Preliminary study on the inhibitory effect of seaweed Gracilaria verrucosa extract on biofilm formation of Candida albicans cultured from the saliva of a smoker

Background: Candida albicans is an opportunistic fungus that might infect the oral cavity. Increased colony numbers of C. albicans in the mouth can be caused by multiple factors, such as smoking, weakened immune system, antibiotics use and immune-compromised condition. Smoking can increase expression of virulence factors of C. albicans and make it stronger. One virulence factor of C. albicans is biofilm formation. The ability of creating biofilm makes C. albicans more tolerant to commercial antifungal agents. The objective of this preliminary study was to examine the ability of the seaweed G. verrucosa extracts to inhibit the formation of biofilm by C. albicans isolated from the saliva of a smoker. Methods: The extract of G. verrucosa was prepared by maceration using 96% methanol and subjected for phytochemical analysis. C. albicans was isolated from the saliva of a smoker who voluntarily participated in the study after providing informed consent. In triplicate, the fungus was cultured in the growth medium containing increased concentrations of G. verrucosa (6.25, 12.5, 25, 50, 75 and 100% ).The same reaction using fluconazole 0.31 µg/ml C. albicans was prepared as positive control. Biofilm formation was accessed based on optical density of cell mixtures using an ELISA reader. The data obtained were subjected to Kruskal-Wallis test at a significance limit of 0.05. Results: Methanol extract of seaweed G. verrucosa contained three bio-active compounds namely steroids, terpenoid, and tannins. Inhibitory activity of seaweed extracts on C. albicans biofilm formation increased as their concentration increased. The highest inhibitory effect was recorded at fungus culture treated with seaweed concentration of 25% at 24 hours of time exposure. Conclusions: Seaweed G. verrucosa extract contained steroids, terpenoids and tannins that were able to effectively inhibit the formation of biofilm by C. albicans at the concentration of 25% after 24 hours of time exposure.


Introduction
Smoking is a common problem in both developed and developing countries, including in Indonesia. Based on a survey by the Tobacco Atlas in 2015, Indonesia has the highest number of smokers in Asia, with 66% of men in Indonesia being active smokers 1 . Smoking can lead to addiction owing to the nicotine contents, and harm due to the presence of toxic compounds such as carbon monoxide, ammonia and tar contents in tobacco 1 . Substances in cigarettes can also contribute for the occurrence of oral candidiasis, infection of in the mouth cavity caused by the fungus Candida albicans 2 . This fungus is part of the normal flora of the human mouth, but it can become pathogenic in certain conditions, for example, due to nicotine exposure 3 .
The C. albicans that infects human tissues generally form biofilm 3 , an extracellular matrix consisting of C. albicans colonies 4 . The size of the biofilm increases when the fungus was exposed to substances in cigarette smoke because cigarette contains chemicals that can initiate the growth of and nourish C. albicans 5,6 .
Currently, fluconazole and nystatin are the most effective drugs for treating oral candidiasis. Unfortunately, these drugs could result in undesired side effects. Prolonged use of fluconazole, for example, can leads to resistance 7 whereas high dosages of nystatin give gastrointestinal discomfort and increase plaque formation 8 . Therefore, plant-derived antifungals may be a viable oral treatment option for candidiasis. One of these potential plants is Gracilaria verrucosa. This seaweed contains several bioactive compounds, including alkaloids, flavonoids, phenolics, saponins, steroids and terpenoids 9 . Aceh Province, Indonesia, has large G. verrucosa resources, although this aquatic plant has not been commonly used for medicinal purposes. Hence, the objective of the present study was to examine the ability of seaweed extract to inhibit the biofilm formation of C. albicans isolated from the saliva of smoker.

Time and site
The study was conducted in August 2017 at the Laboratory of Microbiology, Veterinary Faculty, Syiah Kuala University. The G. verrucosa seaweeds were collected from a farmer in Pulo Aceh, Aceh Province.

Ethics
All research protocols used in this study were approved by the Research Ethics Committee of the Dentistry Faculty of Syiah Kuala University No. 1741/UN11.1.21/TU/2017.

Saliva collection
The saliva was collected from an active smoker who worked as administrative staff at the Faculty of Dentistry Medicine of Syiah Kuala University and voluntarily participated in this study after completing informed consent. Inclusion criterion of the volunteer was active smoker who smoked 20 cigarettes per day. Saliva was collected once by spitting into a glass jar (15 ml) right after the subject finished smoking, and added with 10 ml of PBS (0.01 M, pH 7.2). The jar was centrifuged at 10,000 rpm for 10 minutes. The precipitate was stored for the microbiological examination.

Candida albicans isolation and preparation
Precipitate was cultured in ChromAgar Candida medium and incubated at 37°C for 2 days. The C. albicans fungus grew as green colonies. One colony of C. albicans was mixed with 5 ml of peptone in a tube and incubated at 37°C for 24 hours. Turbidity of medium was compared to a 0.5 McFarland solution standard, which was equivalent to 1.5 × 10 8 CFU/ml.

Seaweed extraction
Seaweed extraction was performed based by maceration using 96% methanol 10 . In brief, a total of 500 g of fresh seaweeds were washed with tap water then with distilled water. Seaweed samples were at 25°C for 24 hours, chopped into small pieces (2 mm), and soaked in 1.5 liters of 96% methanol. Macerate was filtered using Whatman filter paper No. 42. Filtrate collected was concentrated using a vacuum rotary evaporator (Laborta 4003 control, Heildolph) 60°C and at a speed of 80-90 rpm for 3 days. Concentrated extract was put in a sealed dark bottle and stored at 4°C.

Phytochemical tests
Flavonoid test. A 0.5 cm magnesium plate was rinsed in 5 ml of seaweed extract, mixed with two drops of HCl, and heated by passing it over a Bunsen flame. Red or purple coloration formed on the heating indicated the presence of flavonoids 11 . Alkaloid test. Seaweed extract (5 ml) was mixed with 8 ml of HCl and filtered. Filtrate was subjected to Mayer, Wagner and Dragendroff tests for alkaloids 11 . This was done by mixing 2 ml of filtrate with 5 g potassium mercuric iodide (Mayer test), 2 ml of Wagner reagent, or 2 ml of bismuth potassium iodide solution (Dragendroff test). The formation of white or pale precipitates (Mayer test), brown or reddish-brown precipitates (Wagner test) and red precipitates (Dragendroff test) indicated the presence of alkaloids.
Tannin/phenolic test. Two drops of 1% FeCl 3 was added to 1 ml seaweed extract. The change in the color to a blackish green indicated the presence of tannin/phenolic 12 .

Amendments from Version 2
Revisions, corrections and details addition have been done throughout the manuscript as a response to the last comments/ questions of reviewers. Significant improvements made are including an explanation about the reasons why fluconazole 0.31 mg/ml did not show good antimycotic properties as expected. References supported this explanation were also provided. Some parts of this explanation also answers a question from reviewer about the reason for determining dose of fluconazole. Correction and enrichment in methods were also performed to provide better clarity and readability. The method used was a modification of those previously used by two different researchers in analyzing biofilm formation by bacteria. Further details, especially those related to the low respondent involved, could not be made because research presented was still in the preliminary stage.

REVISED
Saponin test. Seaweed extract, 1 ml, was diluted in 20 ml of distilled water and shaken vertically for 15 seconds. Persistent foaming indicated the presence of saponin content.
Steroid test. Two milliliters of seaweed extract was added with 2 ml of CHCl 3 , 2 drops of H 2 S and 1 ml of CH 3 COOH. The formation of green or blue precipitates indicated the presence of steroid 11 . Terpenoid test. Seaweed extracts (5 ml) was mixed with 2 ml of chloroform. Concentrated H 2 SO 4 , 3 ml, were carefully added. The formation of reddish brown layer at the interface of extract and chloroform solution indicated the presence of terpenoids 13 .

Examination of biofilm formation
Casein-peptone lecithin polysorbate broths (Merck-1117230500), 100 µl, were poured in each well of a 96-well plate, incubated at room temperature for 5 minutes and discarded by blotting the plate on paper towels 2-5 times. Fifty microliter C. albicans culture had turbidity was equal to 0.5 McFarland standard were added to each well and incubated for 5 minutes to attachment of fungal cells on casein. Cell mixtures were washed by aspiration. In triplicate, 50 µl of decreased concentrations of seaweed extracts (100, 75, 50, 25, 12.5 and 6.25%) were added. The same reaction using fluconazole 0.31 µg/ml was prepared as positive control. The plates were then incubated at 37°C for 24, 48 or 72 hours. Each well was added with 200 µl of 0.1% violet crystal, incubated at 25°C for 15 min, and washed three times with 200 µl of 0.01 M PBS. The crystal violet in each well was then removed by adding 100 µl of 96% ethanol for 2 min. The biofilm formation was analyzed by reading optical density of mixture using an ELISA reader at 620 nm 14,15 .

Data analysis
The data obtained were subjected to Kruskal-Wallis test using SPSS software v20.0 for windows.

Results
The results of phytochemical tests in Table 1 show that methanol extract of seaweed G. verrucosa contained bioactive compounds belonged to steroids, terpenoids, and tannins/polyphenols. In general, the inhibitory effect was reduced as seaweed concentration increased (Figure 1). The highest inhibition after 24 and 48 hours time exposures was recorded from C. albicans culture treated with 25% seaweed extract, followed by those with 50% and 75%. The best inhibition after 75 hours time exposure, however, was found in C. albicans culture treated with 25% seaweed extract. In all time exposures, the inhibitory effect caused by fluconazole (control) was the worst, followed by those caused by 100% seaweed extracts, but there was no difference between these two groups. Results of Kruskal-Wallis analysis showed that seaweed extract significantly (p<0.05) inhibited the formation of biofilm by C. albicans, indicated potential of the extract to inhibit the growth of the fungus.

Discussion
The study showed that 25-75% seaweed extracts are promising for inhibiting the growth of C. albicans as shown by much lower biofilm formation after 24, 48 and 72 hours exposure compared to those caused by positive control (fluconazole) and 100% seaweed extract. This indicated potential of seaweed extract as natural anti-fungus to treat oral candidiosis caused by against C. albicans in smokers.
C. albicans is a normal micro-organism in the human mouth. This fungus, however, can be pathogenic in certain circumstances 3 such as in the mouth of smokers 2 . Smoking can stimulate synthesis of HWP1, EAP1 and SAP2 proteins in C. albicans, causing higher virulence of the fungus. This can lead to increased formation of biofilm and finally cause oral candidiasis 6 . Smoking also cause a decreased immune function, making individuals more susceptible to oral infections, including candidiasis 4,6 .
The low effectiveness of fluconazole 0.31 µg/ml, the leading choice of therapy against C. albicans, against this fungus was probably caused by several factors such as low therapeutic dose and resistance of the fungus to the drug. Pfaller et al., who Fluconazole dose of 0.31 µg/ml used in this study was slightly higher than minimum dose ideally used to test antifungal resistance of the antifungal. This also meant that C. albicans strain isolated from smoker respondent involved in this study already developed resistance to the dose used. Increasing resistance of C. albicans against this fluconazole, one of the most azole antimycotics have been reported 18,19 . This resistance, that is assumed related to prolonged or repeated exposure to low-dose of the antifungus 20 , must be confirmed by further study.
The stronger ability of 25-75% seaweed extracts to inhibit the formation of C. albicans biofilm significantly compared to those of fluconazole (positive control) was probably caused by bioactive compound presence in the extracts. These included steroids, terpenoids, and tannins/polyphenols, all of which have been known their benefit for human health. According to Sampaio et al. 21 , antifungal activity of a substance strongly depends on the composition of its bioactive compounds. Steroids can kill C. albicans through their lypophilic properties, interfering with the formation of fungal spores and mycelium 22 . This activity weakens C. albicans, inhibiting the formation of the biofilm. To function optimally, steroids require oligosaccharides that are also present in the seaweed content 23 Terpenoids are derivatives of saponins that may act as an antifungals by damaging organelles of the fungus and by inhibiting secretion of enzymes, leading to growth the inhibition C. albicans cells. Terpenoids can also damage the morphology of C. albicans 24 . Tannins may inhibit chitin synthesis in C. albicans cell walls, leading to lost of membrane cell protection and disrupted cellular metabolism. Tannins also can inhibit ergosteron activity of C. albicans 25 .
The effectiveness of seaweed extracts in inhibiting fungal growth is influenced by at least three factors, namely concentration, exposure time, and contact surface media 26 . The present study showed that diluted concentration (25-75%) of extracts showed better inhibitory effect on the growth of C. albicans than the more concentrated 100% extract. Concentrated extract usually has lesser effectiveness in vitro due to solubility and import problems. In more aqueous condition plant extracts generally show better medicinal properties with increased concentration as shown by a study testing antifungal activity of ethanol extracts of Syzygium jombolanum, Cassia siamea, Ordina wodier, Momodica charantia and Melia azedarach as well as two algal species Sargassum wightii and Saulerpa scalpellformis against 25 C. albicans isolates in vitro 27 .
This study also showed the best inhibitory effect of seaweed extracts was recorded at 24 hour of exposure. This is probably because the farnesol, a quorum-sensing molecule that has the potency to inhibit C. albicans growth 28 , works effectively after 48-72 hour of exposure.
This study, however, was still preliminary due to limited number of subject and unavailability of non-smoker, control. Virulence factor of C. albicans analyzed was also only biofilm formation. Since more virulence factors are possibly synthesized by the fungus under certain environmental condition, further studies are need to be conducted to investigate effect of individual bioactive compound contained in the seaweeds on the formation of biofilm and on the expression other virulence factors of C. albicans isolated form both smoker and non-smoker individuals.

Conclusion
Extract of Gracilaria verrucosa seaweed could inhibit the growth of C. albicans isolated from the saliva of a smoker. The inhibitory effect decrease with the increase of concentration, and reached the highest at concentration of 25% and time exposure of 24 hours.

Grant information
The author(s) declared that no funding was involved in supporting this work.
I cannot comment. A qualified statistician is required.

Version 1
Author Response 05 Aug 2018 , Syiah Kuala University, Indonesia Zaki Mubarak Some revisions and improvements have been done throughout the manuscript as responses to the good comments and suggestions from reviewers. Major revisions performed include grammar check and corrections; sentence improvements; and reorganization and adding appropriate details in each section of the material and methods part. Grammar issues, were concerned by all reviewers, had been addressed. The ways to present results were also changed as response to comment from Indonesia reviewer about the meaning of OD values in presenting the growth of and how to interpret them. Restructure of C. albicans result and discussion, therefore, was also performed. These changes became the basis for synthesizing more appropriate conclusion sentences. One reference was also added to support discussion about the importance of extract concentration for resulting in expected medicinal effects, in this study was growth inhibition that reflected by biofilm formation. Since this study is still in preliminary step, no non-smoker participant was participated. As consequence, no comparison could be made between smoker and non-smoker groups. Yet the study was still able to confirm potential of seaweed extract Gracia verrucose as anti candidiasis in smokers. Potential of doing further studies was mentioned in relation to this weakness of our study.
No competing interests were disclosed.

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