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Abstract
In Mozambique, the bulb of Gladiolus psittacinus Hook is used in treating diarrhea, dysentery,
gonorrhea, renal and rheumatic pains, etc. In this study, was evaluated the antimicrobial activity
of different extracts and fractions of dried and fresh bulb against 6 bacterial strains:
Staphylococcus aureus ATTC 25923, Escherichia coli ATCC 25922, Klebsiella pneumoniae
ATCC 15380, Pseudomonas aeruginosa ATCC 27953, Shigella flexneri ATCC 12022 and 2
fungi: Candida albicans and Saccharomyces cerevisiae, and the interaction Ciprofloxacin
Extract. Most of the bulb extracts and fractions showed strong inhibitory activity against Candida
albicans, Saccharomyces cerevisiae and Pseudomonas aeruginosa. The aqueous extract revealed
antagonism with ciprofloxacin while the juice (from the fresh bulb) showed additive effect.
Keywords: Gladiolus psittacinus; Iridaceae; Antimicrobial; Bulb.
Introduction
Since the antiquity, the man uses natural resources such as vegetables, for various
purposes, mainly food and medicine. In this constant man-environment interaction, the need has
become an important factor in the development of folk medicine. Plants have been used as the
basis of many traditional medicine systems throughout the world for thousands of years. They
continue to provide mankind with new remedies. Every region has its own history of traditional
medicine, for example traditional Chinese medicine, Arabic traditional medicine and African
traditional medicine.
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Fresh samples of Gladiolus psittacinus Hook bulbs were collected in Chiboene, Moamba
district, Maputo province in July 2006 and in Bilene district, Gaza province in March 2011. The
samples authentication was made in the herbarium of the Department of Biology Eduardo
Mondlane University and in the herbarium of the Botany Department Institute of agricultural
Research of Mozambique (voucher specimen Nr. 48529).
Samples preparation
Fresh Juice:
After collection, the fresh bulbs were washed, cut into small pieces and mechanically crushed
with the help of a centrifuge juice in order to extract the fresh juice.
Dried powdered bulb:
The fresh bulbs were washed, cut into small pieces and were dried in the oven for 7 days at a
temperature of about 50 C and then ground with the help of an electric grinder.
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300 g of dried powdered bulb was percolated for 4 days in a 500 ml percolator using as
solvent a mixture of methanol and water (9:1). The resulting extract (3 l) was filtered with suction
on a borosilicate glass funnel fitted with a filter membrane and stored at 4 C in a fridge for 48
hours for precipitation of the fatty material.
The extract with precipitated material was left at room temperature for 1 h and then
decanted and filtrated with Whatman filter paper (No. 1) and concentrated under reduced
pressure in a rotary evaporator BUCHI at 40 C until the formation of a pasty mass (methanol
extract). 100 g of the methanol extract was suspended in a volume of 200 ml of mixture methanol
/ water (1:1) in 250 ml Beaker and quantitatively transferred to a separatory funnel of 500 ml and
then submitted to liquid / liquid partition using solvents with increasing polarities (n-hexane,
chloroform, ethyl acetate and n-butanol).
The hexane fraction (HEX) was obtained by liquid / liquid partition using 3x100 ml of nhexane. And then separated into a 500 ml flask and concentrated in a rotary evaporator under
reduced pressure at 40oC. The chloroform fraction (CHL) was also obtained by liquid / liquid
partition with 3x100ml of chloroform. And then separated into a 500 ml flask and concentrated
to dryness in a rotary evaporator at 40o C. The hydromethanolic solution was transferred to a
round bottom flask of 500 ml and concentrated until elimination of methanol.
The aqueous extract was further entirely subjected to liquid / liquid partition with 3x100
ml of ethyl acetate, followed by 3x100 ml of n-Butanol, yielding three fractions: ethyl acetate
fraction (EA), n-butanol fraction (Bu) and aqueous fraction (Aq.Fr.) respectively. The two
organic fractions were also concentrated in a rotary evaporator under reduced pressure at 40 C.
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20 g of the dried powdered bulb was introduced into a cellulose cartridge Whatman (30
mm x 100 mm) and subjected to Soxhlet extraction with 400 ml of the mixture dichloromethane methanol (1:1). After 24 hours the extract was filtered by suction on a glass funnel with a
membrane filter, then through a filter paper, and concentrated in rotary evaporator at low pressure
until dryness. The dried extract was placed in a desiccator until obtaining constant weight, and
after 7 days the yield of extraction was determined
30 g of the fresh bulb, pre-treated with distilled water and cut into small fragments, was
milled in Kenwood blender and transferred quantitatively to the cellulose cartridge. Thereafter,
the material was extracted in a Soxhlet using as solvent 400 ml of distilled water. After 24 hours,
the extract was recovered from the flask, cooled to room temperature and filtered with cotton
wool in glass funnel, followed by filter paper and taken to carry out biological testing, after a
preliminary purification by filtration with sterile cellulose acetate filter (GVS Filter Technology,
USA) 0.45 mm in porosity and 25-mm diameter.
50 g of the fresh bulb treated with fresh distilled water was mixed with 50 g of water and
milled in Kenwood blender. The resulting sample was transferred to a 250 ml Beaker and filtered
with cotton wool in a glass funnel. The filtrate was centrifuged and the resulting extract was
purified with cellulose acetate filter (GVS Filter Technology, USA) porosity of 0.45 mm and
diameter of 25 mm, and transferred to a sterile glass container and taken to carry out biological
testing.
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In that extraction, 20 g of fresh juice was treated with methanol (600ml) for 24 hours.
After filtration, the filtrate was evaporated to dryness in rotary evaporator, leading to a yellow
residue after total evaporation of the solvent. This residue was dissolved in distilled water (20ml)
and then proceeded to the successive extractions (3 times respectively), first with the solvent
petroleum ether (PE), then with chloroform (CHL), followed by ethyl acetate (EA ) and finally
with n-butanol (Bu) with equal volumes of 100ml each. Petroleum ether (PE), chloroform (CHL),
ethyl acetate (EA) and n-butanol (Bu) fractions were concentrated on the rotary evaporator to
give petroleum ether residue ( white ), chloroform residue (pale yellow), ethyl acetate residue
(yellow and butanolic residue (intense yellow color), which have been subjected to preliminary
phytochemical tests.
Phytochemical Analysis
The extracts and fractions were subjected to qualitative phytochemical tests for alkaloids,
tannins (hydrolysable and condensed), coumarins, iridoids, anthraquinones, flavonoids, saponins,
steroids and triterpenoids, amino acids, proteins and carbohydrates adopting the procedures
described by Gomes & Silva (2007), Duarte; Fonte & Santos (2010) and Khan et al (2010).
Antimicrobial assay
Bacterial cultures reference standard (ATCC) and the yeast Candida albicans (isolated
from vaginal discharge) were obtained from the Microbiology Laboratory of the Central Hospital
of Maputo. The yeast Saccharomyces cerevisiae was obtained from commercial packaging yeast.
The tests were performed using a standard method recommended for this purpose (NCCLS). Six
bacterial strains were used in the tests, Staphylococcus aureus (ATTC 25923), Escherichia coli
(ATCC 25922), Klebsiella pneumoniae (ATCC 15380), Pseudomonas aeruginosa (ATCC
27953), Shigella flexneri (ATCC 12022) and Salmonella typhimurium (ATCC 14028). The
culture media used were Nutrient broth, Sabouraud Dextrose Broth and Mueller Hinton Agar.
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The assay was performed using the disk diffusion method where each sterile disk of
Watman filter paper of 6.0 mm in diameter was impregnated with 10 l aliquots of the extract at
concentrations of 1 mg / disk to the total extract and the respective fractions.
The aqueous extract and juice were placed in Petri dishes containing sterile paper discs
and allowed to soak for two hours prior to testing. The suspensions containing the bacteria were
prepared separately suspending a colony of each microorganism taken from stock samples with
the aid of a platinum loop in test tubes containing nutrient broth and incubated for 24 hours at 37o
C.
After were adjusted according to the turbidity of 0.5 in McFarland scale, by visual
comparison
with
standard
tube
containing
standardized
suspension.
The adjusted suspensions were separately dispersed in a Petri dish containing Mueller Hinton
Agar.
The Petri dish was divided into four equal portions and placed aseptically one disc of each
impregnated
with
plant
extract
in
the
center
of
each
division.
Plates were also placed in the disks impregnated with ciprofloxacin, DMSO and pure distilled
water respectively. The plates were incubated for 24 h at 37 C and then the reading of the
diameter of zones of inhibition. All tests were done in triplicate.
The suspensions of the yeasts Candida albicans and Saccharomyces cerevisiae were
prepared separately by suspending a colony of each microorganism in test tubes containing
Sabouraud Dextrose Agar (SDB) incubated at 37 C for 48 hours, then standardized according to
the turbidity 0 5 in McFarland scale for visual comparison with standard test tube. The dispersion
of the suspension in the plate containing the Mueller Hinton Agar, was cleaned with sterile
cotton swab across size of Petri dish (90 mm) up to obtain a uniform inoculum. The yeast
Saccharomyces cerevisiae was isolated by suspending 1 g of yeast in 20 ml of sugar solution.
Was allowed to stand for five minutes to allow the activation of yeast and subsequently taken 200
l of it to a test tube containing 15 ml of SDB and incubated for 48 h. Assays were performed
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The aqueous extract (Aq.Extr.) and juice were evaluated for the ability to interact with
ciprofloxacin. Disks were initially impregnated to saturation with the extracts and dried in sterile
medium for 30 minutes. Thereafter foi taken 10 uL of ciprofloxacin 2 mg / ml and was also added
on the disks containing extracts and aseptically transferred to Petri dishes previously inoculated
with bacteria and incubated for 24 h at 37 C. All tests were done in triplicates and the respective
percentages of interaction determined using the following formula: Interaction% = (Di-Dii) / Dii
x100 where: Di: mean zone of inhibition of ciprofloxacin combined with extract. Dii: mean zone
of inhibition of ciprofloxacin alone.
Phytochemical Screening
The phytochemical tests carried out on the extracts, fractions and fresh juice of the bulb,
allowed the identification of the following metabolites: alkaloids, anthraquinones, flavonoids,
coumarins, saponins, hydrolysable tannins, steroids and triterpenoids, amino acids, proteins and
reducing sugars.
The phytochemical screening results showed that there is not much difference between
the chemical composition of juice and powdered dry bulb, but in practice it has been proved to be
easier to work with the dry powdered bulb than the juice in particular during the solvent / solvent
partition.
The extraction solvent / solvent of the juice extracted from the fresh bulb and powdered
dry bulb allowed to obtain less complex fractions aiming semi-purification of substances through
their polarity and solubility.
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Methanol 90%
Extract ( fresh
bulb)
Aqueous
extract (24h
Soxhlet fresh
bulb)
Juice
(fresh bulb)
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
The results of the antimicrobial screening realized in this study with extracts and fractions
less complex of Gladiolus psittacinus Hook against Escherichia coli, Pseudomonas aeruginosa,
Staphylococcus aureus, Shigella flexneri, Salmonella typhimurium, Klebsiella pneumoniae,
Candida albicans and Saccharomyces cerevisiae showed interesting antimicrobial activities of
this plant as shown in table 3a and 3b.
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BU
230,6
200,1
70,0
130,6
BE
-
(_ _): Mean and standard deviation of triplicates; concentrations of extracts - 100 mg / disc, (-) No zone
of inhibition was observed; Positive Control : Ciprofloxacin (CIP) 2 mg / disc for bacteria and nystatin (NT )
3.2 cg / disk for C. albicans; DMSO: solvent control; MET: crude methanol extract; HEX: hexane fraction;
CHL: chloroform fraction; EA: ethyl acetate fraction; BU: butanol fraction; Aq.frac.: aqueous fraction;
Aq.Extr.: aqueous extract; BE - bacteriostatic activity. The yeast Saccharomyces cerevisiae had not
control.
NT
Candida albicans
E.coli ATCC25922
P. aeruginosa
ATCC 27953
K. pneumoniae
ATCC 15380
S.aureus
ATCC 25923
Saccharomyces
cerevisiae
S. typhimurium
ATCC 14028
Shigella flexneri
ATCC 12022
180,6
CIP
DMSO
320,6
CH2Cl2CH3OH
210,3
-
320,0
301,0
320,6
Aq.Frac.
Juice
Aq.Extr.
160,3
190,4
160,1
151,0
121,0
140,3
120,3
150,0
320,0
330,6
(_ _): Mean and standard deviation of triplicates; concentrations of extracts - 100 mg / disc, (-) : No zone
of inhibition was observed; Positive Control : Ciprofloxacin (CIP) 2 mg / disc for bacteria and nystatin (NT )
3.2 cg / disk for C. albicans; DMSO: solvent control; MET: crude methanol extract; HEX: hexane fraction;
CHL: chloroform fraction; EA: ethyl acetate fraction; BU: butanol fraction; Aq.Frac.: aqueous fraction;
Aq.Extr.: aqueous extract; BE: bacteriostatic activity. The yeast Saccharomyces cerevisiae had not
control.
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25
26
27
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Diameters of inhibition of ciprofloxacin when tested in combination with the juice show a
slight increase. Only in the case of Staphylococcus aureus and Salmonella typhimurium, the
combination showed indifference. However, the aqueous extract had an opposite effect, the
diameters of inhibition of the control showed a reduction when used in combination with the
aqueous extract for all tested microorganisms.
According to Nwze & Eze (2009), the convention suggests to use additive when the
percentage of the increase of the diameter of inhibition of the plant extract combination with the
antibiotic is less than or equal to 19%; indifferent, when the diameter of inhibition of the
antibiotic combination with the extract is equal to the diameter of inhibition when the antibiotic
is tested alone, and there is an antagonistic effect in case of loss of inhibition diameter when the
antibiotic control is used in combination with the extract.
This work was based on the same convention. An additive effect was observed in the
following microorganisms: Escherichia coli with 6.45% addition, Shigella flexneri with addition
of 6.25%, Klebsiella pneumoniae with 3.45% of addition and Pseudomonas aeruginosa with
addition of 13.33% when the juice combined with ciprofloxacin. Moreover, the bacteria
Salmonella typhimurium and S. aureus were indifferent to the combination. The combination of
the antibiotic with the aqueous extract showed an antagonistic effect for all the microorganisms
tested.
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Conclusion
Qualitative phytochemical analysis of the extracts and fractions of the bulb revealed the
presence of: alkaloids, anthraquinones, tannins, saponins, flavonoids, terpenoids, steroids,
cumarins, reducing sugar, aminoacids and proteins.
Most of the bulb extracts and fractions showed strong inhibitory activity against Candida
albicans, Saccharomyces cerevisiae and Pseudomonas aeruginosa. The determination of the
interaction Ciprofloxacin - aqueous extract and Ciprofloxacin - juice revealed an antagonism
between ciprofloxacin and the aqueous extract and an additive effect between Ciprofloxacin and
juice.
Phytochemical and antimicrobial test results obtained in this work in combination with
the previously reported in literature by different authors validate to some extent the use of the
bulb of Gladiolus psittacinus Hook in traditional medicine in treating diseases of microbial
origin, thus demonstrating the potential of this plant.
References
Ameh, S., Obodozie, O., Olorunfemi, P., Okoliko, I., Ochekpe, N. (2011). Potentials of gladiolus
corms as an antimicrobial agent in food processing and traditional medicine. Journal of
Microbiology And Antimicrobials, 3(1), 8-12.
Dixon, R.A., Dey, P.M., Lamb, C.J. (1983). Advances In enzymology and relates areas of
molecular biology. New York: John Wlley & Sons.
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