in traditional
medicine was used to study its mutagenic properties,
and antimicrobial activity [8,19]. It was reported that
water is almost universally the solvent used to extract
activity, either cold or boiling [20]. The validity of
both aqueous and organic solvents for extraction of
plant materials of antimicrobial activity was also
reported [21].
The use of organic solvents to extract plant
materials of antimicrobial activity was reported
[7,9,10 &22].
In accordance with these findings, it was reported
that in most cases organic solvent plant extracts have
higher antimicrobial activity than aqueous extracts
[10].
Conclusion
Plecturanthus tenuiflorus (Labiatae) plant is
widely distributed in Saudi Arabia. The plant has
several substances with antimicrobial activity against
a group of disease causing microorganisms including
bacteria, yeast and fungi. The essential oil of the
plant (containing 85% thymol) has a complete
bactericidal effect on the tested organisms at
concentrations less than 20 mg /100ml with higher
activity than pure thymol. Both aqueous and organic
solvents extracts showed antimicrobial activity
against the test organisms, but their efficiency can't
be compared with the results of essential oil of the
plant. Generally, aqueous extraction succeeded to
extract substances from the stand point of quality and
quantity more efficient to inhibit the growth of P.
aeruginosa, A. fumigatus and A. niger, while organic
solvents extracts were the best to inhibit the growth
of E.coli, S. aureus and C. albicans.
The results revealed that both aqueous and
organic solvents were efficient for extraction of
antimicrobial substances from P. tenuiflorus. Their
activity was depending on the tested organism.
However, essential oil extracts showed promising
bactericidal and fungicidal effect on the tested
organisms.
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Saleh Kabli