Anticandidal activity of Cassia fistula and its effect on ergosterol biosynthesis

Abstract

Context: Cassia fistula Linn. (Caesalpiniaceae) has been used in folk medicine. Anthraquinone derivative rhein having antimicrobial properties is actively present in C. fistula fruit. Although, as yet there has been no study of its anticandidal potential.

Objective: The present study was conducted to determine the phytochemical composition of fruit pulp and seed extract and their effect on Candida albicans ATCC 10261, Candida glabrata ATCC 90030 and Candida tropicalis ATCC 750, respectively.

Materials and methods: The fruit pulp and seed extracts were tested for phytochemicals by various standard methods and rhein was identified by thin-layer chromatography. The anticandidal activity was determined by minimum inhibitory concentration (MIC), growth curve studies, cytotoxicity and ergosterol estimation assay.

Results: The fruit pulp and seed extracts showed high content of phenolic compounds. Rhein was identified in both extracts, Rf 0.38. MICs of seed extract obtained with C. albicans, C. tropicalis and C. glabrata is 350, 300 and 300 μg/ml. However, for fruit pulp extract, these values significantly reduced to 150, 250 and 100 μg/ml, respectively. Comparative MIC values for fluconazole were 16, 16 and 04 µg/ml. At MICs, pulp reduced ergosterol content in cell membrane of C. albicans, C. tropicalis and C. glabrata by 54.42, 48.78 and 68.0%, seed extract by 38.11, 47.0 and 45.0%, whereas, fluconazole showed 93.56, 89.21 and 98.0%, respectively.

Discussion and conclusion: C. fistula fruit pulp and seed extract possessed anticandidal activity. The result was significantly correlated between the MICs, cytotoxicity and ergosterol inhibition. It was concluded that the crude extract is a promising source for anticandidal compounds.

Keywords::

• Cassia fistula
• Candida albicans
• Candida glabrata
• Candida tropicalis
• anticandidal activity
• ergosterol inhibitor

Introduction

Infectious diseases have been cured with herbal medicine throughout the history of mankind. Natural products either as pure compounds or crude extracts provide unlimited opportunities for new drugs due to the chemical diversity. The increasing failure of chemotherapeutics and antibiotic resistance exhibited by pathogenic fungal infection has led to the screening of several medicinal plants for their potential antifungal activity (Ficker et al., 2003; Ken-Ichi et al., 2008; Miyasaki et al., 2010). In recent years, secondary plant metabolites have been extensively investigated as a source of medicinal agents (Michal & Klaus, 2009).

Cassia fistula Linn. (Caesalpiniaceae) (Indian Laburnum or Golden shower) is frequently used as folk medicine in various disorders like hematemesis, pruritus, leucoderma, diabetes and laxatives (Moshahid et al., 2009). Scientifically, the fruits parts of C. fistula have been reported as antidiabetic (Singh & Bharadwaj, 1975), antitumor (Gupta et al., 2000), hypolipidemic (Gupta & Jain, 2009), sedative (Mazumder et al., 1998), antioxidant (Siddhuraju et al., 2002), antimicrobial (Dhar & Qasba, 1984; Duraipandiyan & Ignacimuthu, 2007), anthelmintic (Irshad et al., 2010), hepatoprotective (Das et al., 2008), abortifacient and purgative (Elujoba et al., 1999).

Rhein (Figure 1), a major anthraquinone derivative and sennidin-like compound, identified in fruit pulp, seed, flower and leaves (Modi & Khorana, 1952; Kapadia & Khorana, 1966; Bahorun et al., 2005). Antibacterial and antifungal properties have been reported as (Duraipandiyan & Ignacimuthu, 2007; Phongpaichit et al., 2004; Perumal et al., 1998).

Figure 1.  Rhein (1,8-dihydroxyanthraquinone-3-carboxylic acid), an active phytochemical of Cassia fistula.

Candida is a fungus frequently found on skin and also mucosal flora. In humans with impairment of the immune system, it can lead to severe diseases from superficial mucosal to life threatening systemic disorders (Kaleli et al., 2007). In the last 10 years, there has been a considerable increase in mycoses and systemic infections caused by Candida albicans, particularly among immunocompromised patients (Eggimann et al., 2003). Studies have indicated C. albicans resistance to azoles or hepatonephrotoxic or nephrotoxic linked to polyene use, particularly amphotericin-B (White & Goetz, 1994; Rex et al., 1995). Therefore, searching for the sources of new anticandidal is required from the medicinal plant derivatives owing to their versatile application.

The aim of this study was to investigate the anticandidal activity of the fruit pulp and seed extract of C. fistula against three most prevalent species causing candidiasis, C. albicans ATCC 10261, Candida glabrata ATCC 90030 and Candida tropicalis ATCC 750. Anticandidal activity is investigated by determining minimum inhibitory concentration (MIC), growth curve studies, cell cytotoxicity and membrane associated property: ergosterol extraction and estimation assay. The present study is designed to investigate new therapeutic treatments for Candida spp. infections by using plant derived principles which are low cost and readily available.

Materials and methods

Reagent and chemicals

All media constituents were obtained from HiMedia (Mumbai, India). All chemicals and solvents were of analytical grade and obtained from Merck (Mumbai, India). Fluconazole was obtained from HiMedia.

Collection and preparation of plant extract

The fresh ripe fruits of C. fistula were collected in June 2010 from the campus of Jamia Millia Islamia University, New Delhi and authenticated by Prof. M. P. Sharma. A voucher of specimen (PM 21) was stored in the laboratory for further reference. The fruit pulp and seed was separated and ground to coarse powder. The samples were defatted with hexane and then extracted with methanol in a Soxhlet apparatus for 72 h. It was filtered with Whatman filter paper and the solvent was evaporated. The crude extracts were used for the phytochemical investigation and anticandidal potential by clearance of biosafety and ethical committee of the institute.

Phytochemical analysis

The extracts were subjected to various phytochemical tests to determine the active constituents. Tests were performed for alkaloids (Mayer’s reagent and Dragendorff’s reagent test), flavanoids (Shinoda test), cardiac glycoside (Keller Killiani test), tannins (Braemer’s test), anthraquinones (Borntrager’s test), steroids and terpenoids (Salkowski test, Liebermann–Burchardt test) by standard methods (Harborne, 1973; Trease & Evans, 1978; Sofowora, 1993). The compound rhein was identified by the thin-layer chromatography on silica gel 60 F₂₅₄ thin-layer chromatography aluminium sheet having mobile system of ethyl acetate: methanol: water mobile (5:1:0.5). Spot of rhein on the chromatograms was visualized by sparing with 10% methanol potassium hydroxide solution.

Growth conditions

C. albicans ATCC 10261, C. glabrata ATCC 90030 and C. tropicalis ATCC 750 used in this study were obtained from the Indian Institute of Integrative Medicine (formerly Regional Research Laboratory, Jammu). Stock cultures were maintained on slants of nutrient agar (yeast extract 1%, peptone 2%, d-glucose 2% and agar 2.5%) at 4 °C. Cells from primary culture (10⁸ cells/ml) were inoculated in 100 ml fresh YEPD (yeast extract, peptone and dextrose) medium and grown for 8–10 h, i.e., up to mid-log phase (10⁶ cells/ml). Microbial cultures grown at 37 °C were appropriately diluted in sterile YEPD broth to obtain the cell suspension at 10⁶ CFU/ml.

Determination of MIC

Microtiter assay

Cells were grown for 48 h at 30 °C to obtain single colonies, which were resuspended in a 0.9% normal saline solution to give an optical density at 600 nm (OD₆₀₀) of 0.1. The cells were then diluted 100-fold in yeast nitrogen base medium containing 2% glucose and the respective auxotrophic supplements. The diluted cell suspensions were added to the wells of round bottomed 96-well microtiter plates (100 µl/well) containing equal volumes of medium and different concentrations of test extracts (CLSI/NCCLS, 2008). Fluconazole was included as a positive control. In addition to this, a drug free control was also included. The plates were incubated at 30 °C for 24 h. The MIC test endpoint was evaluated both visually and by observing the OD₆₂₀ in a microplate reader (Bio-Rad, iMark, Hong Kong) and is defined as the lowest concentration that gave complete inhibition of growth compared to the controls.

Growth curve studies

For growth studies, 10⁶ cells/ml (optical density A₅₉₅ = 0.1) culture of Candida cells were inoculated and grown aerobically in YEPD broth for control and along with various concentrations of test extracts (1000–3500 μg/ml) in individual flasks. Growth was recorded turbidometrically at 595 nm using LaboMed spectrophotometer (LaboMed Inc., CA). The growth rate of different Candida species in the absence and presence of inhibitor was performed in triplicate for each concentration, average of which was taken into consideration.

WST1 cytotoxicity assay

The assay was monitored with a spectrophotometer using adherent or cultured cell suspension in microplate followed by incubation with 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-2H-tetrazolium, mono sodium salt (WST1). Appropriate concentrations of the test extract was added to cell culture and incubated for 24 h. WST1/CEC assay dye solution 10 µl was added to each well. Fluconazole was included as positive control. Plates were incubated for 30 min in a cell culture incubator with intermediate shaking for 1 min. The reaction was stopped by the addition of 10 µl of 1% sodium dodecyl sulfate. WST1 tetrazolium salt is reduced to formazan by cellular dehydrogenases there by generating a deep red colored formazan, measured at 450 nm and directly correlated to cell number (Perez et al., 1993).

Ergosterol extraction and estimation assay

A single Candida colony from an overnight sabouraud dextrose agar (Difco, KS) plate culture was used to inoculate 50 ml of sabouraud dextrose broth (Difco) for control and for various concentrations of test extracts. Fluconazole was included as a positive control. The cultures were incubated for 16 h and harvested by centrifugation at 2700 rpm for 5 min. The net weight of the cell pellet was determined. Alcoholic potassium hydroxide solution 25% (3 ml) was added to each pellet and vortex mixed for 1 min. Cell suspensions were transferred to sterile borosilicate glass screw-cap tubes and were incubated in a 85 °C water bath for 1 h. Following incubation, tubes were allowed to cool. Sterols were then extracted by the addition of a mixture of 1 ml of sterile distilled water and 3 ml of n-heptane followed by vigorous vortex mixing for 3 min. The heptane layer was transferred to a borosilicate glass screw-cap tube and stored at −20 °C. Prior to analysis, 20 µl aliquot of sterol extract was diluted five-fold in 100% ethanol and scanned spectrophotometrically between 240 and 300 nm with a spectrophotometer (Systronics UV-Visible Spectrophotometer 117, Delhi, India). Ergosterol content was calculated as a percentage of the wet weight of the cell as described by Breivik and Owades (1957).

Statistical analysis

The mean value ± standard error of mean (SEM) was determined from triplicate samples for each experimental group. Statistical significance was examined with Student’s t-test by comparing experimental groups with appropriate controls.

Results and discussion

Phytochemical analysis

The result from the present study showed that the C. fistula fruit pulp and seed extract have a significant amount of phenolic compounds like flavonoids, rhein, anthraquinone and tannins (Table 1). Thin-layer chromatography study also shows the pink color spots obtained at Rf 0.38 in the both extracts, indicating presence of rhein.

Table 1.  Phytochemical analysis of Cassia fistula fruit pulp and seed extract.

Groups of compound	Phytochemical test	Fruit pulp	Seed
Phenolic compounds	Anthraquinones	+++	++
	Flavonoids	++	+++
	Tannis	+	+
	Rhein	++
Terpenoids	Terpenoids	+	+
	Steroids	—	+
Cardic glycosides	Cardic glycosides	+	+
Saponins	Saponins	—	++
Alkaloids	Alkaloids	+	+

The test was based on the color intensity.

Negative (absent), + positive (slightly present), ++ positive (moderate concentration), +++ positive (high concentration).

Minimal inhibitory concentration (MIC)

The MICs values of fruit pulp extract against tested organisms ranged from 100 to 150 μg/ml, while that of seed extract ranged from 300 to 350 μg/ml (Table 2). The MIC results showed that the fruit pulp is more active against C. glabrata (100 μg/ml) followed by C. albicans (150 μg/ml) and C. tropicalis (250 μg/ml), although seed extract showed almost similar effects on these organisms. In comparison, the fruit pulp extract showed lowest MICs against tested organism than the seed extract. All the standard strains were fluconazole-susceptible with MIC of 16 µg/ml (C. albicans), 16 µg/ml (C. glabrata) and 4 µg/ml (C. tropicalis), respectively. The t-test significant correlation (0.0621) was observed between anticandidal activity of fruit pulp and seed extract. The anticandidal potential of crude extract of fruit pulp and seed extract is due to the presence of the phenolic compound like anthraquinone and rhein. The antimicrobial properties of these compounds are already reported (Agarwal et al., 2000). Similarly, the crude extract of medicinal plants reported as antimicrobial and antifungal activity in which anthraquinone and rhein is actively present (Ibrahim & Osman, 1995; Hatano et al., 1999).

Table 2.  Minimum inhibitory concentrations (MIC) (µg/ml) of fruit pulp and seed extract against different Candida species.

Cassia fistula	MIC (μg/ml)
	Candida albicans ATCC 10261	Candida tropicalis ATCC 750	Candida glabrata ATCC 90030
Pulp extract	150	250	100
Seeds extract	350	300	300
Fluconazol	16	16	4

Growth studies (turbidometric measurement)

The test extracts suppressed growth and delayed exponential phases of all three tested organisms in a concentration-dependent manner (Figures 2A–C and 3A–C). The fruit pulp and seed extract effect the growth pattern of C. albicans, C. tropicalis and C. glabrata in a similar manner. However, the control group have a normal pattern of growth with lag phase of 4–5 h, active exponential phase of 8–10 h, before attaining stationary phase. In the presence of fruit pulp and seed extract, the growth pattern decreases with increasing concentration and finally a flat line was observed at MIC values. After 24 h incubation, extract-treated Candida species were highly reduced, whereas, no such effect was seen in control groups under the microscope.

Figure 2.  Growth curve in the presence of different concentration of fruit pulp extract. (A). Concentration range of 50–150 μg/ml against Candida albicans. (B). Concentration range of 100–250 μg/ml against Candida tropicalis. (C). Concentration range of 30–100 μg/ml against Candida glabrata.

Figure 3.  Growth curve in presence of different concentration of seed extract. (A). Concentration range of 100–350 μg/ml against C. albicans. (B). Concentration range of 100–300 μg/ml against C. tropicalis. (B). Concentration range of 100–300 μg/ml against C. glabrata.

WST1 cytotoxicity assay

Test extracts were investigated for their anticandidal activity by WST1 cytotoxic assay. The toxicity effects on tested Candida organism increased with the increase of drug concentration. Fruit pulp extract results showed the cytotoxicity on C. tropicalis (52.31%), followed by C. albicans (56.48%) and C. glabrata (63.55%), at the minimum inhibitory concentration. Cytotoxicity of the seed extract was observed against C. albicans (39.78%) followed by C. glabrata (46.58%) and C. tropicalis (50.78%) under the same conditions (Table 3). Killing activity of fluconazole under the same experimental conditions was, however, significantly higher: 89% (C. albicans), 95% (C. glabrata) and 87% (C. tropicalis), respectively. The fruit pulp extract was seen to have greater potential compared to that of the seed extract. At MICs, fruit pulp and seed extract showed t-test significant correlation 0.099. It was concluded that crude extracts are a promising source of anticandidal compounds and the killing activity may be due to the presence of certain active compounds present in these extracts.

Table 3.  Percentage cytotoxicity in the presence of different concentrations of fruit pulp and seed extract. The data represents mean ± standard error of mean.

Pulp
Candida albicans ATCC10261		Candida tropicalis ATCC 750		Candida glabrata ATCC 90030
Extract	%Cytotoxicity	Extract	%Cytotoxicity	Extract	%Cytotoxicity
50 μg/ml	9.6 (± 0.04)	100 μg/ml	11.25 (± 0.01)	30 μg/ml	14.55 (± 0.10)
100 μg/ml	31.8 (± 0.05)	200 μg/ml	27.42 (± 0.05)	60 μg/ml	27.45 (± 0.07)
150 μg/ml	56.48 (± 0.20)	250 μg/ml	52.31 (± 0.09)	100 μg/ml	63.55 (± 0.05)
Seed
100 μg/ml	7.71 (± 0.14)	100 μg/ml	10.35 (± 0.00)	100 μg/ml	10.98 (± 0.17)
200 μg/ml	17.4 (± 0.15)	200 μg/ml	24.4 (± 0.03)	200 μg/ml	23.2 (± 0.17)
350 μg/ml	39.78 (± 0.11)	300 μg/ml	50.78 (± 0.14)	300 μg/ml	46.58 (± 0.11)
16 μg/ml*	89.32 (± 0.32)	16 μg/ml*	87.53 (± 1.03)	4 μg/ml*	95.21 (± 0.60)

*Fluconazole-positive control.

Sterol assay

Sterol assay was performed for the mechanistic analysis of the tested drug. Most antifungal drugs target the ergosterol biosynthesis pathway and disrupt membrane fluidity, asymmetry and integrity. Ergosterol also contributes to proper functioning of membrane bound enzymes (Lupetti et al., 2002). Total sterol content of samples treated with varying concentrations of test extracts was studied. At MICs, fruit pulp extract showed %ergosterol inhibition in C. albicans, C. tropicalis and C. glabrata was 54.42, 48.78 and 68.0%. However, the seed extract showed 38.11, 47.0 and 45.0%, respectively (Table 4). %Ergosterol decrease in ergosterol content by fluconazole at MIC was 93.56, 89.21 and 98% with C. albicans, C. tropicalis and C. glabrata, respectively. Ergosterol inhibition was significantly correlated with the cytotoxicity. The experimental result further compared at a constant concentration 100 μg/ml, showed that fruit pulp extract had a greater effect on C. glabrata followed by C. albicans and C. tropicalis. The seed extract has less effect at the same concentration (Figure 4). The finding showed that increase in concentration of extracts increased the cytotoxicity as well as %ergosterol inhibition. The decrease in ergosterol content indicates that these organisms show trailing growth in the presence of tested extracts. The correlation between the MICs, cytotoxicity and ergosterol content for all the species suggest that ergosterol biosynthesis is the primary target of the active compound of these extracts.

Table 4.  Effect of fruit pulp and seed extract on %Ergosterol content in different Candida species. The data represents the mean of three experiments.

Candida albicans ATCC 10261		Candida tropicalis ATCC 750		Candida glabrata ATCC 90030
Extract	% Ergosterol content	Extract	% Ergosterol content	Extract	% Ergosterol content
Pulp
Control	100	Control	100	Control	100
50 μg/ml	94.34	100 μg/ml	95.66	30 μg/ml	93.11
100 μg/ml	82.30	200 μg/ml	85.89	60 μg/ml	79.41
150 μg/ml	45.58	250 μg/ml	51.22	100 μg/ml	32.00
Seed
Control	100	Control	100	Control	100
100 μg/ml	98.2	100 μg/ml	96.13	100 μg/ml	95.78
200 μg/ml	91.37	200 μg/ml	86.60	200 μg/ml	89.46
350 μg/ml	61.89	300 μg/ml	53.00	300 μg/ml	55.00
16 μg/ml*	06.44	16 μg/ml*	10.79	04 μg/ml*	0 2.00

*Fluconazole-positive control.

Figure 4.  Effect of fruit pulp and seed extract (100 µg/ml) on % cytotoxicity and ergosterol biosynthesis inhibition in different Candida species.

Conclusion

C. fistula displayed anticandidal activities against the tested organisms. The minimum inhibitory concentration of crude extract of fruit pulp and seed varies with the organism tested. Hence, at MICs, the cytotoxic effect and ergosterol inhibition was significantly correlated. Therefore, it was concluded that the crude extract has a promising source of anticandidal compounds. The anthraquinone and rhein was actively present in both crude extract, it may provide anticandidal potential and effects ergosterol biosynthesis of membrane.

Acknowledgements

This work was financially supported by the Life Science Research Board (LSRB-139), DRDO, Govt. of India. The authors acknowledge Prof. M. P. Sharma (Department of Botany, Jamia Hamdard, New Delhi, India) for the identification of plant materials. The assistance of Mr. Irfan Ahmad and S. Jafar Mehdi is acknowledged with thanks.

Declaration of interest

The authors report no conflict of interest. The authors alone are responsible for the content and writing of the article.