Objective: To investigate the effect of methanolic extract of Cassia fistula Linn. stem bark against different virulence factors of Candida albicans and its phytochemical, GC-MS analysis.

Methods: For phytochemical analysis available standard tests were done. Standard methodologies of susceptibility testing were used to evaluate the efficacy of methanol extract of Cassia fistula Linn. against planktonic growth and virulence factors of Candida albicans. Antibiofilm activity of extract was analyzed in microplate based in vitro biofilm model, using XTT-metabolic assay. Light and Scanning electron microscopy (SEM) were used to observe biofilm structure. GC-MS and FT-IR analysis were used to identify phytocompounds in the extract.

Results: Extract exhibited minimum inhibitory concentration (MIC) of 2.0 mg/ml for planktonic growth and 4.0 mg/ml for biofilm of C. albicans ATCC 3017. Extract inhibited yeast to hyphal dimorphism at low concentrations (1–4 mg/ml), while adhesion to a solid surface was prevented at 2–4 mg/ml. Extract arrested cell cycle of Candida at S and G2/M phase. The extract reveals the significant inhibition (p< 0.05) of biofilm formation, planktonic and Yeast to Hyphal morphogenesis respectively against Candida albicans. Phytochemical, GC-MS and FT-IR analysis showed the presence of bioactive compounds such as; Ethanone, 1-[4 – (1, 1 – dimethylethyl) phenyl], Diethyl Phthalate, 3, 6-Bis-dimethylaminomethyl -2, 7-dihydroxy-fluoren-9-one, Cyclopentasiloxane, Hentriacontane, Cyanoacetylurea and o-Veratramide etc.

Conclusion: The efficacy of the bark extract of Cassia fistula Linn. against the drug resistant biofilm of the human pathogen Candida Albicans and Ten Phytochemical compounds are detected in the extract by GC-MS and FT-IR analysis.

•    Cassia fistula Linn
•    Biofilm
•    Candida albicans
•    Drug resistance
•    Adhesion

Bansode B, Ghule N, Mortale S, Kolhe R, Pohare S, et al. (2018) Efficacy of Methanol Extract of Cassia Fistula Linn Stem Bark against Different Virulence Factors of the Human Pathogen Candida albicans. Ann Clin Med Microbiol 3(1): 1013.

MIC: Minimum Inhibitory Concentration; SEM: Scanning Electron Microscopy ; YPD: Yeast-Peptone-Dextrose; PBS: Phosphate Buffer Saline; MOPS: (3-[N-morpholine] Propane Sulphonic Acid); RPMI: Roswell Park Memordium Institute; DMSO: Dimethyl Sulphoxide; XTT: 2, 3-bis (2- methoxy-4-nitrosulfophenyl)-2H-tetrazolium-5-carboxanilide; CLSI: Clinical and Laboratory Standards Institute; RMA: Relative Metabolic Activity; GC-MS: Gas Chromatography Mass Spectrometry; FT-IR: Fourier-transform infrared spectroscopy.

Candida albicans is an opportunistic fungal pathogen of the humans. It colonizes the gastrointestinal (GI) tract, reproductive tract, oral cavity and skin of the most humans [1-4]. Candida can form biofilms on the surface of various solid materials, which were used for medical prostheses, cardioverter defibrillators, urinary and vascular catheters, and cardiac devices [5,6]. The National Institutes of Health estimated that the biofilms were responsible for 80% of all the microbial infections in the United States [7]. Mainly, with healthy immune systems Candida albicans is often harmless and kept balance with other members of local microbiota. However, alterations in the host microbiota (e.g., due to antibiotics), changes in the host immune response (e.g., during stress, infection by another microbe, or immunosuppressant therapy), or variations in the local environment (e.g., shifts in pH or nutritional content) can enable Candida albicans to overgrow and cause infection. These infections range from superficial mucosal and dermal infections, such as thrush, vaginal yeast infections, and diaper rash, to hematogenously disseminated infection with sizable mortality rates (approaching 40% in some cases) [8]. Candida biofilms shows the resistance to anti-fungal agents including the widely prescribed anti fungal drug fluconazole [9]. Emergence of resistance to conventional antibiotics and side effects of the synthetic chemical drugs have increased the problems in antimicrobial therapy [10,11]. In such circumstances use of products of natural origin for anti-infective and therapeutic purposes is more attractive.

Cassia fistula Linn. commonly known as Amaltas and in English popularly called “Indian Laburnum”. It is a native of India [12-14] grown in Mauritius, South Africa, Mexico, Brazil, China, Nepal, West Indies and East Africa as an ornamental plant due to its beautiful bunches of yellow flowers [15-17]. It has been used in the treatment of various ailments in ancient India, dating back to Sushruta Samhita and Charaka Samhita [13,18]. In the Ayurvedic medicinal system, Cassia fistula Linn. was used against various disorders such as haematemesis, pruritus, leucoderma, diabetes and other ailments [19,20]. Mainly, the stem bark has been used against amenorrhoea, chest pain and swellings. The bark possess tonic and antidysentrica properties, also used for skin complaints. The bark powder or decoction of the bark is administered in leprosy, jaundice, syphilis and heart diseases [21]. Antibacterial and antifungal properties of Cassia fistula Linn. have been reported [22-24]. Kulkarni et al., identified different compounds in the methanol extract of Cassia fistula Linn. plant, whereas citronellol and linoleic acid have already reported as anticancer agents from different sources [25-28].

The aim of the study is to investigate the antibiofilm activity of Cassia fistula Linn bark. The present research identifies the phytochemical compounds in the methanol extract of Cassia fistula Linn. GC-MS analysis reveals the presence of Ten different compounds such as (1) Ethanone, 1-[4 – (1,1 – dimethylethyl) phenyl), (2) Diethyl Phthalate, (3) 3,6-Bis-dimethylaminomethyl -2, 7-dihydroxy-fluoren-9-one, (4) Cyclopentasiloxane, (5) Hentriacontane, (6) Cyanoacetylurea, (7) o-Veratramide, (8) 1,5-Diphenyl-2H-1, 2, 4-triazoline -3-thione, (9) Sarcosine, (10) 2-cyano-2- (E) –Heptenoic acid. In the earlier reports among ten compound three compounds were showed biologically activity, such as Hentriacontane acts as an anti-inflammatory agent [29], diethyl phthalate as an antimicrobial agent [30] and Sarcosine was tested for antifungal activity against Aspergillus spp. respectively [31]. Herein, we have studied the antibiofilm and antivirulence properties of methanolic extract of Cassia fistula Linn bark against the human pathogen, Candida albicans.

Collection, authentication and preparation of extracts of tree barks

Tree bark was collected from Kinwat, Maharashtra State of India was authenticated by a Botanist Dr. R. M. Mulani. Bark was sliced into small pieces; shade dried and then grinds into a powder using an electrical blender. The 25 gm of bark powder was subjected for solvent extraction by Soxhlet apparatus in 250 ml of methanol for 5 h. The extract was filtered and concentrated under vacuum by rotary evaporated; the remained powder was weighed and kept in sterile bottles under refrigerated condition until use. The stock solution of dried bark powder was prepared 200 mg/ ml in DMSO and used for testing of respective activities and further dilutions were prepared in RPMI for experiments.

Phytochemical analysis

Phytochemical analysis of the extract was carried out according to standard methods [32,33].

Culture, media, chemicals and culture conditions

Candida albicans, ATCC 3017 received from the Institute of Microbial Technology, Chandigarh, India. The strain was maintained on Yeast-Peptone-Dextrose (YPD) agar slants at 4°C. A single colony from the yeast extract-peptone-dextrose (YPD) agar slant was inoculated in 50 ml of YPD broth (pH 6.5) in a 250 mL Erlenmeyer flask. The flask was incubated at 30°C on Remi cooling orbital Shaking Incubators (Capacity 215 Ltr) Cis-24 Plus by Remi at 120 rpm for 24 h. The cells from the activated culture were harvested by centrifugation for 5.0 min at 2000 rpm. Collected cells were washed three times and resuspended with phosphate buffer saline (PBS) (10.0 mM phosphate buffer, 2.7 mM potassium chloride and 137 mM sodium chloride, pH 7.4). For susceptibility testing, RPMI-1640 medium with L-glutamine, without sodium bicarbonate and buffered with 165 mM MOPS (3-[N-morpholine] propane sulphonic acid) pH 7, was prepared and filter-sterilized using 0.2 µm filters. Various concentrations of extracts were prepared in RPMI-1640 medium by double dilution. The Concentration of the solvent i.e. Dimethyl Sulphoxide (DMSO), never exceeded >1 % during dissolving the extract. A fluconazole was used as a standard antifungal drug was purchased from Forcan, Cipla Pvt. Ltd., Mumbai, India). 2, 3-bis (2- methoxy-4-nitro-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and menadione were purchased from Sigma-Aldrich Chem. Ltd. Mumbai, India.

Minimum inhibitory concentration (MIC) for planktonic growth

Effect of extract on the growth of planktonic cells of Candida albicans was studied by using the standard broth microdilution methodology, as per Clinical and Laboratory Standards Institute (CLSI) guidelines [34]. Briefly, various concentrations of the extract (ranging from 0.25 to 4.0 mg ml-1) were prepared in RPMI-1640 medium in 96 well plates. The wells without test extract served as a control and inoculum from washed Candida albicans cells were added to each well so that to get 1×103 cells ml-1 [35]. The plates were incubated at 35°C for 48 h. To analyze the growth absorbance was read using a microplate reader (at 620 nm) (Multiskan EX, Thermo Electron Corp., USA). The lowest concentrations of extract which caused 50% reduction in the absorbance compared to that of control and considered as minimum inhibitory concentrations (MICs) for growth of Candida albicans [36].

Adhesion assay

Effect of extract on the adherence of Candida albicans to a solid surface (i.e. polystyrene) was studied using a microplate based assay and prepared various concentrations (0.25-4 mg ml−1) of extract in PBS. Wells without extract was kept as controls, while fluconazole was used as a standard drug. The cell suspension 50 µl was added to each well to obtain 1 × 107 cells ml– 1. The final volume of the assay system in each was kept at 100 μl. The plates were incubated at 37°C for 90 min at 100 rpm in an orbital shaking incubator to allow attachment of cells to the surface.

After the incubation, wells were washed with PBS to remove non attached cells. Density of the adherence in each was analyzed as relative metabolic activity (RMA) using the XTT-assay. More than a 50% reduction in RMA compared to the control was considered significant [37].

Morphogenesis

Serum induced yeast to hyphal morphogenesis in Candida albicans was studied in a microplate-based assay. Various concentrations of test molecules were prepared in 20% serum with deionized water. Cells were inoculated to get 1 × 106 cells/ ml, in test and control wells. Final volume of assay system in each well was kept at 200µl. The plate was incubated at 37°C at 200 rpm, on an orbital shaker for 2 h. Cells were examined microscopically after 2 h to confirm the formation of germ tube and counted 100 cells and yeast and hyphae were noted. Concentration which inhibited ≥ 50 % of hyphae compared with that of control was considered the MIC for morphogenesis [38].

Biofilm formation

Candida albicans biofilms were developed on polystyrene surface of 96 well plates as per standardized in vitro biofilm model. Cell suspension of 1×107 cells ml-1 was prepared in PBS and 100 μl was inoculated in each well. In the adhesion phase, plate was incubated at 37°C for 90 min at 100 rpm to allow attachment of cells on the surface. Non-adhered cells were removed by washing the wells with sterile PBS, two to three times. The 200 µl of the RPMI-1640 medium was added to each well and the plates were incubated at 37°C for 48 h to allow biofilm formation. To observe the effect on development of biofilms, RPMI-1640 medium along with various concentrations (0.25-4 mg ml-1) of the extracts were added to each well immediately after adhesion phase and incubated for 48 h at 37°C. To analyze activity against mature Candida albicans biofilms, the extracts were added to 24 h mature biofilms and further incubated for 48 h at 37°C. After incubation wells were washed to remove any nonattached planktonic cells. The presence or absences of biofilms structure in the wells was observed using an inverted light microscope (Metzer, India) and photographs were taken by Labomed microphotography system (Labomed, India) at x 200 magnification. Biofilm growth was analyzed and confirmed with XTT metabolic assay [39].

Biofilm quantitation by XTT assay

Biofilm growth was quantitated using XTT i.e. [2, 3-bis (2-methoxy-4-nitro-sulfophenyl)-2H-tetrazolium-5-carboxanilide] metabolic assay. The solution of XTT was prepared by mixing 1.0 mg ml-1 XTT salt in PBS and stored at 20°C prior to use, meanwhile the solution of Menadione solution was prepared in acetone. The menadione solution was added to the XTT solution to get 4 µM final solution. The wells containing biofilms were washed with PBS to remove non adhered cells. The 100 µl of XTT-menadione solution was added to the wells and incubated for 5 h in dark at 37°C. The development of colour indicates the formation of water soluble Foramzon product and was measured at 450 nm using a microplate reader (Multiskan EX, Thermo Electron Corp., USA). The wells without extract were considered as control, while those without biofilms are the blank. The concentration of extract which caused ≥ 50 % lowering in RMA was considered MIC for biofilm [40,41].

Microscopic analysis

Germ tube formation and biofilms were observed under an inverted light microscope (Metzer, India). Photographs were taken by a Labomed microphotography system (Labomed Korntal, Germany) at x 200 magnification. The scanning electron microscopy (SEM), samples were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 24 hour at 4°C. Samples were post-fixed in 2% aqueous solution of Osmium tetraoxide for 4 h and dehydrated in a series of graded alcohols and finally dried to a critical drying point with a Critical Point Dryer unit. The samples were mounted over stubs and drop casted on silica wafers, further gold coated by using an automated gold coater (model JOEL JFC-1600) for 3 min. Photographs were taken under a scanning electron microscope (Model JOEL-JSM 5600) [40,41].

Cell cycle studies

Candida albicans yeast phase cell from the lag phase of growth were harvested, washed and incubated the cells in sterile water for 1 h, 103 cfu / ml of these cells were inoculated to each flasks containing 50 ml YEPD broth and MICs of selected plant extracts. YPD broth without test extracts considered as control. All the flasks were incubated at 30°C for 3 h. The cells were centrifuged for 1000 rpm for 5 min, washed and fixed in 1 ml of 70 % ethanol by incubating at room temperature on rotary shaker for 30 min. Cells were harvested by centrifugation at 1000 rpm for 5 min, washed twice with 1ml of 50 mM Tris (pH 7.8), resuspended in 500 μl of 50 mM Tris containing 10 μg RNase A and incubated 2 h at 37°C. The cells were harvested by centrifugation and resuspended in 500 μl FACS-buffer (200 mM Tris/HCl pH 7.5; 200 mM NaCl; 78 mM MgCl2 ). 15 μl of propidium iodide (1.0 mg/ ml) was added to each tube and incubated for 30 min and stored at 4°C. Before analysis, cells were sonicated for 10 s and analyzed using FACS Calibur cytometer and cell Quest 3 software. All the experiments were done in triplicates [42].

GC-MS analysis

The GC-MS analysis was performed using an Agilent 6890 gas chromatograph (Agilent Technologies, Palo Alto, CA) equipped with a 5973N mass selective detector (a single quadrupole instrument) and a HP-5MS capillary column of 30 m length, 250 μm internal diameter and 0.25 μm film thickness was used.

Statistical analysis

The values mentioned are the mean with standard deviations obtained from three different observations. The obtained values in the control and treatment groups for various molecules as well as results obtained in XTT were compared using Student’s t -test. A value of P < 0.05 was considered statistically significant [43].

Phytochemical analysis

The standard protocols have been used for analyzing the phytochemical constitutes from the methanol extract of Cassia fistula Linn. The extract gave a positive result towards flavonoids, phenolic, carbohydrates, steroids and glycosides, whereas it gave negative results towards amino acid, starch and alkaloids. Saponins are found to be very less concentration in the extract.

Moreover, the phytochemical analysis reveals that Cassia fistula Linn. posses good extent from phenolic and flavonoids would be imply for biological activities.

Methanol extract of Cassia fistula Linn inhibits planktonic growth, morphogenesis, adhesion and Biofilm mode of growth in Candida albicans

Methanol extract of Cassia fistula Linn inhibited planktonic growth of Candida albicans. Extract inhibited more than 65 % planktonic cells at 4 mg per ml and 60 % at 2 mg/ml. Whereas extract prevented more than 65 % biofilm growth at 4 mg/ ml. MIC is considered as a concentration at which the extract showed more than 50 % inhibition as compared to control. Extract showed MIC at a concentration of 2 mg/ml for planktonic growth and 4 mg/ml for Biofilm of Candida albicans ATCC 3017. In the present study methanol extract of Cassia fistula Linn. did not showed any inhibitory effect on mature biofilm of Candida albicans ATCC 3017 (Figure 1).

Figure 1: Effect of methanol extract of Cassia fistula Linn. on Planktonic growth, Developing Biofilm and Mature Biofilm of Candida albicans ATCC 3017. The percentage biofilm formation by C. albicans ATCC 3017 in the presence of methanol extract of Cassia fistula Linn. was analyzed as a function of the Relative Metabolic Activity in an XTTassay.

Prevention of biofilm was also confirmed with microscopic observations which showed the absence of characteristic biofilm structure at the MICs of the methanol extract of Cassia fistula Linn (Figure 2).

Figure 2: Effect of Methanol extract of Cassia fistula Linn. against biofilm formation by Candida albicans. Where, (A) Control (C. albicans ATCC 3017) (B) represent biofilm preventive activity of methanol extract of Cassia fistula Linn. at 4.0 mg per ml.

Treatment with effective concentrations of methanol extract of Cassia fistula Linn. caused removal of Candida albicans biofilm cells. Only a few hyphae and yeast forms were seen to remain on the solid surface compared to the dense network of cells in the control. Formation of a dense network of filamentous forms and yeast cells were completely prevented. The antibiofilm activity was confirmed by Scanning Electron Microscopy (SEM) (Figure 2).

Yeast to hyphae switching in Candida was inhibited with an MIC at concentration of 1 mg/ ml with treatment of methanol extract of Cassia fistula Linn. about 30 % Hyphal cells observed at 4 mg/ml and 44 % at 2 mg/ml, it reveals that the extract inhibited Yeast to Hyphae morphogenesis (Figure 3).

Figure 3: Effect of methanol extract of Cassia fistula Linn. against inhibition of yeast (Y) to hyphae (H) morphogenesis of Candida albicans 3017.

Methanol extract of Cassia fistula Linn. was found to prevent adhesion of Candida albicans to polystyrene surfaces. Analysis of metabolic activity by the XTT assay revealed that methanol extract of Cassia fistula Linn. was the most active inhibitors of adhesion. It exhibits the antiadhesion activities with an MIC at concentration of 1 mg/ ml of Candida albicans ATCC 3017 (Figure 4).

Figure 4: Anti adhesion activity of methanol extract of Cassia fistula Linn. against Candida albicans ATCC 3017.

Cell cycle inhibition by methanol extract of Cassia fistula Linn bark

Methanol extract of Cassia fistula Linn also found to inhibit Candida albicans cell cycle at S phase and G2/M phase. Cells treated with extract were arrested majority of the cells that is 45 % in S phase and minimum in G2/M phase (Figure 5).

Figure 5: Effect of methanol extract of Cassia fistula stem bark on cell cycle of Candida albicans ATCC 3017

GC-MS analysis

GC-MS analysis identified the Ten different compounds namely (1) Ethanone, 1-[4 – (1,1 – dimethylethyl) phenyl] , (2) Diethyl Phthalate, (3) 3,6-Bis-dimethylaminomethyl -2, 7-dihydroxy fluoren-9-one, (4) Cyclopentasiloxane, (5)Hentriacontane, (6) Cyanoacetylurea, (7) o-Veratramide, (8) 1,5-Diphenyl-2H-1, 2, 4-triazoline -3-thione, (9) Sarcosine, (10) 2-cyano-2- (E) – Heptenoic acid (Figure 6).

Figure 6: Gas chromatogram mass spectrum (GC-MS) of methanol extract of Cassia fistula Linn. stem bark.

The retention time, name, molecular formula, molecular weight and structure of the compounds are given in Table 1.

Three compounds out of Ten already showed biological activities in the literature. Hentriacontane have already reported as an anti-inflammatory agent and diethyl phthalate as an antimicrobial agent. Whereas Sarcosine were tested for antifungal activity against Aspergillus spp

FT-IR analysis

The vibrational frequency of FT-IR observed at 3400-3231 cm-1 represents the presence of alcoholic (-OH) and stretching frequency observed at 1616 cm-1 assign the presences of carbonyl group (-C=O) analogous to carboxylic acid and Ketonic group respectively. While stretching at lower wave number at 1506, 1451 cm-1 and frequency in the range of 1284-1160 cm-1 corresponding to symmetric and asymmetric of (-C=C-, -C-H, -C-N) assigns the presence of aromatic and aliphatic compounds. Furthermore, the stretching at 1069 cm-1 corresponding to (-C-O) assigns the presence of alcoholic, carboxylic acids, and ester respectively (Figure 7) [43,44].

Figure 7: FT-IR spectrum of methanol extract of Cassia fistula Linn. Stem bark.

Cassia fistula is a mild laxative suitable for children and pregnant women [19]. It has been reported to treat intestinal disorders like ulcers [13]. The stem bark is used against amenorrhoea, chest pain and swellings. The bark possesses tonic and antidysentrica properties and also used for skin complaints, the powder or decoction of the bark is administered in leprosy, jaundice, syphilis and heart diseases [22]. Cassia fistula bark has potential to protect the liver against CCl4 induced hepatotoxicity in rats [45]. Ilavarasan et al., has studied the mortality of methanolic extract of Cassia fistula up to 2000 mg/kg and which would be considered safe [46]. Similar results were found for a single dose at 2000 mg/kg oral administration of C. spectabilis leaf extract that was shown to be non-toxic tested on mice [47]. Cassia fistula Linn fruit pulp and seed extract possessed anticandida activity [48]. Surprisingly, the activity of stem bark of Cassia fistula Linn. against Candida albicans biofilm has been not unexplored till date. By considering these results, Herein, we have studied the antibiofilm activity of Cassia fistula Linn stem bark against the human pathogen Candida albicans.

In this study we found that methanol extract of Cassia fistula Linn. is a good inhibitor against different virulence factors of the human fungal pathogen Candida albicans such as growth, adhesion property, yeast to hyphal form and biofilm formation. Yeast to hyphal morphogenesis and filamentation is an important virulence factor in Candida albicans. It is supposed to play a crucial role in tissue invasion and the spread of infection. The present study revealed the strong antimorphogenetic activity of extract (Figure 3). The ability of extract to interfere in the adhesion phase is important for the prevention of Candida biofilm [49]. It inhibited the adhesion property of Candida albicans (Figure 4). Extract inhibited the biofilm formation of Candida albicans which is normally resistant to antifungal agents like fluconazole (Figure 1, Figure 2) The prevention of specific virulence attributes rather than killing the pathogen would be a novel strategy to circumvent the emergence of drug resistance [49,50]. MIC values of the bark extract of Cassia fistula Linn. is established against the virulence factors of Candida albicans. The cell cycle study reveals that Cassia fistula Linn arrested cell cycle at G2/M and S phase (Figure 5).

Mature biofilms of Candida albicans were consisted of multiple layers of a dense network of filamentous and yeast form cells. Extracellular polymeric substances secreted by biofilm cells are deposited along the entire cellular network which gives a thick biofilm structure. In mature stages, biofilms are more resistant to environmental factors as well as antifungal drugs [51]. Phytochemicals of the extract could be a good alternative to the available antifungal agents for mitigation of biofilms [52]. The methanolic extract exhibits the significant inhibition of biofilms at 2-4-fold more concentration than the planktonic MIC (Figure 1). The biofilm inhibition was evident, with a significant reduction in the metabolic activity analyzed by XTT assay. There is a necessity for more in vivo studies where various concentrations of extract could be tested. This may help in the identification of extract concentrations which may deferentially inhibit both bacteria as well as Candida albicans.

The analyzed chemical constituents of methanol extract of the bark of Cassia fistula Linn. by GC-MS analysis was identified Ten different compounds (Figure 6, Table 2).

Table 2: Qualitative phytochemical analysis of Cassia fistula Linn. methanol bark extract.

These molecules are reported from the extract of the bark of Cassia fistula Linn. among three were reported as anti inflammatory, antifungal and antimicrobial activities [29-31]. These phytocompounds may be alone or in combination are responsible for the antibiofilm activity against Candida albicans. Phytochemical analysis supports the data for the presence of phytocompounds in the extract (Table 1). The FTIR analysis reveals the presence of phenolic, alcoholic, alkanes, carboxylic acids and aliphatic compounds (Figure 7). This study opens up the possibility of exploring the bioactive potential of these molecules and the use of methanol extract of the bark of Cassia fistula Linn for the treatment of candidiasis caused by Candida albicans. Experiments in animal models are required to confirm the in vivo antibiofilm potential of Cassia fistula Linn. bark extract.

In conclusion, the present study systematically evaluated the efficacy of extract against the growth, virulence factors and biofilms of Candida albicans. The minimum inhibitory concentration of Methanolic extract of Cassia fistula Linn. with the Candida albicans is tested. Cassia fistula Linn. gives the inhibitory activity against different virulence factors of Candida albicans such as Yeast to Hyphae switching, Biofilm formation and adhesion property. This study demonstrate the use of crude methanolic extract of Cassia fistula Linn is the promising source of anticandidal compounds. The phytochemical analysis identified ten different phytocompounds in the methanol extract by GC-MS analysis and the functionality of the compounds were confirmed by FT-IR spectroscopy method. Out of ten compounds three are already biologically active viz. anti-inflammatory, antimicrobial and antifungal agents. In addition three compounds are also responsible for anticandidal activities. Whereas other seven compounds also responsible for anticandidal activity. This in vitro study revealed Cassia fistula Linn. may be an alternative therapy against the available drug. Nevertheless, detailed toxicity studies are necessary before proposing it as a therapeutic strategy against candidiasis.

BSB is thankful to RGSTC Project (Letter No.APDA/RGSTC/ Proposal-ASTA/2014-15/2994) for financial assistance.

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