Qualitative Phytochemical Screening and Antimicrobial Activity Evaluation of the Bulb Extracts of...

International Network Environmental Management Conflicts http://www.igetecon.org/revista/index.php/inicio/index

International Network Environmental Management Conflicts, Santa Catarina – Brasil, 2(1), pp. 14-31, jan. 2013.

Qualitative Phytochemical Screening and Antimicrobial Activity Evaluation of

the Bulb Extracts of Gladiolus psittacinus Hook (Iridaceae)

François Munyemana

PhD, Professor Auxiliar, Departamento de Química, Universidade Eduardo Mondlane, Maputo, Moçambique

E-mail: [email protected] ou [email protected]

Ana Paula Mondego Mestre, Assistente, Curso de Farmácia, Instituto Superior de Ciências e Tecnologia de

Moçambique, Maputo, Moçambique

Paulo Cumbane Licenciado, Assistente Estagiário, Curso de Farmácia, Instituto Superior de Ciências e

Tecnologia de Moçambique, Maputo, Moçambique

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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Qualitative Phytochemical Screening…


Such practices are traditional because they are deeply rooted in a specific socio-cultural

context, which varies from one community to another. Each community has its own particular

approach to health and disease even at the level of ethno-pathogenic perceptions of diseases and

therapeutic behavior (Rukangira; Ochoa; Ocampo; Muñoz, 2001).

In Africa, herbal medicine gained popularity as alternative and complementary therapies,

largely as a result of cultural traditions and excessive cost of modern medicines. Traditional

remedies made from plants play an important role in the health of millions of people. Low-

income people such as subsistence farmers, people of small isolated villages and native

communities use folk medicine for the treatment of common infections (Rojas et al, 2006).

The interest in medicinal plants has grown considerably in recent years because they have

been a valuable source of products for maintaining human health, becoming potential candidates

for many applications in the pharmaceutical industry. Medicinal plants are the best sources to

obtain a variety of drugs (Santos; Ferreira; Damiao, 2010).

Mozambique, with its rich and varied flora, is one of the countries where people rely

much on the use of traditional knowledge and medicinal plants to treat various diseases.

Gladiolus psittacinus Hook (synonyms in southern Africa collections: Gladiolus natalensis and

Gladiolus dalenii) is an herbaceous and bulbous plant belonging to the family Iridaceae which

comprises about 1800 species grouped in 88 genus. Of these genus, Gladiolus and Iris are the

most representative with about 260 and 250 species respectively (Machado, 2007 & Manning,

2004). The greatest economic importance of the Iridaceae family resides in ornamentals and

medicinal use.

Phytochemical study of several species of the family Iridaceae has led to the isolation of

several secondary metabolites with high biological activity, especially, antimicrobial,

antiinflammatory, antidiarrhoeal, antidysenteric, antioxidant, antinociceptive, antifungal etc.

In the Iridaceae family occur mainly the following secondary metabolites: Flavonoids, alkaloids,

saponins, terpenoids, anthraquinones, naphthoquinones and coumarins.

The plants of the genus Gladiolus are used in different pharmacopoeias for treating

various diseases. In some regions in southern Africa plants of the genus Gladiolus are used to

treat a variety of diseases, including acute diarrhea, intestinal parasitic infections, asthma,

diabetes, constipation, impotency, headaches, relieve rheumatic pains and hemorrhoids.

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In Mozambique the plant Gladiolus psittacinus Hook is used to treat diarrhoea, dysentery,

gonorrhea, regulate the blood flow of women and as a sedative. For the treatment of diarrhea and

dysentery it is an infusion that can be administered orally or enema. As sedative, the plant is used

to calm patients with mental disorders in case of extreme agitation: two drops of fresh juice of the

bulb are applied in the nostrils of the patient.

In the present study was carried out the qualitative phytochemical screening of the bulbs

extracts of Gladiolus psittacinus Hook grown in Mozambique and evaluated the antimicrobial

activity of different extracts and fractions of dried and fresh bulb against gram-positive, gram-

negative bacteria and fungi.

Materials and Methods

Plant Material and Extraction

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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Preparation of extracts

Crude extracts were prepared by maceration, percolation or Soxhlet extraction using

following solvents: Methanol 90%, Methanol, Dichloromethane – methanol (1:1), Distilled

water. Fractions were obtained by solvent-solvent partition with increasing polarities (hexane or

petroleum ether, Chloroform, ethyl acetate, n-butanol).

Extraction of dried sample by percolation

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 n-

hexane. 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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All fractions were stored in dry desiccator until constant weight is obtained, and after 7 days the

yield of extraction was determined.

Extraction of the dried sample by Soxhlet

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

Extraction of the fresh sample by Soxhlet

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.

Extraction of juice in fresh bulb

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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Solvent-Solvent Partition of the fresh juice

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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Bacterial sensitivity test by disc diffusion method

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 µl 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 a standard tube containing a 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.

Fungal sensitivity test by disc diffusion method

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

µl of it to a test tube containing 15 ml of SDB and incubated for 48 h. Assays were performed

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using the same procedure followed for antibacterial tests, with the exception of the positive

control, having been used in this nystatin.

Interaction Extract – Ciprofloxacin

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.

Results and discussion

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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The results of the phytochemical screening of the crude extracts are summarized in the

table 1, and the results of the phytochemical screening of the fractions from solvent-Solvent

partition were summarized in table 2.

Table 1: Phytochemical tests of Crude extracts

Phytoconstituents

Dichloromethane-

Methanol(1:1)

Extract

(24h soxhlet dried

bulb)

Methanol 90%

Extract ( fresh

bulb)

Aqueous

extract (24h

Soxhlet fresh

bulb)

Juice

(fresh bulb)

Alkaloids + + + +

Anthraquinones + + - -

Coumarins - + - -

Flavonoids + + - -

Saponins + + - +

Tannins + + - -

Amino acids + + + +

Proteins - - - +

Reducing Sugars - + + +

+: present; -: absent

Table 2: Phytochemical tests of fractions from solvent – solvent Partition

Phytoconstituents Juice (Fresh Bulb) Powdered dry bulb

PE/HEX CHL EA Bu PE/HEX CHL EA Bu

Alkaloids - + - - - + + -

Anthraquinones - - + - - + + -

Coumarins - + + - - + + -

Flavonoids - + + + - + + +

Iridoids - - - - - - - -

Saponins - - - + - - + +

Steroids/Terpenoids + + + + + + + +

+: present, -: absent, PE: petroleum ether fraction, HEX: hexane fraction, CHL: chloroformic

fraction, EA: ethyl acetate fraction, Bu: butanolic fraction.

Results of antimicrobial assay

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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Table 3a. Results of antimicrobial activity tests mean diameter of inhibition zone (mm).

Micro-

organisms NT CIP MET HEX CHL EA BU

Candida

albicans 18±0,6 20±0,6 28±0,6 25±0,6 24±0,6 23±0,6

E.coli ATCC25922

32±0,6 - - - - -

P. aeruginosa

ATCC 27953 32±0,0 18±0,6 22±1,0 23±0,3 25±0,0 20±0,1

K. pneumoniae ATCC 15380

30±1,0 - - - - -

S.aureus

ATCC 25923 32±0,6 - - - - 7±0,0

Saccharomyces

cerevisiae 10±0,6 - - 20±0,3 13±0,6

S. typhimurium ATCC 14028

32±0,0 BE - BE BE BE

Shigella

flexneri

ATCC 12022 33±0,6 - - BE 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 cµg / 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.

Table 3b. Results of antimicrobial activity tests (cont.) Mean diameter of inhibition zone (mm)

Micro-organisms NT CIP DMSO CH2Cl2-

CH3OH Aq.Frac. Juice Aq.Extr.

Candida albicans 18±0,6 - 21±0,3 - 16±0,3 -

E.coli ATCC25922 32±0,6 - - - 19±0,4 16±0,1

P. aeruginosa ATCC 27953

32±0,0 - 15±1,0 - 12±1,0 14±0,3

K. pneumoniae ATCC 15380

30±1,0 - - - - -

S.aureus

ATCC 25923 32±0,6 - - - - -

Saccharomyces

cerevisiae - 12±0,3 - 15±0,0 -


32±0,0 - - - - -

Shigella flexneri

ATCC 12022 33±0,6 - - - - -

(_ ± _): 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 cµg / 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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Preparations of the bulb of Gladiolus psittacinus Hook are used in traditional medicine

for the treatment of various diarrheal diseases. The presence of secondary metabolites active

against microbial pathogens in the extracts of this plant has been reported.

Ameh et al (2011) demonstrated antimicrobial activity against Pseudomonas aeruginosa

and Aspergillus niger of the aqueous extract of Gladiolus bulb. The phytochemical screening of

the same extract resulted in the identification of some metabolites namely: alkaloids, tannins,

cardiotonic glycosides, flavonoids and carbohydrates.

Nguedia et al (2004) showed antifungal activity of the hydroethanolic extract of the bulb

of Gladiolus gregasius Baker against Candida albicans and Candida krusei. However, the same

extract was not active against Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris,

Staphylococcus aureus and Streptococcus faecalis. The phytochemical screening revealed the

presence of saponins, glycosides, polyphenols, phenols, triterpenes and steroids.

The crude methanolic extract exhibited an antibacterial effect against Pseudomonas

aeruginosa with an inhibition zone of 18 ± 0.6 mm and a bacteriostatic effect against Salmonella

typhimurium. An antifungal effect was shown against the yeasts Saccharomyces cerevisiae and

Candida albicans with inhibition zones of 20± 0.6 mm and 10 ± 0.6 mm respectively.

The activity of that extract against Pseudomonas aeruginosa was low compared to

ciprofloxacin used as control. However, it showed a high antifungal activity against Candida

albicans when compared to the control nystatin. The antifungal activity displayed against candida

albicans was higher than with the other yeast Saccharomyces cerevisiae. This extract was not

active against the rest of the microorganisms tested.

The hexane fraction exhibited an antibacterial effect against Pseudomonas aeruginosa

with an inhibition zone of 22 ± 0.6 mm. An antifungal effect against Candida albicans has been

shown with a zone of inhibition of 28 ± 0.6 mm. This fraction was not active at the same

concentration against other microorganisms tested. The activity of the extract against

Pseudomonas aeruginosa was low compared to the antibiotic used as control; but that was higher

than the activity of the crude methanolic extract. This fraction exhibited also an antifungal

activity against Candida albicans higher than the control nystatin and than the crude methanol

extract. In phytochemical studies performed on this fraction were identified terpenoids.

According to literature, the activity of these compounds is related to its lipid solubility

which allows them to interact with the biomembranes of microorganisms, causing rupture of the

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cells. The solubility of these compounds in the biomembranes enables their interaction with ion

channels, carrier molecules, membranes receptors, which leads to changes in conformation and

loss of function (Lopez, 2010).

The chloroform fraction also showed an antibacterial effect against Pseudomonas

aeruginosa with a zone of inhibition of 23 ± 0.6 mm and a bacteriostatic effect against Shigella

flexneri and Salmonella typhimurium. It was also shown antifungal activity against Candida

albicans with a zone of inhibition of 25 ± 0.6 mm. This fraction was not active at the same

concentration against other microorganisms tested. The activity of the extract against

Pseudomonas aeruginosa was low compared to the control. However, the activity of this fraction

was superior to the activity of the crude methanol extract and the hexane fraction. This fraction

also showed antifungal activity against Candida albicans higher than the control, the crude

methanol extract, and the hexane fraction.

In phytochemical tests performed on this fraction by precipitation methods (Mayer and

Dragendorff) and thin layer chromatography were identified alkaloids. The presence of alkaloids

in this plant have been reported by Odhiambo et al (2010) as the active constituents present in the

extract CH2Cl2: CH3OH (1:1) against Aspergillus niger.

The ethyl acetate fraction exhibited an antibacterial activity against Pseudomonas

aeruginosa with an inhibition zone of 25 ± 0.0 mm and a bacteriostatic effect against Shigella

flexneri and Salmonella typhimurium. This fraction was also active against the yeasts Candida

albicans and Saccharomyces cerevisiae with zones of inhibition of 24 ± 0.6 ± 0.3 and 20 mm

respectively. This fraction was not active at the same concentration against other microorganisms

tested. The antibacterial activity against Pseudomonas aeruginosa of this fraction was low

compared to the control, but greater than the activity of the crude methanol extract, the hexane

and chloroform fractions. This fraction was more active against Candida albicans than against

the yeast Saccharomyces cerevisiae. It also showed antifungal activity against Candida albicans

higher than the control nystatin, crude methanol extract, hexane and chloroform fractions.

Phytochemical tests realized on this fraction showed the presence of alkaloids, coumarins and

saponins that may be associated with antimicrobial activity shown by this fraction.

The butanol fraction showed antibacterial activity against Pseudomonas aeruginosa with

a zone of inhibition of 20 ± 0.1 mm, a low activity against Staphylococcus aureus and a

bacteriostatic effect against Salmonella typhimurium. This fraction also showed antifungal

26



activity against Candida albicans with a zone of inhibition of 23 ± 0.6 mm and a moderate

activity against the yeast Saccharomyces cerevisiae with a zone of inhibition of 13 ± 0.6 mm.

The activity of this fraction against Pseudomonas aeruginosa was low compared to the

antibiotic used as control; but it was higher than the crude methanol extract. It also showed

antifungal activity against Candida albicans higher than the control nystatin and the crude

methanol extract. The phytochemical tests of this fraction made possible the identification of

saponins. The presence of these compounds in the extracts of this plant has also been evidenced

by Odhiambo; Siboe; Lukhoba; Dossaji, (2010).

The extract CH2Cl2: CH3OH (1:1) showed antibacterial activity against Pseudomonas

aeruginosa with an inhibition zone of 15 ± 1.0 mm. It also showed activity against candida

albicans and Saccharomyces cerevisiae with zones of inhibition of 21 ± 0.3 mm and 12 ± 0.6 mm

respectively. The activity of the extract against Pseudomonas aeruginosa was low compared to

the antibiotic used as control. Furthermore, their activity against Candida albicans was higher

than the control.

The activity demonstrated by the extract against Candida albicans was higher than that

displayed against Saccharomyces cerevisiae. Phytochemical Tests carried out on this extract

made possible the identification of alkaloids, amino acids, anthraquinones, flavonoids, tannins

and saponins.

The quinolinic compounds are capable of forming irreversible complexes with

nucleophilic amino acids in proteins, often leading in inactivating proteins and consequent loss of

function, which explains the high antimicrobial effect of these compounds (Lopez, 2010). The

biological role of tannins in plants has been investigated and it is believed that they are involved

in chemical defense of plants against attack by herbivores and against pathogens (Oliveira et al,

2007). Not surprisingly, extracts containing tannins show activity against microorganisms.

Flavonoids are compounds synthesized by the plants in response to microbial infection (Dixon;

Dey; Lamb, 1983), so it is not surprising to demonstrate antimicrobial activity in vitro against a

broad range of microorganisms. This activity is probably due to the ability to form complexes

with extracellular soluble proteins and the bacterial cell wall. The more lipophilic flavonoids can

also disrupt microbial membranes.

In studies realized by Odhiambo et al (2010), was also identified the presence of alkaloids

in the extract CH2Cl2 – CH3OH (1:1) which were attributed the potent antifungal activity

27



demonstrated by this extract against Aspergillus niger. The activity exhibited by this extract

against Candida albicans can probably also be associated with these compounds identified.

The juice from the fresh bulb showed an antibacterial activity against Escherichia coli and

Pseudomonas aeruginosa with inhibition zones of 19 ± 0.4 and 12 ± 1.0 mm respectively. It was

also active against candida albicans and Saccharomyces cerevisiae with respective zones of

inhibition of 16 ± 0.3 and 15 ± 0.0 mm. The activity of the extract against Escherichia coli and

Pseudomonas aeruginosa was low compared to the control. Its activity against Candida albicans

was also lower than control.

Phytochemical tests carried out on this extract, have made possible the identification of

amino acids, proteins, alkaloids and saponins. The activity shown by this extract may be

associated with these compounds.

The aqueous extract of the fresh bulb was active against Pseudomonas aeruginosa and

Escherichia coli with the respective zones of inhibition of 16 ± 0.1 and 14 ± 0.3 mm. However

was inactive against the rest of the tested bacteria. No antifungal effect was demonstrated in this

extract. The activity of the extract against Escherichia coli and Pseudomonas aeruginosa was

low compared to the antibiotic used as control.

Phytochemical tests carried out on this extract made possible the identification of

alkaloids. The study realized by Ameh et al (2011), reported the presence of alkaloids in the

aqueous extract of the bulb of Gladiolus obtained by the same method. However, the above

extract was found to be slightly active against Pseudomonas aeruginosa and Aspergillus niger

and inactive against Candida albicans.

The bacteria Escherichia coli was susceptible to aqueous extract and juice, which are

generally used in traditional medicine for the treatment of diarrhea, gonorrhea and other intestinal

disturbances. This activity may explain its traditional use in the treatment of diarrhea of various

origins.

The results of the interaction Extract-Ciprofloxacin show an additive effect for the

combination Juice + Ciprofloxacin in case of E. coli, S. flexneri, K. pneumoniae and P.

aeruginosa; Indifference in case of S. aureus and S. typhimurium. In the case of the combination

of Ciprofloxacin + aqueous extract, the antagonistic effect was observed for all microorganisms

tested. The results of the interaction Extract – Ciprofloxacin are summarized in table 4.

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Table 4 - Results of the interaction Extract - Ciprofloxacin Mean diameter of the inhibition zone (mm)

Microorganism Distilled

water CIP Juice Juice+CIP Aq.Extr.

CIP+

Aq.Extr.

E.coli ATCC25922

- 31±0,6 19±0,6 33±0,6 16±0,0 28±0,3

S.aureus ATCC 25923

- 33±0.3 - 33±0,6 - 30±2,6

Shigella flexneri ATCC 12022

- 32±1,0 - 34±1,0 - 30±0,6


- 32±0.3 - 32±0,3 - 31±0,6

K. pneumoniae

ATCC 15380 - 29±0,0 - 30±1,6 27±1,0

P. aeruginosa ATCC 27953

- 30±1,0 28±0.5 34±0,6 19±1,0 25±0,3

Purified distilled water: Negative control; CIP: Ciprofloxacin - positive control; CIP + Aq.Extr.: ciprofloxacin combined with aqueous extract; Juice + CIP: ciprofloxacin combined with juice.

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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It is important to note that the action of medicinal plants can never be fully understood by

analyzing its components separately. Proponents of this theory argue that the properties are due to

interactions between multiple components (Wink, 2008). For example, the saponins present in

many medicinal plants, enhance the absorption and activity of other substances. The saponins of

Sapindus mukorossi increase the intestinal absorption of certain natural antibiotics. Therefore,

positive interaction found in the combination of the juice with ciprofloxacin can be associated

with the presence of saponins in the plant, which can essentially increase the absorption of

ciprofloxacin through the biological membranes of the bacteria, thereby enhancing its effect.

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

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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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