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Cabbage leaf extract (Brassica oleracea var. capitata alba) as a herbal medicine for leukorrhea

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Objective:This study was aimed to examine antifungal activity of cabbage leaf extracts against Candida albicans, determined the minimum inhibitory concentration (MIC), analyzed the comparative value of the extracts with ketoconazole and studied the fastest contact time to eliminate Candida albicans. Methods: The extraction of fresh cabbage leaf was done using the maceration method. The antifungal activity test and its comparative analysis against ketokonazole were assessed using the agar diffusion method. The extracts were tested for determining minimum inhibitory concentration value (MIC) using solid medium. While the fastest contact time test was performed using turbidimetric method.Results: Based on its inhibitory diameter, cabbage leaf extracts gave potent antifungal activity against Candida albicans. The MIC concentrations of testing extracts were various ranged from 1.50 to 1.75%w/v. In a comparative analysis of the extracts with ketoconazole, showed that ketoconazole gave greater antifungal activity than the extract at the same concentration. Cabbage leaf extract with concentration 2.5% gave the fastest contact time (2.5 minutes) for eliminating Candida albicans, while Cabbage leaf extract with concentration 0.4% gave the longest contact time (15 minutes) for eliminating Candida albicans.Conclusion:Cabbage leaf extract has a potential antifungal activity against Candida albicans, and prospective to be developed as a topical herbal medicine for treating candidiasis.
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Special Issue (May)
Online - 2455-3891
Print - 0974-2441
II-Indonesian Conference on Clinical Pharmacy
CABBAGE LEAF EXTRACT (BRASSICA OLERACEA VAR. CAPITATA ALBA) AS A HERBAL
MEDICINE FOR LEUKORRHEA
AMI TJITRARESMI*, SRI AGUNG FITRI KUSUMA, DEWI RUSMIATI
Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Padjadjaran, Indonesia. Email: ami.tjitraresmi@unpad.ac.id
Received: 05 November 2016, Revised and Accepted: 24 January 2017
ABSTRACT
Objective: This study was aimed to examine antifungal activity of cabbage leaf extracts against Candida albicans, to determine the minimum inhibitory
concentration (MIC), to analyze the comparative value of the extracts with ketoconazole, and to study the fastest contact time to eliminate C. albicans.
Methods: The extraction of fresh cabbage leaf was done using the maceration method. The antifungal activity test and its comparative analysis against
ketoconazole were assessed using the agar diffusion method. The extracts were tested for determining MIC value using solid medium, while the fastest
contact time test was performed using turbidimetric method.
Results: Based on its inhibitory diameter, cabbage leaf extracts gave potent antifungal activity against C. albicans. The MIC of the cabbage leaf extract
was between range 1.50% to 1.75%. A comparative analysis of the extracts with ketoconazole showed that ketoconazole gave greater antifungal
activity than the extract at the same concentration. Cabbage leaf extract with concentration 2.5% gave the fastest contact time (2.5 minutes) for
eliminating C. albicans, while cabbage leaf extract with concentration 0.4% gave the longest contact time (15 minutes) for eliminating C. albicans.
Conclusion: Cabbage leaf extract has a potential antifungal activity against C. albicans and prospective to be developed as a topical herbal medicine
for treating candidiasis.
Keywords: Brassica oleracea var. capitata alba, Cabbage, Candida albicans.
INTRODUCTION
Leukorrhea or flour albus or vaginal discharge was a symptom related
to female genital disorders. It was white, yellowish-white, or gray
color excessive fluid that discharges from vagina and sometimes
smells offensive. Normally, women from all ages can have flour albus,
but it could not be taken lightly, because this could lead to dangerous
diseases. There are many causes of vaginal discharge (flour albus), it
could be non-pathogen or pathogen condition. Pathologically, it might
be caused by bacteria, fungus, virus, or parasite infection [1-3].
Flour albus that caused by bacteria could be treated by antibiotic;
however, antibiotic consumption in a long term could be decreased
vaginal normal flora, and in this condition, the fungus could grow in that
area. Candida sp. was reported to be the most widely fungus that could
be found at vaginal discharged, and from a hundred species of Candida
sp that isolated from this discharge, 50-60% was Candida albicans [1].
Candidiasis is a fungal infection caused by yeasts that belong to the genus
Candida. There are over 20 species of Candida yeasts that can cause
infection in humans, the most common of which is C. albicans. Candida
yeasts normally reside in the intestinal tract and can be found on mucous
membranes and skin without causing infection; however, overgrowth of
these organisms can cause symptoms to develop. Symptoms of candidiasis
vary depending on the area of the body that is infected [3]. Patients with
impaired immunity, such as those who have AIDS or are neutropenic as
a result of cancer therapy, are at particular risk of developing C. albicans
infections, which may become systemic [4-7].
Successful therapy for serious systemic Candida infections requires
initiation of antifungal therapy as early as possible, as soon as adequate
culture results are obtained [7,8]. Different classes of antifungals
are now available to manage any type of Candida infection. Azoles,
fluconazole in particular have become the mainstay of therapy over the
past few years. Azoles inhibit the cytochrome P450 enzyme 14-α-sterol-
demethylase. This enzyme is implicated in the biosynthetic pathway of
ergosterol, which is an essential molecule of the fungal cell membrane.
Inhibition of this enzyme leads to accumulation of 14-a-methylsterols
on the fungal surface, which results in the arrest of fungal growth [9].
However, it had been reported that C. albicans was resistant against
azole drugs so that it was necessary to seek the new drugs that were
effective to treat candidiasis [2,10-12].
White cabbage (Brassica oleracea var. capitata alba) is a cruciferous
vegetable used worldwide as a food and in traditional medicine. People
had been using cabbage leaf to treat flour albus. Conventionally, people
boiled cabbage leaf, and the boiling water was used to wash the vagina,
and it could reduce itchiness that caused by Candida infection [13-15].
This study was aimed to determine antifungal activity, and minimum
inhibitory concentration (MIC) from cabbage leaf extract against
C. albicans, to analyze the comparative value of the extracts with
ketoconazole, and to study the fastest contact time for cabbage leaf
extract to eliminate C. albicans.
MATERIALS AND METHODS
Materials
The fresh cabbage leaf was obtained from cabbage leaf plantation in
Lembang, West Java, Indonesia, and submitted to Plant Taxonomy
Laboratory, Department of Biology, Universitas Padjadjaran for
authentication. C. albicans ATCC 10231 and sabouraud dextrose agar
(SDA) were obtained from Oxoid, Tokyo, Japan. Dimethyl sulfoxide
(DMSO)and toluene were analytical grade and were produced by
Merck, Germany; Ketoconazole was made by PT. Kimia Farma Jakarta,
Indonesia.
Preparation of extracts
The fresh cabbage leaf that obtained from vegetables plantation in
Lembang, West Java, and the plants was determined at Plant Taxonomy
Research Article
© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.
org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ajpcr.2017.v10s2.19483
40
Special Issue (May)
Tjitraresmi et al.
Laboratory, Department of Biology, Universitas Padjadjaran. The fresh
cabbage leaf was cut into small pieces and then macerated using ethanol
70% as a solvent, for 3×24 hrs, and the solvent was replaced every
24 hrs. The liquid cabbage leaf extract was collected, and the solvent was
evaporated using rotary evaporator to obtain thick cabbage leaf extract.
Antifungal activity test of cabbage leaf extract against C. albicans
Antifungal activity from cabbage leaf extract against C. albicans was
examined using agar diffusion method. SDA was used as a medium for
C. albicans. SDA (65 g) was suspended with one liter purified water
and then heated with frequent agitation and boiled for 1 minute to
completely dissolve the medium. SDA medium was sterilized in the
autoclave at 121°C for 15 minutes, and then, let it cool to 45-50°C.
C. albicans was added into the SDA medium and then poured the mixture
of SDA medium and C. albicans into petri dishes. Sterile filter paper discs
(about 6 mm in diameter) were impregnated with 10 μl of cabbage leaf
extracts with concentration 20%, 40%, 60% dan 80% in DMSO. The
above 4 discs were applied into the mixture SDA medium C. albicans and
then incubated at 37°C for up to 48 hrs. When growth took place, the
size of zones of inhibition was measured for each antifungal agent [16].
Determination of MIC
Cabbage leaf extract was diluted with SDA medium to obtain cabbage
leaf extract concentration 10; 5; 2.5; 2.25; 2; 1.75; 1.5; 1.25; 1; 0.5; and
0.25%, then 5 ml of each mixture were poured into petri dishes. Allow
the agar to set and then dry the surface of the plates. The C. albicans
inoculant (5 µl) was applied to the plates using spreader. Then, let the
petri dishes at 37°C for up to 48 hrs incubation. The growth of C. albicans
colonies was observed. MICs of cabbage leaf extract are defined as the
lowest concentration of cabbage leaf extract that will inhibit the visible
growth of C. albicans colonies [16-18].
Antifungal activity ratio against C. albicans of cabbage leaf extract
compared to ketoconazole
Antifungal activity ratio from cabbage leaf extract against C. albicans
compared to ketoconazole was also examined using agar diffusion
method. SDA medium was sterilized in the autoclave at 121°C for
15 minutes, and then, let it cool to 45-50°C, added with C. albicans and
poured into petri dishes. Sterile disks were impregnated with 10 µl of
cabbage leaf extracts or ketoconazole with various concentrations in
DMSO. The diameter of bacterial growth inhibition zone was measured
by calipers and then plotted as an ordinate (Y) at the graphic and the
axis (X) was the log of extracts concentration. Based on the graphic, the
linear equations for both cabbage leaf extract and ketoconazole were
determined. The comparison of antifungal activity from cabbage leaf
extract and ketoconazole was calculated using this equation [16].
Determination of the contact time for cabbage leaf extract to
eliminate C. albicans
The contact time of the cabbages extract to kill or eliminate C. albicans
was determined using turbidimetric method. Cabbage leaf extract was
diluted in DMSO to form concentration 1.75; 2; 2.25; 2.5; and 3% b/v.
Let the C. albicans inoculant contact with each cabbage leaf extract
concentration at the various time from 2.5 to 15 minutes. The inoculants
then were added into liquid medium consisting broth and dextrose.
Then, let the mixture at 37°C for up to 18-24 hrs incubation. The growth
of C. albicans in the liquid medium was observed and compared with
positive control (C. albicans inoculant added into liquid medium) and
negative control (liquid medium only) [16].
RESULTS AND DISCUSSION
Preparation of extracts
Based on the result of the determination of the plant, the plant material
was from B. oleracea var. Capitata alba, Family Brassicaceae. The
rendemen of fresh cabbage leaf extract that obtained from maceration
method with ethanol 70% as a solvent was 5.54%. The amount of
the extract is small, because the material plants that we used in this
experiment was fresh cabbage that still contains a large amount of
water. The water content of the thick cabbage leaf extract was 16.6%.
Antifungal activity test of cabbage leaf extract against C. albicans
The cabbage leaf extract at the concentration 20%, 40%, 60%, and
80% has antifungal activity against C. albicans. The higher cabbage leaf
extract concentrations yielded bigger diameter of inhibition against
C. albicans. Table 1 shows the diameter of inhibition zone from various
concentrations of cabbage leaf extract from day 1 to day 4.
Determination of MIC of cabbage leaf extract
MICs of cabbage extract are defined as the lowest concentration of
cabbage leaf extract that will inhibit the visible growth of C. albicans
colonies. The MIC test in this experiment was carried out using solid
method. The concentration of cabbage leaf extract for this MIC test was
in range 0.25-10%. The C. albicans inoculant was spreaded at the top of
the mixture of SDA medium and various concentration of cabbage leaf
extract. Table 2 shows the results of observation of C. albicans growth
at the mixture of SDA medium and cabbage leaf extract at various
concentrations.
There was still the growth of C. albicans colony on the SDA medium
with 1.50% cabbage leaf extract, but there were no sign of C. albicans
colony on the SDA medium with 1.75%. Based on this data, the MICs
of cabbage leaf extract against C. albicans were between concentration
range 1.50-1.75%.
Antifungal activity ratio against C. albicans of cabbage leaf extract
compared to ketoconazole
The comparison test of cabbage leaf extract and ketoconazole antifungal
activity against C. albicans was carried out to compare the cabbage leaf
extract with antifungal drugs that had been widely used in the society.
Ketoconazole is one of the antifungal drugs of choice. Table 3 shows the
diameter of inhibition zone of ketoconazole against C. albicans. Fig. 1
shows the effect log of various doses of ketoconazole against diameter
of inhibition zone of Candida albicans.
The linear equation of ketoconazole antifungal activity from the
data above was y=1.3 x +19. The diameter of inhibition zone for
ketoconazole against C. albicans at the dose 400 µg/50 µl or log dose
2.602, based on the linear equation, was (Y) 22.5 mm. The antifungal
Table 1: The diameter of inhibition zone of cabbage extract
against C. albicans
Day Diameter of inhibition zone (mm)
20% 40% 60% 80%
1 0.00 0.00 0.00 0.00
2 16.10 17.10 18.50 20.20
3 16.20 17.20 18.50 20.20
4 16.20 17.20 18.60 20.20
C. albicans: Candida albicans
Table 2: The observation results on C. albicans growth at various
concentrations of cabbage leaf extract
Concentration of cabbage leaf
extract (%)
The growth of C. albicans
colony
0.25 +
0.50 +
1.00 +
1.25 +
1.50 +
1.75 -
2.00 -
2.25 -
2.50 -
5.00 -
10.00 -
“+” means there were colony of Candida albicans on the media, “-” means there
were no colony of Candida albicans on the media, C. albicans: Candida albicans
41
Special Issue (May)
Tjitraresmi et al.
activity of cabbage leaf extract was treated the same as the data of
antifungal activity of ketoconazole. The linear equation of cabbage
leaf extract antifungal activity was y=1.183x + 14.92. To produce the
same diameter of inhibition zone (Y=22.5 mm), this Y value was put in
cabbage leaf extract linear equation, and the log dose (X) was 6.1961.
The antilog of 6.1961 was 1570766.32. Hence, the dose of cabbage
leaf extract that will produce 22.5 mm diameter of inhibition zone
was 1570766.32 µg/50 µl. Hence, the comparison of the doses from
ketoconazole and cabbage leaf extract was 1: 3926,9158.
Determination of the contact time for cabbage leaf extract to
eliminate C. albicans
The contact time was the time period for certain concentration of
cabbage leaf extract to eliminate C. albicans. Table 4 shows the impact of
various concentrations of cabbage leaf extract toward the contact time
to eliminate C. albicans.
Based on the data in Table 4, cabbage extract with concentration 2.5%
gave the fastest contact time (2.5 minutes) for eliminating C. albicans,
while cabbage extract with concentration 0.4% gave the longest contact
time (15 minutes) for eliminating C. albicans.
CONCLUSION
The result of the experiment showed that cabbage extracts with
concentration 20%, 40%, 60%, and 80% gave antifungal activity; the
MICs of cabbage extract against C. albicans were between 1.5% and
1.75%, cabbage extract’s antifungal activity ratio against ketoconazole
was 1: 3926,9158. Cabbage extract with concentration 2.5% gave the
fastest contact time (2.5 minutes) for eliminating C. albicans, while
cabbage extract with concentration 0.4% gave the longest contact time
(15 minutes) for eliminating C. albicans. From the results above from
this experiment, cabbage leaf extract has a potential antifungal activity
against C. albicans and prospective to be developed as a topical herbal
medicine for treating candidiasis.
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Table 3: The diameter of inhibition zone of cabbage extract
against Candida albicans
Concentration (%) Doses (µg/50 µl) Log of
dose
Diameter of
inhibition
zone (mm)
0.8 400 2.602 24.00
0.6 300 2.477 23.00
0.4 200 2.300 22.00
0.2 100 2.000 20.00
C. albicans: Candida albicans
Table 4: The impact of various concentrations of cabbage leaf
extract toward the contact time to eliminate C. albicans
Cabbage extract
concentration (%)
Contact time (minutes)
2.5 5 7.5 10 12.5 15
3.00 - - - - - -
2.75 - - - - - -
2.50 - - - - - -
2.25 + + - - - -
2.00 + + + - - -
1.75 + + + + + +
- = clear, + = muddy because of the growth of Candida albicans,
C. albicans: Candida albicans
Fig. 1: Graphic of effect log dose of ketoconazole against diameter
of inhibition zone of Candida albicans
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The causative strains in 22 patients with recurrent oral candidiasis were examined using two DNA probes (a Histoplasma capsulatum DNA probe that cross-hybridizes with Candida albicans and a C. albicans strain-specific probe derived from repetitive sequence DNA). C. albicans was the causative organism in an 22 initial episodes of infection and was also obtained from 17 patients with recurrent oral disease. Molecular analysis showed that in 11 cases, the same isolate was identified in each episode. Six patients had a clearly different isolate of C. albicans causing a later episode of candidiasis. Five patients had different Candida species causing recurrent disease: 4, Torulopsis glabrata; 1, Candida parapsilosis. Patients with a new isolate (either new species or a new C. albicans strain) were more immunosuppressed and were significantly more likely (P < .001) than patients with the same recurrent strain to have received suppressive azole antifungal agents. These data indicate that the epidemiology of recurrent candidiasis in individual patients seropositive for the human immunodeficiency virus is complex and that both failure of eradication of Candida from the oral cavity and new infection occur.
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The dimorphic yeast Candida albicans has been recognized as an increasingly important human pathogen particularly in immunocompromised hosts because of advanced age, infection or immunosuppressive therapy. This review outlines the history, taxonomy and epidemiology of this medically important yeast as well as discussing some of characteristics which are purported to be related to its virulence. Methods utilized for strain differentiation in the study of the epidemiologic relationship of members of this species are discussed.
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Major developments in research into the azole class of antifungal agents during the 1990s have provided expanded options for the treatment of many opportunistic and endemic fungal infections. Fluconazole and itraconazole have proved to be safer than both amphotericin B and ketoconazole. Despite these advances, serious fungal infections remain difficult to treat, and resistance to the available drugs is emerging. This review describes present and future uses of the currently available azole antifungal agents in the treatment of systemic and superficial fungal infections and provides a brief overview of the current status of in vitro susceptibility testing and the growing problem of clinical resistance to the azoles. Use of the currently available azoles in combination with other antifungal agents with different mechanisms of action is likely to provide enhanced efficacy. Detailed information on some of the second-generation triazoles being developed to provide extended coverage of opportunistic, endemic, and emerging fungal pathogens, as well as those in which resistance to older agents is becoming problematic, is provided.