Efficacy of Bajakah Tampala Ethanol Extract, A Typical Plant Section A-Research paper

of Kalimantan Island (Borneo), Against Candida albicans

Biofilm

EFFICACY OF BAJAKAH TAMPALA ETHANOL EXTRACT,

A TYPICAL PLANT OF KALIMANTAN ISLAND (BORNEO),
AGAINST CANDIDA ALBICANS BIOFILM

Hasyrul Hamzah[a], Sylvia Utami Tunjung Pratiwi[b, c]*, Asriullah Jabbar [d],
Erika Nandini[e]

Article History: Received: 12.06.2022 Revised: 11.07.2022 Accepted: 08.08.2022

Abstract: The emerging resistance to anti-fungal agents encourage the exploration of new and effective natural product showing
anti-fungal activity to significantly impact the and the treatment management of biofilm associated fungal infections. In nature,
most of microbes live in form of biofilms, and up to 80% of microbial infections on human body are biofilms associated. This
research aimed at finding out antibiofilm activity of Bajakah tampala extract against Candida albicans. The inhibition and
eradication activities toward C. albicans biofilm were tested using microtiter broth method by measuring the minimum value of
biofilm inhibition concentration (MBIC50) and minimum biofilm value eradication concentration (MBEC50). The C. albicans
biofilm structure in front of and absence of the extracts was analysed using SEM. At concentration of 1% w/v Bajakah tampala
extract showed activity against C. albicans mid-phase biofilm formation and maturation phase (77.00% ± 0.01, 70.31% ± 0.01)
and breakdown the established biofilms as much as 65.21% ± 0.01. The biofilm architecture analysis by SEM provided evidence
that Bajakah tampala ethanol extract disturbed the extracellular polymeric substance (EPS) matrix of the C. albicans biofilm. The
result obtained clearly indicate that Bajakah tampala exthanol extract can developed as a new antibiofilm candidate for the treatment
of C. albicans biofilm infection.

Keywords: Bajakah tampala, biofilm, C. albicans, infection, antibiofilm

[a]. Faculty of Pharmacy, Universitas Muhammadiyah albicans infection cases studied in hospitals in Spain, the
Kalimantan Timur, Samarinda, Kalimantan Timur, mortality rate was 51% due to C. albicans infection (Almirante
75124, Indonesia. et al., 2005). Meanwhile, in Germany, the number of deaths due
[b]. Department of Pharmaceutical Biology, Faculty of to necrosectomy caused by Candida reached as much as 62%
Pharmacy, Universitas Gadjah Mada, Yogyakara, 55281, [3].
Indonesia. Biofilms are groups various microbial cells that bind
[c]. Medicinal Plants and Natural Products Research Center, irreversibly to the surface , enclosed in a extracellular polymer
Faculty of Pharmacy, Universitas Gadjah Mada, subtances (EPS) matrix, and express phenotypic changes such
Yogyakarta, 55281, Indonesia. as growth rates and plankton cell gene changes transcription or
[d]. Department of Pharmacy, Faculty of Pharmacy, free radicals [4]. According to data from the US National
Universitas Halu Oleo, Kendari, 93232, Indonesia. Institutes of Health (NIH), more than 60% of microbial
[e]. Faculty of Pharmacy, Universitas Muhammadiyah infections are associated with biofilms [5], and up to 80%
Kalimantan Timur, Samarinda, Kalimantan Timur, human infections are biofilm associated [6]. Biofilms as part of
75124, Indonesia. the microbial defense system to quite difficult of destroy for
antibiotics, as a consequence pathogenic bacteria in biofilms
*Corresponding Author can cause serious problems for human health [7].
Indonesia is an archipelagic country with a rich diversity of
Email: [email protected] flora [8]. This wealth must be appreciated as a national asset to
increase the nation's resilience and sovereignty. One of the
DOI: 10.31838/ecb/2022.11.05.009 islands in Indonesia that has varieties of plants is the island of
Kalimantan (Borneo). Kalimantan has long been known as a
home to a wide varieties of medicinal plants and is one of
INTRODUCTION Indonesia's largest sources of plant diversity. One of the famous
Candida albicans is one of the most pathogenic biofilm-forming native plants of Borneo is Bajakah Tampala (Spatholobus
fungi. In immunocompromised individuals, C. albicans has littoralis Hassk) (Saputera and Ayuchecaria, 2018) [10].
become an opportunistic pathogen, and one of the leading However, Bajakah in the language of the Dayak tribe in
causes of morbidity and mortality worldwide . These fungi Kalimantan means roots, referred to hundreds of species of
invade epithelial tissue, cause superficial infections, and are climbing-twisted rooted plants in the Kalimantan rain forest
life-threatening [1].In the United States, 75% of women during [11]. Based on a preliminary qualitative test conducted by
their reproductive years have Candida vulvavaginitis infection, Anshari (2012), Bajakah Tampala (S. littoralis Hassk) contains
i.e., between 40-50% of patients have recurrent infections and phenolic compounds, flavonoids, and tannins. This is
5-8% of patients have chronic candidiasis [2]. Of the 345 C. reinforced by research conducted by Saputera and Ayuchecaria

Eur. Chem. Bull. 2022,11(5), 59 - 63 59

Efficacy of Bajakah Tampala Ethanol Extract, A Typical Plant Section A-Research paper
of Kalimantan Island (Borneo), Against Candida albicans
Biofilm
(2018), where Bajakah Tampala has been shown to have the used as a terrible manipulate. 1% w/v DMSO become used as
ability to accelerate wound healing. Furthermore, other studies automobile manage, and as high-quality manipulate,
have shown that Bajakah Tampala has antibacterial activity Fluconazole at a attention of 1% w/v turned into used in this
[12]. Research on Bajakah Tampala on C. albicans biofilms has observe. The plates have been then incubated at 37°C for 48
not been reported. Therefore, this study will explore the hours to shape the mid-segment biofilm and for forty eight
bioactivity of the ethanol extract of Bajakah Tampala as an hours to shape the maturation phase biofilm.
antibiofilm of C. albicans . Following biofilm formation, the plate changed into washed
with distilled water three instances to detach nonadherent cells,
and dried at room temperature for 5 minutes to remove the final
MATERIAL AND METHODS water. a total of 125 µL of one% crystal violet solution became
introduced to each well to color the shaped biofilm, each dead
The gear used are Laminar Airflow, incubator (IF-2B) (Sakura,
cells and stay cells, which might be also additives of the
Japan), pipette micropipette (Gilson, France), multichannel
biofilm, then incubated at room temperature for 15 minutes.
micropipette (Socorex, Switzerland), polystyrene nicely ninety
After incubation, the microplate changed into washed below
six flatbed microplate (Iwaki, Japan), microtiter plate reader
walking water three instances to take away the final crystal
(Optical device Ivymen 2100 -C, Spain), spectrophotometry
violet, and two hundred µL of 96% ethanol changed into added
(UV Genesys 10 experiment, 335903) (Thermo scientific
to every nicely to dissolve the formed biofilm. Optical Density
Spectronic, america of the united states), autoclave (Sakura,
(OD) readings had been executed using a microplate reader at a
Japan), analytical stability (AB204 -five, Switzerland). The
wavelength of 595 nm.
fabric used on this have a observe turn out to be the ethanol
The OD cost is then used to calculate the percentage inhibition
extract of the Bajakah Tampala collected from the Forests in
inside the following equation:
East Kalimantan, the same old isolate of C. albicans for biofilm
% inhibition
formation (C. albicans ATCC 10231) from the collection of the
Microbiology laboratory of the college of Pharmacy UGM,
(ODnegative control mean – ODtest sample mean )
Fluconazole 1% w/v, DMSO 1% w/v, NaCl, McFarland X 100
widespread zero.five, Sterile Distilled Water, Sabouraud OD negative control mean
Dextrose Broth (SDB) Media, RPMI Media, PBS (Phosphate The pattern level that may inhibit at the least 50% biofilm
Buffer Saline) solution, 1% w / v Quartz Violet, Disposable formation is considered minimal Biofilm Inhibition
Gloves and masks. concentration MBIC50 [14].

Preparation of test fungal Testing of C. albicans biofilm eradication activity

Candida albicans was cultured for approximately 72 hours at The effect of the ethanolic extract of bajakah tampala was also
37⁰C in Sabouraud Dextrose Broth (SDB). with optical density examined on the preformed biofilm of the C. albicans strain
is 600 microbial culture then meet the standard 0.1 equivalent ATCC 10231 using the previously described method [15].
to the McFarland standard 0.5 ~ 1.5 x 108 CFU/ml). Biofilms were inoculated on microtiter plates as defined above.
After incubation at 37oC for 48 h, each of the plate wells were
Antifungal testing washed with 100 and 100 µL of sterile distilled water 3 times to
tested with wells on microtiter plate flat-bottom polystyrene 96 get rid of nonadherent cells. a entire of 100 µL of media
with various concentrations of bajakah tampala ethanol extract, containing ethanolic extract of bajakah tampala with a attention
i.e 1%, 0.5%, 0.25%, 0.125% w/v. The control used were media collection (1% w/v, 0.5% w/v, 0.25% w/v, 0.125 % w/v) have
control, and infected untreated control (100% growth) for become delivered to each well. Plates had been placed decrease
negative control. Fluconazole 1% w/v is a positive control in again into the incubator at 37oC for 48 hours. Following the
this test, and DMSO 1% w/v used as vehicle control. Culture incubation duration, the plates have been washed 3 instances
plates were Incubation is carried out at 37°C for a period of 72 with 200 mL of sterile PBS to remove adhering cells. The
hours. Optical density readings journey were obtained using a staining strategies are defined above. the share of biofilm
microplate reader at 595 nm. degradation through the extract tested became calculated, as
described earlier than [16].
Test Of Inhibition Of C. albicans Biofilm Formation Mid-
Phase (24 Hours) And Mature-Phase (48 Hours) The Usage Scanning electron microscopy (SEM) test
Of Micro Broth Dilution Approach. For SEM observations, C. albicans biofilms were formed on the
A 96-nicely flat-bottom polystyrene microtiter plate become sterile polyvinyl chloride coverslips (with 0.13—17 mm
used to assess the effect of Bajakah tampala ethanol extract at thickness and 22 mm diameter) inside a 12-well microtiter
the formation of C. albicans biofilm [13]. a complete of 100 µL plates (Corning1 Costar1, Sigma-Aldrich, Missouri, USA) in
C. albicans suspension (107 CFU/mL) was added to every the presence of 0.25 % w/v of ethanolic extract of bajakah
nicely of the microtiter plate after which incubated at ± 37°C tampala for 72 h at 35°C, as described in the previous section.
for 90 minutes for the biofilm attachment phase. After the A biofilm grown in the absence of the test drug served as a
incubation period, each of the the plate wells have been washed control. Briefly, the coverslips were removed, washedtwice
with 100 µL of sterile distilled water three times to get rid of with sterile PBS (0.1 M and pH 7.2) and placed in a primary
nonadherent cells. a complete of 100 µL media containing fixative
ethanolic extract of bajakah tampala with a concentration series solution [glutaraldehyde 0.15 M 2.5 % (vol/vol) in PBS] at 4
(1%, 0.5%, 0.25%, 0.125% w/v) changed into added to every 8C for 60 min. The coverslips were subsequently
nicely. A medium with out microbial increase became used as rinsed 2 times with PBS for 5 min, then treated with the
a medium control, and a microbial suspension changed into secondary fixative (osmium tetroxide OsO4 1% w/v)

Eur. Chem. Bull. 2022,11(5), 59 - 63 60

Efficacy of Bajakah Tampala Ethanol Extract, A Typical Plant Section A-Research paper
of Kalimantan Island (Borneo), Against Candida albicans
Biofilm
for1 h. The samples were subsequently washed with distilled The MBIC50 of the ethanol extract of the of Bajakah tampala
water, dehydrated in an ethanol series (70 % for at concentration of one% w/v against the mid-segment C.
10 min, 95 %for 10 min and 100 % for 20 min) and airdried albicans was 77.00% ± 0.01, whereas the Fluconazole showed
overnight in a desiccator. The coverslip was coated twice with inhibition on C. albicans biofilm formation as much as 80.67 %
platinum vanadium using a sputter ion (BalTec SCD 005), ± 0.01, respectively.. Form the result above, it has been showed
followed by bonding to carbon doubleside tape for examination that the same concentration of extract tested (1% w/v) showed
by SEM [17]. weaker activity against biofilm formation inhibition (77.00% ±
0.01) compare to the planktonic (82,31 % ± 0,01), The result
obtained indicate that microbes in biofilm forms are more
RESULTS AND DISCUSSION difficult to inhibit than in planktonic., This is likely because
microbes in planktonic are single cells, free floating microbes,
Antifungal of Bajakah tampala against C. albicans
while microbes in biofilms tend to live together (many
The results obtained on antifungal testing using microdilution,
colonies), stick, and grow on the surface, and form layered
namely the ethanol extract of Bajakah Tampala was able to
structure encased by an EPS matrix, which make biofilm more
provide inhibitory activity against C. albicans by 82.31% ± 0.01
resistant to antibiotics and antimicrobials.
and control activity of Fluconazole drug 88.10% ± 0.01 (Figure
1 ).
Effect of ethanol extract of tampala Bajakah on mono-
species biofilm C. albicans maturation phase (48h).

Figure 1. The antifungal effect of the ethanol extract of

Bajakah tampala against C. albicans Figure 3. Effect of ethanol extract of Bajakah tampala
on mono-species C. albicans in the maturation phase (48
The results obtained on antifungal testing using microdilution hours)
showed that at 1% w/v concentration of ethanol extract Bajakah
Tampala was able to provide inhibitory activity against C. In this phase, effect of ethanol extract of tampala Bajakah
albicans as much as 82.31% ± 0.01, whereas Fluconazole as decreased in activity compared to the inhibition phase of
positive control gave 88.10% ± 0.01 growth inhibition, planktonic cells and the mid-phase biofilm. This is because, in
respectively. This result provide evidence that the ethanol this phase, the C. albicans biofilm has formed completely so
extract of Bajakah Tampala has an antifungal activity against that C. albicans gets fairly strong protection, this visible from
C. albicans, and the activity was dose dependent. the external mucus produced by maturation phase biofilm more
than the middle phase, not only that the EPS matrix produced
Effect of ethanol extract of Bajakah tampala on the mid- in the maturation phase this was much higher than in the middle
phase C. albicans biofilm mono-species (24h). phase, where EPS was the protector and nutrient provider for
This study examined the antibiofilm potential of the ethanolic the survival of the C. albicans biofilm so that new colonies
extract of Bajakah Tampala against C. albicans biofilm continued to form. This is by research [18]. which states that
formation. these consequences imply that the ethanol extract antimicrobial dealers may also have more trouble penetrating
Bajakah Tampala could inhibit as much as 50% of C. albicans biofilm defenses inside the maturation section. Mature biofilms
biofilm formation (Figure 2). encompass a yeast base with hyphal elements forming a
complicated community encased in EPS and a ways from the
floor [19], ethanol extract of the of Bajakah tampala 1% w/v
gave an inhibitory activity in the ripening phase of 70.31% ±
0.01, while the activity (Fluconazole), which was used as a
control drug, showed a better inhibitory activity of 75.10% ±
0.01 (Figure 3). This shows that in the maturation section (48
hours), the ethanolic extract of the Bajakah tampala can offer
50% interest.
These results provide evidence that the longer the biofilm
growth time, the more the matrix arrangement produced, and
the stronger and more complex the biofilm structure formed.
The test compound or drug control decreased activity in
Figure 2. Effect of ethanol extract of Bajakah tampala inhibiting the biofilm. This result is reinforced by Bjarnsholt's
on C. albicans within the intermediate phase (24 hours) (2013) statement and Hamzah's (2018) [20], who state that
biofilm antimicrobial dealers can have extra trouble penetrating biofilm

Eur. Chem. Bull. 2022,11(5), 59 - 63 61

Efficacy of Bajakah Tampala Ethanol Extract, A Typical Plant Section A-Research paper
of Kalimantan Island (Borneo), Against Candida albicans
Biofilm
defenses within the maturation section. The interaction among three: after remedy). Ethanol extract of Bajakah
species causes colonization and infection dynamics, in addition tampala at awareness of zero.five% w/v can purpose
to a number of other responses [21] lysis in C. albicans biofilms cells and the mobile density
decreases (Figure 5).
Biofilm Eradication activity of ethanol extract of Bajakah The biofilm matrix serves as a hyperlink among adhesive and
tampala against C. albicans biofilm. cohesive interactions, which provides mechanical stability to
Using the crystal violet staining, we have found that at the biofilm, controls cell dispersion of the biofilm, and can also
concentration of 1% w/v the ethanol extract of Bajakah tampala act as a nutrient source provider for cell communication [23].
could eradicate C. albicans biofilm by 65.21% ± 0.01, whereas The C. albicans biofilm matrix additionally acts as a protecting
Fluconazole degrade as much as 69.11% ± 0.01 of C. albicans cellular biofilm and a primary barrier defensive biofilm cells all
biofilm, accordingly (Figure 4). through assaults from the immune system and antifungal drug
treatment

Scanning electron micrograph (SEM) result of C. tropicalis

administrated by 0.5% w/v ethanol extract of Bajakah
tampala
Image from SEM (figure 5) shows that the ethanol extract of
Bajakah tampala was proven to disturb C. albicans biofilm
because the active compound could attack the EPS matrix of the
biofilm. On the other hand, decreased cell attachment, density,
and lysis were observed.

Figure 4. The activity of the ethanol extract of the of CONCLUSION

bajakah tampala in eradicating C. albicans biofilm The ethanolic extract of the Bajakah tampala has an antifungal
At the same concentration, the activity of the extract tested in and antibiofilm activity against C. albicans .and also capable to
degrading C. albicans established biofilms was weaker (65.21% eradicate C. albicans biofilms. The ethanolic extract of the
± 0.01) than the activity in inhibit C. albicans biofilm formation Bajakah tampala is capability to be developed as candidate for
(77.00% ± 0.01). The decrease in the activity of the ethanol antifungal and antibiofilm of C. albicans .
extract of Bajakah tampala in eradicating C. albicans biofilm is
thought to be due to the more complex and structured of
preformed biofilms. Biofilm microbes formed a highly ACKNOWLEDGMENT
structured cell as well as thick and dense EPS substances We would like to thank SIMBLITABMAS KEMENDIKBUD
surrounding the biofilm cells, thatprotect the microbes in the DIKTI INDONESIA 2022 with research contract number
biofilm from antimicrobial and antibiofilm agents. According 136/L11/KM/2022 for the trust of funds given to researchers,
to Stewart and Costerton (200) [22] antibiotic therapy so that this research can be carried out properly.
can eliminate or reduce planktonic bacteria, however bacteria
in the biofilm are more persistent. When treatment with Ethical Approval: This study did not use experimental animals
antibiotics is completed, the biofilm will form planktonic cells so it does not require approval from the ethics committee.
again, which results in acute infection.
Funding Details: SIMBLITABMAS KEMENDIKBUD
Scanning electron micrograph (SEM) result of untreated C. DIKTI INDONESIA 2022 with research contract number
albicans biofilm 136/L11/KM/2022
The activity of Bajakah tampala ethanol xtract in inhibit and
breakdown C. albicans biofilm was confirmed by SEM Conflict Of Interest: The authors have no conflicts of interest
analysis. The results confirmed that untreated C. albicans had a to declare that are relevant to the content of this article.
high cellular density inside the EPS Matrix protecting
C. albicans (figure 5). This shows that a biofilm has formed. Informed Consent: The research focused on the biofilm of C.
albicans, one of the microbes that causes wound infection with
diabetic ulcers on the foot, using a plant from the Indonesian
Borneo Island, Bajakah Tampala.

Authorship
H. Hamzah : main idea of research, biofilm testing,
final
approval, data analysis
S.U.T Pratiwi : main idea of research, biofilm testing
A. Jabbar : Plant determination and extract
Figure 5. Scanning Electron Microscopy (SEM) photo manufacture
of C. albicans treated with zero.five% w/v ethanol E. Nandini : draft writing
extract of Bajakah tampala (1,2: earlier than remedy,

Eur. Chem. Bull. 2022,11(5), 59 - 63 62

Efficacy of Bajakah Tampala Ethanol Extract, A Typical Plant Section A-Research paper
of Kalimantan Island (Borneo), Against Candida albicans
Biofilm
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