Antibacterial and antifungal activities of indican ( indoxyl β-D-glucoside )

Indican (Indoxyl β-d-glucoside), plant pigments found in true Indigo, are present in Indigofera genus plants, fungi and human urine. This study evaluated the antibacterial and antifungal activities of this pigment using the microdilution method. Antibacterial and antifungal activities were performed against Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Candida albicans, Candida tropicalis, Candida krusei, Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum gypseun. Minimum inhibitory concentrations (MICs) were measured according to the broth microdilution protocols by the Clinical and Laboratory Standards Institute (CLSI). The Indican showed only antifungal activity. It had MIC values of 128 μg/mL for yeasts and 512 μg/mL for most of the dermatophytes. The minimum fungicidal concentration (MFC) values of the Indican ranged between 512 μg/mL: C. tropicalis (LM-6) and C. krusei (LM-6 and LM-8) and 1,024 μg/mL: C. albicans (ATCC-76645, LM-108, LM-P20). Further studies are needed to clarify the potential fungicidal activity of the Indican and the possibility for topical applications.


INTRODUCTION
Brazilian medicinal plants have been used by the local population in the treatment of tropical diseases such leishmaniasis, malaria, schistosomiasis, fungal and bacterial infections (Duarte et al., 2005).In this context, the study of products from plant or synthetic substances with antimicrobial activity is gaining high prospects in the medical and pharmaceutical fields (Menezes and Lima, 2013).Plants have several chemical compound classes, including alkaloids.Indigo alkaloids belong to the bisindole alkaloid class, being natural products used by mankind in dyes production and for medicinal purposes, in addition to being widely used in traditional Chinese medicine (Calvo, 2007).These pigments are present in Indigofera genus plants, fungi and human urine (Santos and Torres, 2012).Indican is the precursor of indigo pigments (indigo and indirubin), thus being starting material of these metabolites biosynthesis (Maugard et al., 2002).
Superficial fungal infections affect approximately 20 to 25% of the world population and they are classified into inflammatory and non-inflammatory infections.Disease control precautions involve hygiene and proper treatment, but drugs used to treat these diseases are toxic, and some microorganisms are already resistant to them.Drugs based on natural compounds, isolated or synthetic, appear as a possible solution to cope with these microorganisms resistance to conventional treatments (Oliveira and Silva, 2008;Filho et al., 2010;Dias et al., 2013).
The indigo biosynthesis pathway varies among plant species, but has indican (indoxyl β-D-glucoside) as a common precursor.This compound is mainly located in young leaves and can be extracted into aqueous solution.Indican undergoes hydrolysis through β-glucosidase enzyme action, leading to indoxyl (Sandoval et al., 2011).
Indigo is used in popular medicine, having antiinflammatory, antipyretic, antiviral and antimicrobial activities, and recent studies have proved its anticancer property and efficacy in psoriasis treatment (Chiang et al., 2013).Studies showed that the indirubin is one potent cyclin-dependent kinases (CDKs) with activities such as anti-inflammatory, immunomodulatory, antifungal and antileukemic, in addition to inhibiting several tumoral cell lines (Suzuki et al., 2005).Considering the vast potentiality of plants as sources for antimicrobial drugs, the objective of this study was to assess the antibacterial and antifungal activities of the main precursor of indigo, the indican (indoxyl β-D-glucoside).
Suspension was standardized by the 0.5 Mc Farland scale tube, set through spectrophotometric reading (Leitz-Photometer 340-800) to 90% T (530 nm), corresponding to approximately 10 6 colonyforming units (UFC) per mL.The final concentration confirmation was done by counting the microorganisms in a Neubauer chamber (Sahin et al., 2004).

Chemistry
Indican (Indoxyl-β-D glucoside), Cloranfenicol, Nistatin and Fluconazole were obtained from Sigma Aldrich, Brazil.The drugs da Silva et al. 201 were dissolved in dimethylsulfoxide (DMSO), and sterile distilled water was used to obtain solutions of 1,024 µg/mL for each.The concentration of DMSO did not exceed 0.5% in the assays.

Culture media
To test the biological activity of the products, the strains were maintained in appropriate culture media: Nutrient agar (NA) (Sigma-Aldrich, São Paulo, SP, Brazil) for bacteria, Roswell Park Memorial Institute (RPMI-1640) for yeasts, Sabouraud Dextrose Agar (SDA) purchased from Difco Laboratories (Detroit, MI, USA) for filamentous.They were prepared and used according to the manufacturers' instructions.

Minimum inhibitory concentration (MIC)
MICs were measured according to the broth microdilution protocols by the Clinical and Laboratory Standards Institute (CLSI, 2008).The determination of MIC was performed in duplicate using 96 "U" bottomed well microplates.In each plate orifice, 100 µL of double concentrated nutrient broth (CN) was added for bacteria, with 100 µL solubilized Indican subsequent addition, which was also double concentrated.Through serial dilution, concentrations of 1.024 to 8 µg/mL were obtained, and plates were sealed and incubated at 35°C for 24 h.Bacterial growth control was done with chloramphenicol (100 µg/mL).
In antifungal activity was carried out by wells microdilution technique.Microplates were sealed and incubated at 35°C for 24-72 h for yeasts and at 28°C room temperature for 7-10 days for filamentous fungi.Negative controls (without drugs) were used to confirm strains viability, and sensitivity controls (for DMSO) were also included in the studies.
To determine the MFC, the authors subcultured 1 µL aliquots of MIC, MIC×2, and MIC×4 of Indican, Fluconazole, and Nistatin the control yeast growth onto Petri dishes containing SDA.After 24-48 h of incubation at 35°C, a reading was to to evaluate the MFC, based on the growth of the controls.The MFC was defined as the lowest product concentration that inhibited growth of the yeast or permitted less than three CFUs to occur, resulting thus in 99.9% fungicidal activity (Espinel-Ingroff et al., 2002).Biological activity assays were performed in duplicate, and the results were expressed as the arithmetic mean of the MIC and MFC.

RESULTS AND DISCUSSION
Bacterial resistance is currently one of the most important problems.In clinics, this problem is related to inadequate treatment and indiscriminate antibiotics use.This causes high mortality rates, especially when infections are caused by etiological agents, such as multidrug resistant S. aureus, P. aeruginosa and Acinetobacter baumannii (Gómez et al., 2008).
In vitro antimicrobial activity of indican synthetic compound was determined in this study.Indican showed no inhibition, although having growth on Gram-positive and negative strains (Table 1).In this test, it was possible to observe strains of bacterial growth in indican and Cloranfenicol standard antibiotic, demonstrating these microorganisms resistance to the compounds.Bacteria use several mechanisms to ensure their growth in the presence of antibiotics, among them are: enzymatic inactivation, cell wall permeability changes, cell wall precursor changes, among others (Calvo et al., 2006).The difficulty in finding active drugs has led to a search for more potent drugs (Castro et al., 2006).Multidrug resistant microorganism infections complicates treatment, reduces the number of effective antibiotics, and creates the need for broad activity spectrum drugs use, configuring as a public health problem (Dutra, 2015).
Indican, indoxyl β-D-glucoside, is the main indigo and indirubin formation precursor, which is located in Indigofera genus plant young leaves, and is also obtained by synthesis.This study data related to synthetic Indican antifungal activity with Candida genus yeast-formed strains are shown in Table 2.
Antifungal resistance is a challenge to clinical practice, with infectious agent isolation and MIC of potential drugs used for its treatment being suggested when possible (Colombo, 2003).The determination of MIC for Indican is shown in Table 3. Beginning with 128 µg/mL concentration, there was inhibition of growth and with the 256 µg/mL concentration, there was inhibition of most microorganisms.
The Candida genus, which is responsible for local and systemic infections, showed C. tropicalis, C. parapsilosis, C. glabrata, C. krusei, C. lusitaniae and C. guillermondii species as the most pathogenic agents.These microorganisms have also developed antifungal agent resistance.Taking into account mutations or increased genes expression, these pathogens have become resistant to azole agents (Menezes et al., 2009).
C. tropicalis and C. krusei yeasts are opportunistic pathogens, having the ability to colonize different body parts (skin, gastrointestinal and genitourinary tract and respiratory system), and being resistant to several drugs (Pappas et al., 2009;Praneenararat, 2014).C. albicans exhibit polymorphism that makes species capable of changing its shape according to environment conditions, in addition to favoring different tissues colonization (Modrzewska and Kurnatowski, 2013).
There are few available drugs for candidiasis treatment.In addition to low supply, the drugs used have high toxicity and are not too efficient due to microorganisms acquired resistance (Wong et al., 2014).
Antifungal agents frequent use combined with inadequate treatment are responsible for promoting these pathogens resistance to drugs used in medical routine.The difficulty in finding active drugs has led to a search for more potent drugs (Castro et al., 2006).Researches involving synthetic or natural (extracted from medicinal plants) compounds that have active ingredients against multidrug resistant strains are necessary (Gonçalves et al., 2009).
Results showed Indican antifungal potential on yeasts and dermatophytes.According to Sartoratto et al. (2004) and Houghton et al. (2007), Indican MIC can be considered to have good biological activity against C. albicans, C. krusei, T. rubrum, T. mentagrophyte and M. gypseun.S. aureus, S. epidermidis and P. aeruginose bacteria and C. tropicalis ATCC-13803 yeast showed resistance to the compound.Chiang et al. (2013) proved indigo naturalis antimicrobial activity, which comes from Strobilanthes formosanus Moore, on Staphylococcus spp.strains and non-dermatophytes onychomycosis.Isatis costata indole alkaloids also showed antifungal activity over ten strains, showing best results on Trichophyton simii and Trichophyton Schoen leinii dermatophytes, having 80% growth inhibition, while Candida albicans yeast had approximately 69% growth inhibition (Fatima et al., 2007).I. suffruticosa leaves aqueous extract showed growth inhibition against dermatophytes and bacteria ( Leite et al., 2006).
Studies on Indican synthetic are scarce in the literature.
However, studies on indican isolated from I. suffruticosa leaves showed cell lines growth inhibition in mice with Sarcoma 180 (Maranhão, 2008).Research by Lima et al. (2014) demonstrated that indican has anticancer and hepatoprotective activities.

Conclusions
Indican showed antibacterial activity in the tested strains.However, this compound showed good antifungal activity against different species, genus, candida and moderate activity against Trichophyton and Microsporum genera filamentous fungi, thus suggesting that this compound could be used in antifungal topic agent development.
However, further investigation would be necessary on the use for the treatment of superficial mycoses.