Screening for Anticandidal and Antibiofilm Activity of Some Herbs in Thailand

Purpose: To evaluate the anticandidal activity of the ethanol extracts of 12 herbs from Thailand. Methods: The herbs studied were Alpinia galanga, Curcuma longa, Curcuma zedoaria, Mentha cordifolia, Ocimum africanum, Ocimum basilicum, Ocimum sanctum, Piper betle, Piper chaba, Piper nigrum, Piper sarmentosum and Zingiber officinale. Various Candida spp. were examined for minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) using microdilution method; time-kill assay was also used to assess the plants. Antibiofilm activity was investigated using a 3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium-bromide (MTT assay). Gas chromatography mass spectrometry (GC-MS) analysis, thin layer chromatography (TLC) fingerprinting and TLC-bioautography were used to determine the active anticandidal compounds. Results: All tested herbs, except extracts of P. nigrum and Limiaceae family, showed varying zones of inhibition against Candida albicans ATCC 90028. P. betle revealed the strongest anticandidal activity against all tested strains with MIC ranging from 1.56 to 3.13 mg/ml, and MFC from 3.13 to 8.33 mg/ml. Killing activity depended on time and concentrations of the extract. The concentration of P. betle extract required to inhibit ≥ 90 % biofilm formation of C. albicans ATCC 90028 was 3.13 ± 0.15 mg/ml, while that to remove ≥ 90 % biofilm growth was 12.50 ± 0.69 mg/ml. The result of GC-MS analysis showed the major compound of P. betle extract responsible for anticandidal activity as 4-chromanol. Conclusion: P. betle extract contains 4-chromanol which is a good potential anticandidal agent for the treatment of oral infectious diseases caused by certain Candida spp.


INTRODUCTION
The genus Candida contains more than 150 species, however, only a limited number of these species regularly cause diseases in humans [1].Candida species belong to the normal microbiota of human mucosal surfaces including the oral cavity.There has been a significant increase in incidence of oral candidiasis due to higher numbers of immunocompromised patients, the use of dental devices and the use of broad spectrum antifungal drugs.Candida albicans is the main cause of oral candidiasis, however, non-C.albicans (NCAC) species such as Candida glabrata, Candida krusei and Candida tropicalis are now frequently reported as human pathogens [2].
The pathogenesis of oral candidiasis is facilitated by a number of factors including the ability to adhere to medical devices and/or host cells, and to form biofilm. Biofilm formation is an important virulence factor for a number of Candida species as it confers significant resistance to antifungal therapy by limiting their penetration through the matrix and protecting cells from host immune responses [3].It has been demonstrated that biofilms formed by C. albicans and/or NCAC strains have been associated with high morbidity and mortality rates [4].Chlorhexidine (CHX) has been used as an adjunctive therapeutic option for topical use owing to its broad-spectrum antimicrobial efficacy in oral candidiasis.However, it has been known to have side effects with prolonged use [5,6].
Natural products have increasingly received attentions due to their antimicrobial activity against a wide range of human pathogens.Few studies have reported anticandidal activity from various herbs [7,8].In addition, the information on their active compounds is scarce.The aim of this study was to evaluate of the ethanolic extracts of 12 herbs, which are traditionally used in Thai folk medicine against various Candida spp.The phytochemical profile of the most effective extract was also investigated.

EXPERIMENTAL Preparation of medicinal herb extracts
Twelve herbs included in this study were purchased from the local market, and their details are shown in Table 1 1) were deposited in the herbarium.The dried plants were macerated with ethanol for 3 days and then filtered through Whatman no. 4 filter paper.The filtrate, each case, was concentrated using a rotary evaporator at 40 °C and kept at -20 °C.A 10 % (w/v) stock solution in 10 % dimethyl sulfoxide (DMSO) of each extract was used in this study.

Candida strains and growth conditions
A total of eleven Candida spp.were tested, and the details of strains used shown in Table 2.In this study, all tested strains were cultured on Sabouraud Dextrose Agar (SDA; Difco Laboratories, Detroit, MI, USA) or Sabouraud Dextrose broth (SDB; Difco Laboratories, Detroit, MI, USA) at 37 °C for 24 h.

Agar diffusion test
A 100 µl of inoculum ( 107 cfu/ml) was mixed with 20 ml of warm SDA, and poured into the plate with a 6 mm diameter metal cup.After solidifying the SDA, the metal cups were removed and the well filled with 100 µl of each extract.A 10 % DMSO was included as control.Inhibitory activity was determined by measuring the diameter of the inhibited zone.All tests were performed in triplicates.

Broth microdilution test
Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were determined using the broth microdilution method of the Clinical and Laboratory Standards Institute [9].Each extract was subjected to a two-fold serial dilutions in the SDB to obtain concentrations from 0.02 mg/ml to 25 mg/ml at a total volume of 100 µl per well in 96-well plate, and 100 µl of each tested strain (1 × 10 3 cfu/ml) was added.The medium as well as 0.1 % CHX and 10 % DMSO were used as blank, positive control and non-treated control, respectively.MIC was defined as the lowest concentration of the extract that showed no detectable growth.MFC was defined as the lowest concentration of wells that did not grow when 10 µl of the well contents was plated on agar.All experiments were repeated thrice in duplicates.

Time-kill assay
As the screening of extracts revealed that P. betle extract gave the strongest anticandidal activity, it was used for the time-kill assay [10].Growing culture ( 106 cfu/ml) of representative C. albicans ATCC 90028 was added to SDB and exposed to 1, 2 and 4 × the MIC of extract, and extract free medium was used as growth control.Samples were removed for colony counts at 0, 0.5, 1, 2, 4, 6, 8, and 24 h.The viable counts were determined using the serial dilution method after incubation.The procedure was repeated in triplicate, and the log 10 cfu/ml was plotted against time.A 0.1 % CHX was used as positive control.All assays were carried out in triplicates.

Inhibition of biofilm formation
The effect of P. betle extract on biofilm formation of representative C. albicans ATCC 90028 was examined using the modified microdilution method [11].Two-fold serial dilutions of extract and a cell suspension of the tested strain were prepared as described in the MIC assay.A 0.1 % CHX was used as the positive control.The phosphate buffered saline (PBS) at pH 7.0 and the medium alone were used as the non-treated and blank controls, respectively.After overnight incubation, supernatants were discarded and washed 3 times with PBS.Biofilm formation was quantified using a 3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium-bromide (MTT; Sigma-Aldrich, St Louis, MO, USA) assay.The numbers of surviving Candida were determined by measuring their ability to reduce the yellow tetrazolium salt to a purple formazan product at OD 570 nm.Percentage inhibition was calculated by equation 1.
The biofilm inhibition concentration (MBIC 50 and MBIC 90 ) was defined as the concentrations that showed 50 % and 90 % inhibition of biofilm formation.All experiments were repeated in triplicate [11].

Eradication of biofilm formation
The eradication of biofilm was also examined using minimum biofilm eradication concentration (MBEC) assay.A 200 µl (10 3 cfu/ml) aliquot of C. albicans ATCC 90028 was inoculated into 96well plates to form biofilm overnight.The biofilm was washed 3 times with PBS.The biofilm was exposed to a 200 µl of various concentrations of extract ranged from 0.02 to 25 mg/ml.The number of surviving Candida were determined using MTT assay at OD 570 nm.Percentage eradication was calculated by equation 2. E% = [1-(A 570 of the test/A 570 of non-treated control) × 100]………………………………… .. (2) MBEC value was defined as the concentrations that showed 50 and 90 % eradication of biofilm formation.The medium alone, 0.1 % CHX and PBS were used as the blank control, positive and non-treated, respectively.All experiments were repeated thrice in duplicate [11].

GC-MS analysis of phytoconstituents in P. betle extract
One microliter of P. betle extract was investigated for its phytoconstituents using gas chromatography-mass spectrometer (GC-MS, Trace GC Ultra, USA), the TR-5MS capillary column (0.25 mm × 30 m × 0.25 µm film thickness) was used for the separation of various phytoconstituents.The GC conditions were: injection temperature, 250 °C; column temperature, isothermal at 70 °C for 2 min, then programmed to 305 °C at 10 °C/min and held at this temperature for 5 min.Helium gas with a flow rate of 1.0 ml/min was used as a carrier gas.The effluent of the GC column was introduced directly into the source of MS and spectra obtained in the electron ionization mode with 70 eV ionization energy, the sector mass analyzer was set to scan between 35-500 amu for 2 s.The identification of individual unknown compound is based on comparing its retention time relative to those of the known components by matching spectral peaks available with Wiley 9 Mass Spectral Library [12].

Thin layer chromatography (TLC) fingerprinting of P. betle and TLCbioautography-assisted separation of anticandidal
For TLC-fingerprinting study, P. betle extract (100 mg) and pure eugenol (5 µl) were prepared in 1 ml of 95 % ethanol.A 10 µl of each sample was applied onto the alumina silica gel GF 254 TLC-plate, and was run through a series of solvent systems using a mixture of 90 volumes of toluene and 10 volume of ethyl acetate as the mobile phase.The plates were dried in the air, and were stained with a 20 % (w/v) of phosphomolybdic acid in ethanol [13].
TLC-bioautography was performed using agar overlay method.TLC-plates were placed on plate contained 6 ml of SDA, and 10 ml of top agar with 100 µl of inoculum ( 107 cfu/ml) was covered on TLC plate.After overnight incubation, the inhibited zone was observed as clear zone.The relative front values (Rf) was calculated as: Rf = distance travelled by solute / distance travelled by solvent [14].

Statistical analysis
The data are expressed as mean ± standard deviation (SD) by using Microsoft Excel 2007 12.0.6611.1000(Microsoft Corporation, Redmond, WA, USA) from the three experiments with duplicate independent experiments.The graph of time-kill analysis is plotted using SigmaPlot Version 11.0 (Systat Software, Inc., San Jose, CA, USA).

RESULTS
Anticandidal activity of 12 herbs against C. albicans was shown in Table 1.These extracts showed different inhibited zone against C. albicans ATCC 90028.The diameter of inhibited zone for all the extracts ranged from 1 to 20 mm, except P. nigrum and extracts of Lamiaceae did not show any inhibitory zone.Two herbs P. betle and C. zedoaria, revealed the good anticandidal activity, and were further examined for their MIC and MFC against 11 Candida strains.P. betle exhibited a better anticandidal activity than C. zedoaria against all tested strains with the MICs ranged from 1.56 to 3.13 mg/ml and MFCs ranged from 3.13 to 8.33 mg/ml, respectively (Table 2).Time kill assay of P. betle extract was performed for C. albicans ATCC 90028, it was depended on time and concentrations of extract.At 1 × MIC, 2 × MIC and 4 × MIC, the strain was killed after 8, 2 and 1 h, respectively.The killing of 0.1 % CHX was observed within 30 min (Figure 1).The concentrations of P. betle extract required to inhibit ≥ 50 % and ≥ 90 % biofilm formation of C. albicans ATCC 90028 were 0.78 ± 0.31 mg/ml and 3.13 ± 0.15 mg/ml, respectively.The concentrations of P. betle extract to remove of ≥ 50 % and ≥ 90 % biofilm formation were 1.56 ± 0.44 mg/ml and 12.5 ± 0.69 mg/ml, respectively.CHX could completely inhibit or remove biofilm formation (Figure 2).The GC-MS analysis of P. betle extract showed 2 major chemical components of 4-chromanol (62.33 %) and eugenol (17.10 %), which their retention times were 10.46 and 9.11, respectively (Figure 3A).It was demonstrated that there were several bands of components appeared on the TLC-plate (Figure 3B), and one major band with Rf at 0.38 gave the large inhibited clear zone against tested Candida in TLC-bioautography assay (Figure 3C).Another minor band with Rf at 0.74 matched to the standard eugenol did not show any inhibited zone.

DISCUSSION
Candida species are the most common fungal pathogens of humans and are the causative agents at various locations in the body, giving rise to severe morbidity in millions of individuals worldwide [15].In oral cavity, C. albicans is the organism most frequently implicated a range of oral candidiasis like oropharyngeal candidiasis, oral thrush and denture stomatitis [15], and NCAC have been found dramatically in oral candidal infection.A topical use of CHX mouthwash is the most common antimicrobial substance to control oral candidiasis.However, CHX has been reported as having many unwanted effects including taste disturbance, desquamative lesions, and brown staining of the teeth and oral mucosa [5,6] and having limited use in the pregnant and the young children.
Medicinal herbs are widely employed in folk medicine, mainly in communities with low hygiene and poor sanitation.Plants products are considered to have low side effects compared with synthetic ones.Some studies have demonstrated that herbs possess the anticandidal activity against various Candida strains [7,8].In this study, 7 of 12 Thai herbs, A. galanga, C. longa, C. zedoaria, P. betle, P. chaba, P. sarmentosum and Z. officinale showed potential anticandidal activity against tested C. albicans.However, P. nigrum and plant extracts in family of Lamiaceae did not any activity possible because their major potent compounds are volatile oil which could have evaporated during preparation of extracts by a rotary evaporator.Among those plants tested, P. betle extract exhibited the strongest anticandidal activity against tested Candida species.
In addition, P. betle extract has shown an antibiofilm activity.Biofilm of C. albicans and NCAC are associated with high incidence of hospital morbidity and mortality [4] due to the increased use of medical devices such as lens, implants and dentures.Such biomaterials facilitate Candida strains to colonize and form biofilm leading to the development of fungal resistance to antifugal agents such as amphotericin B, ketoconazole and fluconazole [16].Experiments comparing biofilm of Candida in this study have demonstrated that higher concentrations of MBEC 90 of P. betle extract (4 folds) than of MBIC 90 are required to significantly remove existing biofilm cells.This is an expected result since Candida in the biofilm is strongly protected and less susceptible to antifungal agents than in planktonic form [17].It was shown that P. betle extract was able to remove Candida in the dose and time dependent manner.Our results indicated that P. betle extract acts as a potent antibiofilm agent that has dual actions of preventing biofilm formation and removing existing biofilm.
P. betle has been extensively used in traditional medicines in many countries.It was reported to have various pharmacological activities such as antifungal, antimicrobial, immunomodulatory and anti-inflammatory [18].
The antimicrobial activity has been previously shown to be a broad spectrum of both bacteriostatic and fungistatic effects against C. albicans, Pseudomonas aeruginosa, Aspergillus flavus and Escherichia coli [19,20].Some studies reported an anticandidal effect of P. betle on a wide range of Candida spp.[7,20].Our results are in concordance with the study of Himratul-Aznita et al [20] who demonstrated the anticandidal activity of aqueous crude extract of P. betle leaves against 7 oral Candida species.However, there was no information of antibiofilm or active compounds mentioned in that study.The ethanol extract in this study revealed the lower MICs and MFCs of anticandidal activity compared to the aqueous extract [20].This was also found in a study of the ethanol extract of P. betle leaves with stronger antibacterial activity compared with aqueous extract [21].It indicates that the ethanol may be the appropriate solvent for P. betle leaves extraction.
The results of the GC-MS analysis, TLC fingerprinting and TLC-bioautography in the present study indicated that 4-chromanol could be the active compound responsible for the anticandidal activity.There were only 2 major peaks found in the GC-MS analysis, and the one with the lower peak matched to the eugenol by the TLC fingerprinting.Eugenol did not exhibit any anticandidal activity in TLC-bioautography assay.Therefore, the major band found on the TLC fingerprinting with Rf of 0.38 showing a large inhibited zone against Candida strain should be the highest peak of 4-chromanol found in GC-MS analysis.
A number of phytochemical constituents of P. betle extract have been documented in some studies [18,22]; however, 4-chromanol has not be reported.The finding of the phytochemical profile of P. betle extract in this study is in agreement with Deshpande and Kadam [21], in which the extract revealed the presence of 4-chromanol.However, the presence of 4chromanol was a major component in this study (62.33 %), which was higher than earlier reported that was higher than their report.The finding of chemotypic variations in this study and others may be due to ecological and geographical conditions, age of herb and time of harvesting.The apparent variation in the chemical profile may influence their anticandidal activity.

CONCLUSION
The current study supports the traditional uses of the studied plant and suggests that the P. betle leaves extract is a potential source of natural anticandidal agent.It possesses 4-chromanol with good anticandidal properties that may be used for oral infectious diseases caused by certain Candida spp.Further work should be performed to determine its mode of action as well as in vivo activity.

Table 1 :
Plants and susceptibility of Candida albicans ATCC 90028

Table 2 :
MIC and MFC of tested extracts against Candida

spp Strain Concentration of extracts (mg/ml)
MIC -the minimum inhibitory concentration; MFC -the minimum fungicidal concentration; the values represent data obtained from three independent assays and where the appropriate mean ± S.D. is given