Anti-candidal activity and effect on relative cell surface hydrophobicity of Pongamia pinnata

Pongamia pinnata is used in Sri Lankan traditional medicinal systems to cure diseases including microbial infections. Different parts of this plant were investigated for in vitro anti-candidal activity. Extracts of bark, roots and leaves of P. pinnata were examined for their activity on different Candida spp. using well diffusion assay. The extract with best activity was evaluated for its minimum inhibitory concentrations (MIC) using agar dilution method. Its effects on germ tube formation, germ tube elongation and relative cell surface hydrophobicity were determined. The best activity was shown by the root extract of P. pinnata. Neither bark nor leaf showed positive activity. MICs of the root extracts ranged from 1.6 to 12.8 mg/ml. The treatment with root extract of P. pinnata inhibited the germ tube formation, germ tube elongation and relative cell surface hydrophobicity in a dose dependent manner. The results reveal the significant anti-candidal activity of root extracts of P. pinnata.


INTRODUCTION
Plant material has played a major role in traditional medicinal systems throughout the world for centuries.Antimicrobial activity is one such medicinal property of plants in these medicinal systems.This traditional knowledge on antimicrobial activity in higher plants has been systematically proven in numerous studies throughout the world (Chea et al., 2007;More et al., 2008).Plant compounds such as flavonoids, alkaloids, essential oils, tannins and saponins are proven to have antimicrobial properties (Cowan, 1999;Arif et al., 2009;Duarte et al., 2007).Furthermore, plant derived antimicrobial products have recently come into the picture as a potential solution to counter the rising incidence of drug resistance in microbes (http://www.who.int/mediacentre/factsheets/fs194/en/).
The medicinal plant Pongamia pinnata (L.) Pierre.(Fabaceae), is used extensively in Sri Lankan traditional medicinal systems to cure diseases including microbial infections (Jayaweera, 1981).Fresh bark and leaves of P. pinnata have been used against acute rheumatism, rheumatoid and gonorrheal arthritis, lymphangitis and elephantiasis.Seeds of this plant are employed to cure chronic eczema, psoriasis, scabies and ringworm, while the bark is used internally for diarrhea, dyspepsia and flatulence.The root bark of P. pinnata has been used for the treatment of bleeding piles (Jayaweera, 1981).Furthermore, roots and twigs of P. pinnata have been used as a traditional tooth brush in ancient times and is still in practice in remote areas of Sri Lanka (Uragoda, 2003).The plant is also popular in Indian traditional medicinal practices.It has been used for skin diseases such as eczema, scabies, leprosy, and for ulcers, tumors, piles, enlargement of spleen, vaginal and urinary dis-charges.Juice of root is used for closing fistulous sores and cleaning foul ulcers while flowers are used for the cure of diabetes.Powder of seeds is used for whooping and irritating coughs of children.Seed oil is used in cutaneous affections, herpes and scabies (Khare, 2007).
Candida species are present in oral cavities of more than 50% of the adult population (Zaremba et al., 2006).This relatively innocuous commensal may revert to an opportunistic pathogen when the host immunity is altered (Ellepola, 2005).Lesions caused by Candida spp.termed candidosis or candidiasis manifests as superficial infections (example, oral candidiasis, vaginal candidiasis and skin infections) and / or systemic infections (example, candedimia).Among different Candida spp.C. albicans has been identified as the most common species causing infections (Melo et al., 2004;Babic and Hukic, 2010).The contribution of others, which are called non-albicans (example C. parapsilosis, C. glabrata, C. tropicalis, C. guilliermondii, C. krusei etc), has recently gained prominence in the light of increasing populations with compromised immune status (Samonis et al., 2008).Moreover, clinical and laboratory studies have shown rising incidence of antifungal resistance by Candida spp.(Baily et al., 1994).Resistant strains of C. glabrata, C. albicans, C. tropicalis and C. haemulonii against fluconazole have been reported (Giusiano et al., 2006).Zomorodian et al. (2011) reports that the highest rates of resistance to ketoconazole were seen in C. glabrata (16.6%) and C. albicans (3.2%) among a total of 206 yeast isolates that were assessed.Under these circumstances, it is logical to look into the possible antifungal effects of selected plants.Hence the current study attempted to study the anti-candidal activity of the extracts of P. pinnata against selected candidal species.

Plant materials
Voucher specimens of the plant P. pinnata, were prepared and identified at the National Herbarium at Peradeniya, Sri Lanka.A voucher specimen (NO:DK2009-01) was deposited in the herbarium of Department of Botany, University of Peradeniya, Sri Lanka.The fresh roots, leaves and bark of P. pinnata, were collected during July 2009 from the botanical garden of the Department of Botany, University of Peradeniya, Sri Lanka (latitude 7°26; longitude 80°60), and used for extraction on the same day.

Extraction of plant material
Roots of P. pinnata (20 g) were cut into 0.5 cm x 0.5 cm pieces and soaked in 75% ethanol at 85°C for 2 h (Sathishkumar and Baskar, 2008).The sample was kept in 75% ethanol overnight and extracted by the vacuum infiltration technique (Adikaram and Bandara, 1998) in which the sample was placed in a conical flask (250 ml) with a side arm connected to a vacuum pump.Solvent infiltration was carried out under continuous stirring.The extraction procedure was repeated with bark (20 g) and leaf (20 g) samples.
The extracts were filtered using Whatman No.1 filter paper.The filtrates were evaporated to dryness in vacuo using a rotary evaporator (Stuart RE 300, UK) at 50 rpm and 45°C and the residue was further freeze dried at -54°C.The dried samples were stored in sterile vials at -70°C.

Well diffusion assay
Anti-candidal activity was demonstrated using well diffusion bioassay (Rangama et al., 2009), with some modifications.Inocula of Candida were prepared by suspending 20 to 22 h old cultures in NaCl (0.85%) and adjusting their turbidity to 0.5 McFarland standards.Inocula were flood inoculated onto the surface of Petri plates containing 25 ml of Mueller Hinton Agar (MHA).Wells of 9 mm diameter were cut in the agar using a sterile cork borer.The base of the wells was sealed by adding a drop of molten agar into each well.
Aliquots (180 l) of the extracts were added into separate wells.Sterile distilled water was used as the negative control.After incubation overnight at 37°C, the plates were examined and the diameter of zone of growth inhibition was measured.Results were expressed as mean ± SD.

Determination of the Minimum Inhibitory Concentration
The extracts which showed activity against most of the Candida isolates in the well diffusion bioassay were further tested to determine the MIC (Andrews, 2001).Plant extracts were incurporated in different concentrations to prepare dilution plates of MHA.The inocula (2 l) adjusted to 0.5 McFarland were spotted on the dilution plates.The presence or absence of the growth of each isolate on the dilution plates was observed after incubating the plates overnight at 37°C.The lowest concentration of the extract that inhibited the growth of an isolate was considered as its MIC of the plant extract.MIC of fluconazole used as the positive control was also determined.

Effect of root extract of P. pinnata on germ tube formation by C. albicans
C. albicans (ATCC 90028) was harvested from overnight cultures maintained in SDA at 37°C, suspended in human blood serum and the turbidity was adjusted to 0.5 McFarland standard.The extract equivalent to the MIC was incorporated into 1 ml of prepared inoculum and vortexed for ten seconds.The mixture was further incubated at 37°C for 2 h re-vortexed and examined microscopically.The percentage of germ tube formation was enumerated by counting 100 cells.A cell was considered to have germinated when the germ tube has elongated to the length of a blastospore (Pinto et al., 2009).Length of 30 germ tubes was measured using an ocular micrometer.The test was repeated at varying concentrations (MIC, 2, 3 and 4 MIC) of the root extract of P. pinnata.In addition clinical isolates of C. albicans were subjected to the above procedure.All experiments were repeated three times with each isolate.
Effect of root extract of P. pinnata on relative cell surface hydrophobicity (RCSH) of Candida spp.
The method given by Sweet et al. (1987) was used for the hydrophobicity assay.A 5 ml portion each of a suspension of C. albicans (ATCC 90028) (OD 1± 0.02; 520 nm) was added to two glass tubes (tube A and B).Thereafter, 1 ml of xylene was added to tube B. Both tubes were placed in a water bath at 37°C for 10 min to equilibrate, and vortexed for 30 s and returned to the water bath for a further 45 min to allow the immiscible xylene and aqueous phases to separate.The lower aqueous phase was carefully pipetted out and transferred to a clean test tube.The absorbance of the tube A and B was measured at 520 nm.The hydrophobicity was expressed as the percentage reduction in optical density (OD) of the tube B compared to tube A.

% Reduction in (OD) = OD A -OD B OD A
The entire procedure was repeated incorporating the different concentrations of the freeze dried extract of P. pinnata (MIC, 1/2 MIC, 1/4 MIC, and 1/8 MIC) to the inoculum using the above procedure.
The percentage reduction of the OD of this test experiment was compared with that of previously mentioned control experiment.The RCSH was repeated with five selected isolates which were sensitive to the root extract of P. pinnata.

Statistical analysis
Data were subjected to analysis of variance (ANOVA) and means were separated by Duncan multiple range test at P<0.05 significant levels using the statistical package SigmaPlot Version 12.0.

RESULTS
Root extract of P. pinnata was active against all standard and clinical isolates of C. albicans investigated.Among four standard non-albicans strains subjected to the assay, only C. tropicalis (ATCC 13803) was sensitive to the root extract of P. pinnata.Majority of the clinical nonalbicans isolates were sensitive to the root extract.Extracts of bark and leaf did not show any inhibitory activity (Table 1).
The MICs of P. pinnata against the tested organisms varied between 1.6 and 12.8 mg/ml.C. guilliermondii (2) had the lowest and a clinical isolate of C. albicans had the highest MIC.The extract was inactive against C. tropicalis (ATCC 13803) even at the highest concentration tested.Comparing the MIC values for the plant extract with fluconazole, the plant extract had relatively higher MIC values for all the isolates (Table 2).
A 2 h exposure of C. albicans isolates to the root extract of P. pinnata, resulted in a reduction in their germ tube formation (Figure 1) and elongation (Figure 2) in a concentration dependent manner.Except C. albicans (3), the other four isolates showed a significant reduction (P<0.05) in the germ tube elongation when treated with their MIC values.C. albicans (3) achieved a significant reduction in its germ tube length when treated with 2 x MIC (Figure 2).
Five different isolates were selected to determine the effect of the root extract of P. pinnata, on RCSH.RCSH was reduced greatly by exposing the organisms to subinhibitory concentrations of the extract in a concentration dependent manner (Figure 3).Three isolates, including C. albicans (ATCC 90028) showed a significant reduction (P<0.05) in their RCSH when treated with 1/8 MIC, which was the lowest concentration used in the study.However, 1/4 MIC value was needed to obtain a significant reduction in the RCSH of the other two isolates.
When the root extract was subjected to thin layer chromatography and observed under UV light -254 nm, a dark spot (Rf = 0.8) against a fluorescent green background was observed (Figure 4b).Under UV light 365 nm, a blue fluorescence was observed at Rf = 0.88 (Figure 4c).Spraying with ethanolic AlCl 3 resulted to a yellow spot at Rf = 0.9 (Figure 4d) and the spot fluoresced in green colour when observed under UV -365 nm (Figure 4e).There was no blue colouration when the TLC plate was sprayed with FeCl 3 (Figure 4f).An inhibition zone was visualized as a clear area (Rf value of 0.92) against a red coloured background, on the TLC plate subjected to bio-autography (Figure 4g).

DISCUSSION
The traditional knowledge based drug development reduces the time and cost of the development process (Patwardhan et al., 2004).The production of an enormous number of bio-active compounds in the plant has  led to its versatile applications in the pharmaceutical industry (Fabricant andFarnsworth, 2001a, 2001b).Hence in the present study an attempt was made to identify the potential of P. pinnata as a source of new anti-candidal agents.Among different parts of the plant, only the root extract exhibited anti-candidal activity during the current assay.However, Sharma et al. (2011) have reported the ability of leaf extract of P. pinnata in inhibiting C. albicans.The reason for this difference in the two observations may be the fact that Sharma et al. ( 2011) have used the purified flavonoid fraction of leaf extract of P. pinnata while in the current study the crude extracts have been used.
According to reports on the chemical composition of this plant, furanoflavonoids have been identified as the main component (Tanaka et al., 1992;Pavanaram and Ramachandra Row, 1955).In 2008, Sathishkumar and colleagues have shown that maximum extraction of flavonoids from a plant material can be achieved by using 75% ethanol as the solvent.Hence the furanoflavonoids could be the main active compound contributing to the positive activity of ethanolic root extract.Even though, previous reports have shown the antimicrobial property of different parts of P. pinnata (Baswa et al., 2001;Wagh et al., 2007), this is the first record of anti-candidal activity of roots of this plant.Although MIC values shown by the crude root extract was always higher than that of fluconazole, if the active compound of P.pinnata root is used the MIC values will be lower.
Since the germ tube formation by C. albicans supports the organism to adhere to the host tissue and initiate infections (Kimura and Pearsall, 1980) it is considered as an important feature that contributes to the pathogenicity of C. albicans.The ability of the essential oil extracted from Syzygium aromaticum to inhibit the germ tube production by C. albicans has been previously shown by Pinto et al. (2009b).Melaleuca alternifolia, Aloe vera and Streblus asper are some other plants with a potential of controlling germ tube production by C. albicans (Hammer et al., 2008;Bernardes et al., 2012;Taweechaisupapong et al., 2005).Controlling the germ tube formation will make the organism less adherent to host tissues.Relative cell surface hydrophobicity (RCSH) has been identified as one of the key factors for adhesion of the candidal cell to the host tissue (Hazen et al., 1991).The ability of root extract of P. pinnata to influence the RCSH could be due to its potential in altering the surface characteristics of the organism.The ability of the root extract of P. pinnata to make a significant reduction in the RCSH at the sub-inhibitory concentrations will assist the control of the pathogen instead of its complete inhibition.Since Candida is a commensal fungus, usage of subinhibitory concentrations will help to maintain the balance of the host microflora while controlling pathogenicity of the organism.The ability of different plant extracts to influence the RCSH of pathogenic microbes has been identified by previous scientists.The ability of Piper betle and Psidium guajava to reduce the CSH of oral pathogenic bacteria was shown by Razak and Rahim (2003).However, an increment in the cell surface hydro-phobicity of C. albicans, when they were treated with the aqueous extract of Azadirachta indica have also been shown (Polaquini et al., 2005).
It is possible that despite not showing significant anticandidal activity in the well diffusion bio assay, extracts of leaf and bark of P. pinnata could have an effect on germ tube formation, elongation and cell surface hydrophobicity in Candida spp.Further investigations should be carried out to identify such effects of bark and leaves of the plant.During bioautography, since similar Rf values were obtained for spots identified as flavonoids and inhibitory spots with Candida sp., it can be assumed that the compound/s responsible for the anti-candidal activity of roots of P. pinnata could be flavonoids.Similarly, earlier researchers have identified flavonoids as the main constituent of roots (Tanaka et al., 1992).In addition, flavonoids have already proven to possess antimicrobial activity against a wide range of microbes including fungi, bacteria and virus.flavanone, possessing significant antifungal activity (Wächtera et al., 1999).In addition, the activity specifically against Candida spp have been recorded by plant extracts which constitute flavonoids.Fruits of Terminalia bellerica contain flavonoid compounds named as termilignan, thannilignan, 7-hydroxy-3', 4'-(methylenedioxy) flavan and anolignan B which can inhibit C. albicans (Valsaraj et al., 1997).It has been proven that Apulian propolis can inhibit C. albicans as well as other fungal pathogens causing dermatosis (Cafarchia et al., 1999).Flavonoids isolated from Hypericum spp., Capsella spp.and Cromolaena spp.are also known to bear inhibitory activity against bacterial human erythrocytes at a concentration of 100 g/ml.However, cytotoxicity assays have to be carried out to identify the toxicity effects of crude extract of P. pinnata against human cells.
In conclusion, the results of our study reveal that roots of P. pinnata possess a significant anti-candidal activity.In addition, the results showed the ability of the root extract of P. pinnata in reducing the germ tube formation, germ tube elongation and the relative cell surface hydrophobicity in vitro.Moreover, the study suggests that the active component of root extract of P pinnata could be compound/s belonging to the group of flavonoids.
Hence this study could lead to the establishment of some compounds that could be used to form novel anticandidal drugs of plant origin.However, further phytochemical studies are required to identify the compound(s) responsible for the anti-candidal activity of this plant extract.

Figure 1 .Figure 2 .
Figure 1.Effect of different concentrations of root extract of P. pinnata on the percentage germ tube production of clinical isolates of C. albicans, expressed as the mean ± SEM (n=3).Bars with the same letter among concentrations are not significantly different (P > 0.05).

Figure 3 .
Figure 3. CSH, after the addition of root extract, of P. pinnata at 1/8 MIC, 1/4 MIC, 1/2 MIC and MIC values.Values plotted are means of percentage reduction of the absorbance ± standard deviations of six determinations.Bars with the same letter among concentrations are not significantly different (P > 0.05).

Figure 4 .
Figure 4. Thin layer chromatograms of ethanolic root extract of P. pinnata (a) under day light (b) under UV light -254 nm (c) under UV light -365 nm (d) sprayed with AlCl3 and observed under day light (e) sprayed with AlCl3, observed under UV -365 nm (f) sprayed with FeCl3 and (g) subjected to bio-autography.

Table 1 .
Average zone of inhibition obtained for extracts of P. pinnata (25 mg/ml), against standard and clinical isolates of Candida spp. in the well diffusion bio-assay.

of the isolate Average zones of inhibition with extract of P. pinnata
*ND, Not Determined

Table 2 .
MIC values of P. pinnata and the positive control obtained from agar dilution method.