Antifungal activity of aerial parts as well as in vitro raised calli of the medicinal plant , Balanites aegyptiaca Del .

1 Department of Microbiology, Era’s Lucknow Medical College and Hospital, Lucknow-226003, India. 2 Department of Microbiology, Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh202002, India. 3 Department of Community Medicine, Teerthanker Mahaveer Medical College and Research Centre, Teerthanker Mahaveer University, Moradabad244001, India. 4 Plant Biotechnology Laboratory, Department of Botany, Aligarh Muslim University, Aligarh-202002, India.


INTRODUCTION
Fungi cause opportunistic infections in patients who are immunocompromised, either by an underlying disease process or immunosuppressive agents.Candidiasis has become a major public health problem as an opportunistic infection of HIV/AIDS and considered as a leading fungal infection in immune-suppressed population (Al Ashaal et al., 2010).Patients with cancer often develop serious fungal infections, especially during periods of cancer treatment.Antibiotic usage for the prevention and treatment of infections in these high-risk patients leads to selection pressures resulting in the emergence and spread of resistant organisms (Panghal et al., 2011).
Nosocomial fungal infections have gained more importance especially in association with or as a consequence of the extraordinary progress in the management of seriously ill patients during the past few decades.However, *Corresponding author.E-mail: drnoorj@rediffmail.com.despite this increase, there has been comparatively little progress in elucidating treatment of such nosocomial and opportunistic fungal infections.The treatment of these infections in the immunocompromised patients is a challenging task.Although many antifungal agents have been developed so far but only a few are clinically effective and safe to use.Also, resistance to antifungal agents has been reported in AIDS patients who suffer from recurrent azole-resistant oropharyngeal or oesophageal candidiasis (Chander, 2002).
This situation has forced the researchers to search for new antimicrobial substance from various sources including medicinal plants (Scazzocchio et al., 2001;Erdogrul, 2002;Bandow et al., 2003;Parekh and Chanda, 2008).Antimicrobials of plant origin have proved effective in the treatment of several infectious diseases while simultaneously mitigating many of the side effects that are often associated with synthetic antimicrobials (Samy and Ignacimuthu, 2000).
But unfortunately, many plant species, possessing medicinally important compounds are disappearing at an alarming rate due to the destruction of their natural habitats (Vanila et al., 2008).Through in vitro cultivation, it would be possible to preserve and conserve these important endangered plant species and also in vitro cultivation of explants may be used to obtain phytotherapeutic compounds, especially, at places where the parent plants cannot be grown because of the adverse atmospheric conditions (Shahid et al., 2009b).
Balanites aegyptiaca Del. (Zygophyllaceae or Simaroubaceae), known as 'desert date' in English, 'Hingoli' in Hindi and 'Angavriksha' in Sanskrit (Iwu, 1993;Gaur et al., 2008), is a savanna tree that grows in Sahel-Savanna regions and drier parts of middle-belt zones of Nigeria, Ghana, Ivory Coast and India.In India, it is particularly found in Rajasthan, Gujarat, Madhya Pradesh and Deccan.The tree produces date-like fruits between March and October (Chothani and Vaghasiya, 2011).
Although, B. aegyptiaca is a versatile medicinal plant its use is restricted in few localities of Indian sub continents and parts of Africa.The present study was carried out to evaluate the antifungal potential of medicinal plant B. aegyptiaca and its in vitro raised callus by testing their activity against an exhaustive range of fungal isolates, including both standard as well as clinical strains.

Collection of plant materials
The fruit pulp of 15 years old plant of B. aegyptiaca was obtained from Tissue Culture Laboratory, Department of Botany, Gujarat University, Ahmedabad (Gujarat) and its in vitro cultivation was done in Botanical Garden, Department of Botany, Aligarh Muslim University, Aligarh.

Sterilization of the collected explants
The young explant was washed under running tap water for 30 min to remove any adherent particles and then washed thoroughly in sterile double-distilled water (DDW).Then these explants were kept in 1% (w/v) Bavistin (Carbendazim Powder, BASF India Limited) for 25 min, followed by thorough washing with 5% (v/v) Teepol (Qualigens Fine Chemicals, India), for 15 min by continuous shaking method (Shahid et al., 2007(Shahid et al., , 2009b)).
The treated explant was washed in sterilized DDW 3-4 times under an aseptic condition, to remove the chemical inhibitors.It was then treated with 70% (v/v) ethanol for 30 -40 s followed by a rapid washing with sterile DDW and then surface sterilized by emersion in a freshly prepared aqueous solution of 0.1% (w/v) HgCl2 (Qualigens Fine Chemicals, India) (Bhojwani and Razdan, 1996), for 4 min under laminar flow.Finally, the explants were washed 5-6 times with sterile DDW with intervals of 5 min to remove all traces of sterilants (mercury ions).

Inoculation of sterilized explants on MS medium
Murashige and Skoog's (MS) medium (Murashige and Skoog, 1962) containing 2.5 µM of 2,4-dichlorophenoxy acetic acid was prepared in culture tubes (25 × 150 mm, Borosil).The pH of the medium was adjusted at 5.8 ± 0.2.1% agar (HiMedia Lab. Ltd., India) was added to the medium and it was autoclaved at 15 lb pressure per square inch, 121°C temperature for 15 min.The sterilized explant was then inoculated aseptically and incubated at 25 ± 2°C with relative humidity of 55 ± 5% and exposed for 16 h photocycle of 2,500 Lux intensity (Shahid et al., 2009b).5 weeks old callus was used for evaluation of the antimicrobial effect.

Preparation of plant extracts
The alcoholic extracts of the plant were tested for antifungal activity.The extracts were prepared according to the method of Singh and Singh (2000) with some modifications (Shahid et al., 2007(Shahid et al., , 2009a, b), b).To prepare alcoholic extracts, fresh fruit (15 g) from parent plant was surface sterilized in 70% ethyl alcohol for 1 min and then washed 3 times with sterilized double distilled water (DDW).The calli of the explants were aseptically removed from the culture tubes and all the plant materials, including calli, were grounded with a sterilized pestle and mortar in 150 ml of 95% ethanol and centrifuged at 5000 rpm for 15 min.The resultant supernatant was filtered and taken as the alcoholic extracts which were immediately used for experimentation.

Fungi tested
The clinical fungal strains included in our study were Candida albicans, Candida krusei, Candida parapsilosis, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Penicillium spp.and Fusarium spp.isolated in the Department of Microbiology, Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh, India.The fungal control strains tested were C. parapsilosis (ATCC 22019), C. krusei (ATCC 6258) and A. fumigatus (ATCC 204305), obtained from New Drug Discovery Research, Ranbaxy, Gurgaon, India.The fungi were grown at 25°C and maintained on Sabouraud's Dextrose agar (SDA) slants.

Antifungal susceptibility testing
Antifungal testing was performed on SDA plates using agar well diffusion method (Vanden-Berghe and Vlietinck, 1991;Akinpelu, 2001), with some modifications (Shahid et al., 2007).An inoculum size of 2 × 10 6 yeast cells or fungal spores was used for inoculating the susceptibility plates.These plates were lawn cultured with fungal suspensions with the help of sterile swabs.Wells of 5 mm diameter were made in each plate using a sterile borer.Plant extracts in a volume of 20 µl were poured in the wells using a micropipette.20 µl of 95% ethanol was used to serve as negative control, whereas, antifungal agent voriconazole (500 µg/20 µl) was used as positive control.The plates were kept upright for 5-10 min until the solution diffused into the medium.SDA plates were then incubated aerobically at 25°C in a biological oxygen demand (BOD) incubator for 2-5 days and the zone of inhibition was measured and recorded.All experiments were performed in triplicate.

Determination of minimum inhibitory concentrations (MIC)
MIC was determined by broth micro-dilution method.It was performed according to Clinical and Laboratory Standards Institute (CLSI), formerly NCCLS (1997) for yeasts and NCCLS (2002) for filamentous fungi, with minor modifications (Shahid et al., 2007).Doubling dilutions of the extract was prepared using RPMI-1640 (HiMedia, India) broth supplemented with 0.3 g/L L-glutamine (HiMedia, India), 0.165 mol/L of 3-[N-morpholino] propanesulfonic acid (MOPS) buffer (HiMedia, India) and 0.01% of dimethyl sulphoxide (DMSO) (Qualigens Fine Chemicals, India).Extracts were dissolved in DMSO, and further diluted 1:50 in RPMI-1640 medium, and each resulting solution was used for a doubling dilution series.Microtitre plates were prepared containing 100 µl of undiluted extracts in the first well, followed by doubling dilutions of extracts from second well.The standardized inoculum of each fungal species was added to the respective dilution wells including the first well.The final concentrations of the extracts ranged from 25 × 10 3 to 48 × 10 -3 µg/ml.For each test there was a sterility control well containing alcoholic extract in RPMI-1640 broth plus DMSO and a growth control well containing fungal suspension without alcoholic extract.The microtitre plates were incubated at 35°C for 48 h with their upper surface covered by sterile sealers.The lowest concentration that did not show any visible growth was considered the MIC of that extract for the tested fungal species.All the MIC experimentations were performed in duplicate.

Statistical analysis
All the experiments of antimicrobial susceptibility testing were performed in triplicate.The results were expressed as the mean ± standard error (SE).Data were analyzed statistically by one way analysis of variance (ANOVA) followed by Tukey's multiple analysis test using SPSS Software, Chicago, III, version 10. P values were calculated by one-sample T-test and P < 0.05 was taken as statistically significant.

RESULTS AND DISCUSSION
Antifungal activity of alcoholic extracts of parent plant as well as its in vitro raised callus against the tested fungal species is shown in Table 1.Negative control (ethanol) showed the zone of inhibition in the range of 0.00 to 8.67 ± 0.33 mm.Positive controls (voriconazole) showed the zone of inhibition in the range of 9.33 ± 0.33 to 13.00 ±0.58 mm.The extracts derived from both parent plants as well as callus gave good antifungal activity (Table 1).The alcoholic fruit extract of parent plant showed significant antifungal activity (P < 0.05) against C. albicans (P = 0.003), C. parapsilosis (P=0.012) and Penicillium spp.(P = 0.017), with MIC ranging from 3.05 to 24.0 µg/ml (Figure 1).
Various studies have been under taken previously by different researchers to analyze the antifungal potential of B. aegyptiaca (Runyoro et al., 2006;Maregesi et al., 2008;Al Ashaal, 2010;Panghal et al., 2011).They showed significant antifungal activity of this plant against C. albicans, which supports our present research findings.
Since we tested an exhaustive range of fungal isolates, both standard and clinical strains as compared to previously studies, we were able to detect a wide spectrum of antifungal activity of this plant extract.In the present study, it was also found that alcoholic fruit extract of B. aegyptiaca also showed good antifungal activity against C. parapsilosis and Penicillium spp., which were not tested in earlier studies.Another study done by Abdallah et al. (2012) showed significant antifungal activity of this plant against A. niger and Fusarium species.These findings are in contrast with our study.This could be due to different concentrations of extracts used in their study as well as variation in active metabolites present in plant extracts derived from different places.
As seen in Table 1, the alcoholic extract of in vitro cultivated callus also showed significant antifungal activity against C. albicans, C. parapsilosis and Penicillium spp.which were comparable to the parent plant extract.In addition, it also showed good antifungal activity against C. krusei.The MIC of in vitro raised alcoholic callus extract ranged from 1.53 to 12.0 µg/ml (Figure 2).To the best of our knowledge, this is the first study analyzing the antifungal potential of in vitro raised calli of this plant; therefore, our findings could not be compared.

Conclusion
Extracts of B. aegyptiaca have remarkable antifungal potential against clinical and standard strains and thus could be used to derive antifungal agents especially against C. albicans, C. parapsilosis and Penicillium species.Hence, it could be used in the treatment of various opportunistic and life threatening fungal infections especially   in diabetics, cancer patients and immunocompromised patients.As the extracts from in vitro raised callus showed significant and even better antifungal potential as compared to parent plant, in vitro cultivation of this plant may be used to obtain phytotherapeutic compounds, even at places where it does not grow naturally because of adverse atmospheric conditions.In the future, bioactive compounds responsible for antifungal activity could further be enhanced by nutritional and hormonal manipulations of the cultivation medium, as depicted in our study.
20 µl of 95% ethanol was used as negative control.∆ = concentration of extracts used in the test, that is, 2 mg/20 µl.£ = concentration of voriconazole used in test, that is, 500 µg/20 µl.Diameter of zone of inhibition is a mean of triplicates ± SE (mm).Differences were assessed statistically using one way ANOVA followed by Tukey's test.P<0.05 was considered as significant.The mean represent ed by same letter is not significantly different within the column.

Figure 1 .
Figure 1.MIC determination of alcoholic fruit extract of B. aegyptiaca against tested pathogenic and opportunistic fungi.

Figure 2 .
Figure 2. MIC determination of alcoholic extract of in vitro raised callus of B.aegyptiaca against tested pathogenic and opportunistic fungi.

Table 1 .
Antifungal activity of alcoholic extracts of parent plant of B. aegyptiaca and its in vitro raised callus against pathogenic and opportunistic fungi.

tested Zone of inhibition (mm) ± SE Alcoholic fruit extract of parent plant ∆ Alcoholic extract of in vitro raised callus
aC. krusei (ATCC 6258) 0.00±0.00