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Pharmacologyonline 1: 731-738 (2009) Kamilla et al.
731
Antimicrobial Activity of Clitoria ternatea (L.) Extracts
L. Kamilla, a S.M. Mnsor, a S. Ramanathan,a and S. Sasidharan,b
aCentre for Drug Research, Universiti Sains Malaysia, 11800 Minden, Penang,
Malaysia.
bInstitute for Research in Molecular Medicine (INFORMM), Universiti Sains
Malaysia, 11800 Minden, Penang, Malaysia.
E-mail: srisasidharan@yahoo.com
Summary
The antimicrobial activities of the methanol extracts of the leaf, stems, flower, seed
and roots of Clitoria ternatea were studied. The extracts of C. ternatea were tested in
vitro against 12 bacterial species, 2 yeast species, and 3 filamentous fungal by the
agar diffusion and broth dilution methods. The leaf and root extracts were found to be
most effective against all of the tested organisms (p < 0.05). The MIC (minimum
inhibitory concentration), MBC (minimum bactericidal concentration) and MFC
(minimum fungicidal activity) values of C. ternatea extracts ranged from 0.3 mg/ml
to 100.00 mg/ml.. The C. ternatea extracts were also screened for tannin,
phlobatannin, flavonoid, antharaquinone, alkaloid, saponin, cardiac glycosides,
volatile oils, steroids and terpenoids. The anthraquinone and saponin were absent in
all the plant material investigated. Hence, C. ternatea can be used to discover
bioactive natural products that may serve as leads in the development of new
pharmaceuticals in food preservation as well as natural plant-based medicine.
Key Words: antimicrobial activity, Clitoria ternatea, food preservation,
phytochemical screening
Introduction
Many extracts from medicinal plant have been known to possess antimicrobial effects
and used for the purpose of food preservation and medicinal purposes (1-3). The
growth of food spoilage and food-borne pathogens in food can lessen nutritional
quality of the food by consuming fat, protein and carbohydrate that are present in the
food for their survival, accordingly lead to food discoloration, heating, mustiness,
biochemical changes, weight loss and accumulation of toxic substance as by products.
Some species of food spoilage and food-borne pathogens are capable to produce
highly toxic compounds in food which can adversely affect the health of humans (4,
5). The way of microbial growth inhibition most appropriate to food is the use of food
preservatives. A perfect food preservative must be inexpensive, corrosion-free, low in
toxicity, and have good antimicrobial activity. The inhibitors of food preservative
available for practical use today are mainly chemical preservations. Nevertheless, the
safety problems with chemical preservatives are receiving extensive attention world
wide, and natural preservatives derived from the natural resources such as medicinal
plants have high potential for the food industry as food preservative (4, 6).
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732
Malaysia, being one of the 12 mega-diversity centres of the world, is reach in
all three levels of biodiversity (7), as species diversity, genetic diversity and habitat
diversity, with many plants used for medicinal and nutritional purpose. In order to
fully tap the natural resources of our country, it is very important to put the
comprehensive utilization and process of natural resources on this agenda. Therefore,
in this study, we focus on the studies on the antimicrobial activities of extracts from
Clitoria ternatea L., belong to the Fabaceae family against general food spoilage and
human pathogens so that new food preservatives can be explored and developed on
the basis of the natural resources.
Materials and Methods
Materials
The leaf, stems, flower, seed and roots of C. ternatea were collected from the
Seberang Jaya, Penang, Malaysia, in January of 2008 and authenticated by the
botanist of the School of Biological Sciences, University Science of Malaysia, where
a specimen was deposited in the herbarium (Voucher number of 11006).
Test microorganisms and growth media
The following Gram-positive and Gram-negative bacteria, yeasts, and molds were
used for antimicrobial activities studies: : Gram-positive bacteria included Bacillus
cereus, Bacillus subtilis, Bacillus thuringiensis, Staphylococcus aureus, Streptococcus
faecalis; Gram-negative bacteria included Escherichia coli, Klebsiella pneumoniae,
Pseudomonas aeruginosa, Salmonella typhi, Enterobacter aerogens, Proteus
mirabilis, Herbaspirillum spp.; yeasts included Candida albicans and Saccharomyces
cerevisiae; molds included Rhizopus spp., Aspergilus niger, Penicillium spp., were
used in this study. The bacterial strains were grown in Mueller–Hinton agar (MHA;
Difco, USA) plates at 37 °C, whereas the yeasts and molds were grown in sabouraud
dextrose agar (SDA; Difco, USA) plates and potato dextrose agar (PDA; Difco, USA)
plates media, respectively, at 28 °C. The stock culture was maintained on nutrient
agar slants at 4 °C.
Extract preparation
The various plant part extracts were prepared by maceration of dried powdered plant
material in methanol solvent (8) for 3 days. 200 grams of powdered leaves, stems,
flowers, seeds and roots were macerated in methanol under stirring conditions for 72
hours. The macerated extracts were then filtered through No. 1 Whatman filter paper.
The methanolic crude extracts were obtained. The crude extracts were then vaporized
to dryness using the rotary evaporator (BUCHI Rotary Evaporator R-110, USA).
Qualitative phytochemical screening
The methanolic crude extracts were subjected to qualitative phytochemical testing for
the detection of major chemical groups. Extracts were screened for the presence of
tannin using Braemer’s test (9, 10), phlobatannin (11), flavonoids using Shinoda test
(9), saponin using frothing test, cardiac glycosides using Keller-Kiliani test (10),
volatile oil (12). The Liebermann-Burchardt test was used to test the presence of
steroids and terpenoids (9). In addition, the salkowski test was used to test the
presence of terpenoids (11).
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Antimicrobial disk diffusion assay
Antibacterial and antifungal activities of the five plant extracts were investigated by
the disk diffusion method (13, 14). The MHA plates, containing an inoculum size of
106 colony-forming units (CFU)/mL of bacteria or 2x105 CFU/mL yeast cells or molds
spores on SDA and PDA plates, respectively, were spread on the solid plates with an
L-shaped glass rod. Then disks (6.0-mm diam.) impregnated with 25 µL of each
extract at a concentration of 100.0mg/mL were placed on the inoculated plates.
Similarly, each plate carried a blank disk by adding solvent control alone in the
centre, and antibiotic disks (6.0-mm diam.) of chloramphenicol, levofloxacin (30
µg/ml, for bacteria) and myconazole (30 µg/ml, for fungal) were also used as a
positive control. All of the plates were incubated at 37°C for 18 hours for bacteria and
at 28°C for 48 hours for fungi.
The zones of growth inhibition around the disks were measured after 18 hours
of in incubation at 37°C for bacteria and 48 hours for fungi at 28°C, respectively. The
sensitivity of the microorganism species to the plant extracts was determined by
measuring the sizes of inhibitory zones (including the diameter of disk) on the agar
surface around the disks. All of the experiments were performed in triplicate. The
results are reported as the average of three experiments. The scale of measurement
was as the following (disc diameter included): ≥ 20 mm zone of inhibition is strongly
inhibitory; 20 – 12 mm zone of inhibition is moderately / mildly inhibitory; and ≤ 12
mm is no inhibitory (Rota et al., 2008). Strains that were inhibited by more than 12
mm were considered for the MIC, MBC and MFC studies.
Determination of minimum inhibitory concentration (MIC)
MIC was determined by broth dilution methods (15). Two-fold serial dilutions
(0.3125 – 100 mg/mlL) of the 5 extracts, with the appropriate antibiotics, were
prepared as positive controls in Mueller-Hinton broth for bacteria and Saboraud
glucose broth for fungi. For broth dilution tests, 0.1 mL of standardized suspension of
bacteria (106 CFU/mL) and fungal cell or spores (5 × 105 CFU/mL) was added to each
tube (containing fractions of 5 extracts at a final concentration of 0.3125 to 100
mg/mL) and incubated at 37 °C for bacteria for 18 h or at 28 °C for fungi for 48 h.
MICs were taken as the average of the lowest concentration showing no growth of the
organism and the highest concentration showing visible growth by macroscopic
evaluation (16). Each assay was performed in triplicate.
Determination of minimum bactericidal (MBC)
A loop full of microbe culture was taken from each test tube and inoculated onto
MHA agar plates. Then, the plates were incubated at 37oC for 24 hours. The MBC
was recorded as the lowest concentration of the extract that gave complete inhibition
of colony formation of the test bacteria at the latter cultivation. Each assay was
carried out in triplicate.
Determination of minimum fungicidal concentration (MFC)
The hyphal growth inhibition test was used to determine the antifungal activity of the
C. ternatea extract against A. niger and Rhizopus sp. The procedure used in the hyphal
growth inhibition test has been described previously (17). Briefly, dilutions of the test
solutions dissolved in methanol were added to sterile melted Potato Dextrose Agar at
45 ºC to give final concentrations of 100, 10, 1, 0.8, 0.6, 0.4, 0.2 and 0.1 mg/mL. The
resultant solution was thoroughly mixed and approximately 15mL was poured onto
Pharmacologyonline 1: 731-738 (2009) Kamilla et al.
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the Petri plate. Plugs of 1 mm of fungal mycelium cut from edge of active growing
colony were inoculated in the center of the agar plate and incubated in a humid
chamber at 25ºC. Control cultures received an equivalent amount of methanol. Three
replicates were used for each concentration. Radial growth was measured when the
control colonies almost reached 1.5cm. Results were expressed as the percentage of
hyphal growth inhibited (18).
Statistical analysis
The triplicate data were subjected to an analysis of variance for a completely random
design using Statistical Analysis System (SPSS version 12) programs. Multiple-range
test was used to compare the difference among means at the level of 0.05.
Results and Discussion
The results of the qualitative phytochemical screening to test the presence of tannin,
phlobatannin, flavonoid, antharaquinone, alkaloid, saponin, cardiac glycosides,
volatile oils, steroids and terpenoids in the extracts from various parts of C. ternatea
are shown in Table 1. The preliminary phytochemical screening study revealed that
the leaf of C. ternatea contains moderate level of tannin, cardiac glycosides and
steroids and mild level of alkaloid. There were no phytochemicals noted in the stem.
Both the flowers and seeds of C. ternatea contain phlobatannin, flavonoid, terpenoid
at moderate levels. In addition, the seeds contain moderate presence of alkaloid and
mild presence of volatile oil. The roots contain small amount of flavonoid, volatile oil
and terpenoid. These findings had provided a general understanding of the
antimicrobial properties of the extracts tested in this study.
Table 1. Phytochemical Screening of Secondary Metabolites from Clitoria ternatea.
Secondary
metabolites Name of the test Leaf Stem Flower Seed Root
Tannins Braemer’s test ++ - - - -
Phlobatannins - - - ++ ++ -
Flavonoids Shinoda test - - ++ ++ +
Anthraquinone KOH test - - - - -
Alkaloid Dragendorff test + - - ++ ++
Saponin Frothing test - - - - -
Cardiac
glycosides
Keller-Kiliani test ++ - - - -
Volatile oils - - - - + +
Liebermann Burchardt test + - - - - Steroids
Steroids test ++ - - -
Liebermann Burchardt test - - ++ ++ + Terpenoids
Salkowski test - - ++ ++ ++
‘++’ Moderate, ‘+’ Present mildly, ‘-’ Absent.
Pharmacologyonline 1: 731-738 (2009) Kamilla et al.
735
The results of the antimicrobial screening assay of the extracts of all parts of C.
ternatea are shown in Table 2. All parts of plant included in the present study were
found to be active on at least one of the selected microbial strains tested. In general,
among the tested microbial strains, bacteria were found to be more sensitive to the test
extracts than fungi. The preliminary disk diffusion assay of C. ternatea extracts
against microbes showed that the leaf and root extracts were more favourably
compared to the rest of the extracts (which inhibited 16 out of 17 test microorganism
with zone of inhibition 10-25 mm).The antimicrobial screening assay of C. ternatea
extracts against yeast showed that, all the extracts inhibited C. albicans (with zone of
inhibition 9-20 mm) tested. As shown in Table 2 no matter what extract was used, of
all the fungal strains included in the test found to be virtually sensitive to all extracts
except the A. niger only sensitive to the leaf extract. The antimicrobial activities of the
herbal drugs (leaf, stems, flower, seed and roots) were found to be less effective to the
positive controls (chloramphenicol, levofloxacin for bacteria and myconazole for
fungus). The negative controls methanol was devoid of any antimicrobial activity.
The different extract of C. ternatea showed different spectrum of activities, especially
by the disk diffusion method where the microorganisms tested produced difference
zones of inhibition. The extracts of difference parts of C. ternatea showed different
efficacy against the tested microorganisms. These differences could be due to the
nature and level of the antimicrobial agents present in the extracts and their mode of
action on the different test microorganisms (19).
Literature review on the phytochemical constituents of these plants revealed
that various secondary metabolites like flavonoids, anthocyanin glycosides,
pentacyclic triterpenoids and phytosterols have been isolated from this plant (20). A
protein designated as ‘finotin’ has been isolated from C. ternatea seeds and reported
to have antifungal, antibacterial and insectidal properties (21). It is possible that this
compound was mainly responsible for the observed antimicrobial effects in this study.
Based on the initial antimicrobial screening assay, those extract showed
positive results were selected for further studies for the determination of MIC, MBC,
and MFC because they were found to be active against bacterial, fungal and yeast
strains tested. The MICs, MBCs and MFCs of the extracts are shown in Table 3. The
MIC, MBC and MFC values of C. ternatea extracts ranged from 0.3 mg/ml to 100.00
mg/ml. The common food borne fungal species such as A. niger and Rhizopus sp.,
showed a lower MBC values compared with bacterial species tested in this study with
the range of 0.4 mg/mL to 0.8 mg/mL. However the reference antibiotics such as
chloramphenicol, levofloxacin and myconazole showed lower MIC, MBC and MFCs
values (10 µg/mL to 1000 µg/mL) compared with extracts tested. But, being crude
extracts, the overall antimicrobial activity screening results are still indicative of the
potential of these herbal drugs for the purpose of food preservation and medicinal
purposes against all tested microorganism.
Conclusion
In conclusion, all the extracts investigated possessed activity against at least
one strain of bacteria and/or fungi. Further studies aimed at the isolation and
identification of active substances from the methanol extracts of C. ternatea could
also disclose compounds with better value for food preservation as well as natural plant-
based medicine.
Pharmacologyonline 1: 731-738 (2009) Kamilla et al.
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Table 2. Antibacterial Activities of Clitoria ternatea.Extracts
Zone of growth inhibition (mm) a
Clitoria ternatea extracts (100.00 mg/ml) Reference antibiotic (30 µg/ml)
Strains
Leaf Stem Flower Seed Root
Chloramphenicol Levofloxacin Myconazole
-
13.7±1.5 12±2 14±1 12.3±0.6 14.3±1.5 19.7±0.6 23.7±1.1 -
11.3±1.5 12±1 12.7±1.1 12±2.6 11.3±3.5 21.6±0.6 22 -
10±1 14.3±1.1 15.7±0.6 14±2.6 19±1 24±1 25.33±9.8 -
11 12±1 13±1 12.7±1.1 12.3±1.5 19.6±2.5 25.7±3.5 -
Gram positive bacteria
Bacillus cereus
Bacillus subtilis
Bacillus thuringiensis
Staphylococcus aureus
Streptococcus faecalis 14.7±1.5 16±1 12±1 12.3±2.5 13.3±0.6 19.33±2.9 26.7±0.6 -
13.3±1.1 14.3±1.1 13.3±0.6 12.7±1.1 15 17±1.7 26.33±1.5 -
13.3±1.1 11.7±1.1 12.7±0.6 13±1 14.7±1.5 18.33±2.5 16.7±0.6 -
13.3±0.6 10.7±2 11.3±1.5 12.3±2 9±2.6 23±2.6 25.33±0.6 -
21±2.3 18.7±2.1 10.3±1.1 11.3±1.7 28.7±4.5 25.6±1.1 25.33±0.6 -
13.3±2.1 12.3±0.6 13±1 12.7±1.5 14 14±1 26.33±0.6 -
18.7±1.5 19.3±0.6 13.7±2.9 15.7±4 23.7±1.5 19±1.7 24.7±2.3 -
Gram negative bacteria
Escherichia coli
Klebsiella spp
Pseudomonas aeruginosa
Salmonella typhi
Enterobacter aerogens
Proteus mirabilis
Herbaspirillum spp. 14.7±1.1 12.7±0.6 11.3±2.3 14.3±0.6 14.3±3.5 11±1 21.7±2.5 -
14 - 19 10.7±1.5 25±2.6 - - 21±1.7
- - - -
11.33±0.6 - -
11
11.67±0.6 11.67±2.1 11±1 9.67±1.1 11.33±1.5 - -
14.33±3.8
19.89±0.5 - - - - - - 20.33±3.2
Fungi
Candida albicans
Saccharomyces cerevisiae
Rhizopus
Aspergillus niger
Penicillium spp. 11 8.7±0.6 8.33±0.6 9 9.67±0.6 - -
10.33±2.1
a Values for zone of growth inhibition are presented as mean±SD from the experiments in triplicate; ‘-‘ inhibition zone was not noted.
.
Pharmacologyonline 1: 731-738 (2009) Kamilla et al.
737
Broth dilution method (mg/ml)
Leaf Stem Flower Seed Root Reference antibiotic (ug/ml)
Strains
MIC MBC/
MFC
MIC MBC /
MFC
MIC MBC /
MFC
MIC MBC /
MFC
MIC MBC /
MFC
Chloramphenicol Levofloxacin Myconazole
MIC MBC MIC MBC MIC MFC
3.125 6.25 >100 >100 >100 >100 25 50 50 25 >1000 >1000 <31.25 <31.25 - -
12.5 25 50 100 >100 >100 50 100 <0.312 <0.312 >1000 >1000 <31.25 <31.25 - -
50 100 >100 >100 12.5 25 3.125 6.25 <0.312 <0.312 500 1000 <31.25 <31.25 - -
50 100 >100 >100 >100 >100 50 100 25 50 >1000 >1000 31.25 62.5 - -
Gram positive bacteria
Bacillus cereus
Bacillus subtilis
Bacillus thuringiensis
Staphylococcus aureus
Streptococcus faecalis 50 100 >100 >100 >100 >100 50 100 25 50 >1000 >1000 <31.25 <31.25 - -
50 100 >100 >100 >100 >100 50 100 0.125 0.25 >1000 >1000 <31.25 <31.25 - -
>100 >100 >100 >100 >100 >100 50 100 50 100 125 250 <31.25 <31.25 - -
50 100 >100 >100 50 100 25 50 50 100 >1000 >1000 <31.25 <31.25 - -
12.5 25 >100 >100 >100 >100 50 100 25 50 >1000 >1000 <31.25 <31.25 - -
50 100 >100 >100 >100 >100 50 100 1.56 3.125 250 500 31.25 62.5 - -
12.5 25 >100 >100 50 100 50 100 25 50 250 500 31.25 62.5 - -
Gram negative bacteria
Escherichia coli
Klebsiella spp
Pseudomonas aeruginosa
Salmonella typhi
Enterobacter aerogens
Proteus mirabilis
Herbaspirillum spp. 50 100 50
100 >100 >100 50 100 50 100 >1000 >1000 <31.25 <31.25 - -
25 50 >100 >100 >100 >100 >100 >100 25 50 - - - - 500 1000
ND ND ND ND ND ND ND ND ND ND - - - - ND ND
0.8 1.6 0.8 1.6 0.8 1.6 0.8 1.6 0.8 1.6 - - - - ND ND
0.4 0.8 ND ND ND ND ND ND ND ND - - - - <10 <10
Fungi
Candida albicans
Saccharomyces cerevisiae
Rhizopus
Aspergillus niger
Penicillium spp. 0.8 1.6 0.8 1.6 0.8 1.6 0.8 1.6 0.8 1.6 - - - - ND ND
a Result was the average of records determined by both agar and broth dilution methods; ND= not determined.
Table 3. Minimum Inhibition Concentration, Minimum Bactericidal Concentration and Minimum Fungicidal Concentration of The Clitoria Ternatea Extracts.
Pharmacologyonline 1: 731-738 (2009) Kamilla et al.
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