Antifungal Activity of Roots Barks Extract of Securinega virosa (Roxb. ex Willd.) Baill and Anogeissus leiocarpa (DC.) Guill. & Perr, Two Plants Used in the Traditional Treatment of Candidiasis in Northern Côte d'Ivoire

Aims: To evaluate the anticandidal activity of some solvent extracts of Securinega virosa and Anogeissus leiocarpa from northern Côte d'Ivoire. Study Design: In vitro assay of antifungal activity. Methods: The herbs studied were examined for diameter of inhibition zone using agar well diffusion method; minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) using microdilution method. Results: All tested plants extracts, except the aqueous extracts, showed varying zones of inhibition against fungi tested. The diameters of inhibition zones for all organic extracts are greater than 10 mm for a sample concentration of 500 mg/ml and were significantly higher than for nystatine (p <0.05; p <0.01). The ethanol extract of Anogeissus leiocarpa revealed the strongest anticandidal activity against all tested strains with MICs ranging from 0.195 to 12.5 mg/ml, and MFCs from 0.390 to 50 mg/ml. The phytochemical screening of extracts shows the presence of polyterpenes and sterols, polyphenols, flavonoids, catechin tannins and alkaloids. Conclusion: S. virosa and A. leiocarpa possesse compounds with good anticandidal properties. This results support their traditional use in treatment of infectious diseases caused by certain Candida species.


INTRODUCTION
Whatever its origins and its civilization, man has sought in his plant or animal environment remedies to overcome his health problems. Research and knowledge of the properties of drugs have spawned real pharmacopoeia [1]. Medicinal plants and their knowledge are for the African continent a rich heritage hitherto insufficiently unexplored. Adjanohoun identified nearly 50.000 species [2]. In Côte d'Ivoire are nearly 800 species of medicinal plants and more than 1.400 drug recipes that have been identified [3]. The study of biological and chemical properties showed that the Ivorian flora has a real therapeutic and nutritional potential and can be used to treat or prevent many diseases [4]. These plants have a real interest in human health for their antibacterial [5][6][7], antifungal [8,9], antiplasmodial [10] and antioxidant [11] activities. Infectious diseases are a serious public health problem in developing countries where they are the main cause of high mortality rates, and in industrialized countries where resistance to existing antibiotics are growing alarmingly. This creates a growing need to find new antimicrobial compounds and/or inhibitory mechanisms of antibiotic resistance. Therefore, some 250-300.000 plants species inventoried, only 5 to 15% have been investigated for bioactive molecules, represent a huge pool of potential new bioactive compounds [12]. According to some authors, the naturally occurring compounds have the advantage of a great diversity of chemical structures and they also have a wide range of biological activities [13]. In this context, the present work was focused on, two plants Securinega virosa and Anogeissus leiocarpa whose root barks are used in traditional treatment of microbial infections in northern Côte d'Ivoire. Various researches have focused on the study of the biological activities of leaves and stem barks of these plants. However, there are very few studies on the evaluation of the antifungal activity of the root bark of plants.
This work intends to evaluate the in vitro antifungal properties of different extracts of both plants of the Ivorian flora on three species of Candida namely Candida albicans, Candida tropicalis and Candida glabrata.

Plant Material
The plant material consisted of the root bark of A. leiocarpa and S. virosa. These organs were harvested in January 2013 to Kouto, town located to 725 km north of Abidjan after an ethnobotanical survey of traditional healers in the community. Plants used in this study were identified by Professor AKé-Assi Laurent of the National Floristic Center, University of Félix Houphouet Boigny Cocody Abidjan.
These barks were dried out of the sun for two weeks before being ground into a fine powder by grinding. From the powder obtained after spraying, the different extracts to be tested were prepared.

Fungal Strains
The fungal strains tested were C. albicans, C. glabrata and C. tropicalis, yeasts isolated to pathological products from consulted patients with candidiasis in the Laboratory of Bacteriology-Virology University Hospital Center of Treichville (Abidjan, Côte d'Ivoire). These strains were maintained in a refrigerator at 4°C for the duration of the experiment.

Preparation of aqueous total extract
The total aqueous extract was obtained by dissolving 50 g of the plant powder in 2 l of distilled water. The macerate was mixed for 48 h using a magnetic stirrer type IKAMAG RCT at a temperature of the laboraty. The homogenate was filtered two times successively on cotton wool and then once on Whatman filter paper Nº3. The filtrate obtained was reduced using a rotary evaporator type IKA Labortechnick before being evaporated at 50ºC using an oven type Med Center Venticell. The powder obtained for each plant was the total aqueous extract and were stored at 5ºC.

Preparation of the organic extracts
The extraction method used was that described by Manga et al. [14]. The plant material was subjected to a first out with solvents of increasing polarity of dichloromethane, ethyl acetate and ethanol.
The liquid-liquid chromatography also called partition chromatography was based on the sharing of the solute in the two immiscible liquid phases. This chromatography required the use of three different polarity solvents namely dichloromethane, ethyl acetate, ethanol. To achieve this chromatography, 20 g of aqueous extract plant were added to 100 ml of water. This solution was transferred into a separatory funnel of 500 ml in which 100 ml of dichloromethane were then added. The vial was stirred for 2 min and then allowed to sedimentation. The lower aqueous phase was then collected. This operation was repeated four times and the fractions collected dichloromethane (400 ml) were evaporated to dryness. The aqueous phase was then extracted with 100 ml of ethyl acetate four times. The ethyl acetate fractions (400 ml) were combined and then evaporated to dryness. The aqueous phase is then extracted with 100 ml of ethanol 4 times then ethanol fractions (400 ml) were combined and then evaporated to dryness. The aqueous phase is evaporated to dryness. These different fractions then were kept in refrigerator at 5ºC before use.

Preparation of Inoculum
Fungal strains tested were seeded on Petri dishes containing Sabouraud agar and incubated for 48 to 72 h to obtain young cultures and isolated colonies. From these boxes, using a platinum loop two well-isolated colonies and identical were perfectly picked and put in 10 ml of Sabouraud broth and incubated for 3-5 h at 37ºC for pre-culture. A volume of 1 ml of this broth was taken and added to 10 ml of Sabouraud's broth. This fungal suspension produced was valued approximately at 10 5 cells/ml and was the pure inoculum.

Preparation of Concentration Ranges
The range of extracts concentrations was prepared by double dilution method according to a geometric progression of ratio 1/2 with concentrations ranging from 500 mg/ml to 7.81 mg/ml. These tubes were then autoclaved at 121ºC for 15 min.

2.6.1Sensitivity test
Antifungal activity was screened by agar well diffusion method described by Bakkiyaraj and Pandiyaraj [15], Irshad et al. [16] and Adekunle et al. [17]. The principle of this method was based on the spread of antimicrobial in solid media into a Petri dish by creating a concentration of gradient after a certain time of contact between the drug and the tested microorganisms. The antimicrobial effect of the drug on the tested microorganisms was assessed by the measurement of a zone of inhibition; and depending on the diameter of inhibition.
Sabouraud medium was poured into sterilized Petri dish to a thickness of 8 mm. After inoculation by flooding adequate dilution (approximately 10 5 cells/ml) of the tested strains made, wells of 6 mm diameter were formed concentrically in the agar. Each well was then filled with 20 μl of a given concentration of the extract. After a pre-diffusion of 45 min at room temperature in the hood, the strains were incubated at 37ºC for 24 h after which the diameters of the zones of inhibition were determined. Alongside Nystatin a standard antifungal was used.

Determination of antifungal parameters: minimum inhibitory concentration (MIC) and minimum fungicide concentration (MFC)
The incorporation of the different extracts was made in Sabouraud in inclined tubes according to the double dilution method previously described by Yayé et al. [18] and Ouattara et al. [19].
A serie of 7 tubes were prepared according to the method of double dilution with concentrations ranging respectively from 50 to 0.390 mg/ml.

Phytochemical Screening
Vegetable extracts obtained were subjected to a phytochemical screening to reveal the major chemical groups it contains. The methods used are those described by Bekro

Statistical Analysis
Analysis and graphical representations of data were performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego California USA, www.graphpad.com) software. The values expressed were the means of three experiments with the standard error of the mean (mean ± SEM).

Sensitivity Tests
The results of susceptibility testing of plant extracts against the strains investigated were shown in Figs. 1 and 2.  Comparing the diameters of inhibition zones, extracts of A. leiocarpa (Fig. 1) showed that the effect of the extracts at the same concentration was more marked with ETHA (ethanol extract of A. leiocarpa). It induced inhibition zone diameters of 19.67±0.88 mm; 18.33±0. and 20.00±1.15 mm respectively on C. albicans, C. glabrata and C. tropicalis. EDMA (dichloromethane extract of A. leiocarpa) was the less active extract with diameters of inhibition of 14.23±0.66 mm; Furthermore the average diameters of inhibition induced by ETHA was significantly greater than those induced by NYS (p <0.05; p <0.01).

C . a l b i c a n s C . g l a b r a t a C . t r o p i c a l i s
As for S. virosa (Fig. 2), the diameters of inhibition zones induced by its various extracts were lower than those of A. leiocarpa. At the same concentration, all extracts of S. virosa induced any inhibition zones except ETAS (total aqueous extract of S. virosa), which had no effect on all the microorganisms tested.   a l b i c a n s  C  . g l a b r a t a  C  . t r o p i c a l Fig. 2

. Activity of S. virosa on growth of Candida
MICs for S. virosa ranged 1.562 to 25 mg/ml and MFCs 3.125 to 50 mg/ml. The fungicidal effect appreciated by the ratio MCF/CMI showed that ETHA, ETHS, EAS (ethyl acetate extract of S. virosa) and EDMS (dichloromethane extract of S. virosa) were fungicidal because this ratio was between 1 to 2 and EAA was funcicidal on C. albicans and C. tropicalis but fungistatic on C. glabrata. EDMA was fungistatic on C. albicans and C. glabrata.

DISCUSSION
We performed the extraction of the plant powder with solvents of increasing polarity. The polar extracts have the highest yields with 7.80% for aqueous extract of A. leiocarpa and 4.50% for aqueous extract of S. virosa. The extraction yields reveal that root barks of these plants are richer in polar compounds than unpolar because yields increase with polarity of the solvent used.
In this study the antifungal activity of the extracts of root bark of A. leiocarpa and S. virosa was evaluated on three species of Candida which are: C. albicans, C. tropicalis and C. glabrata. These saprophytes and opportunistic yeast are present in the mucosa where they take advantage of an imbalance of existing flora or immune deficiency to multiply and cause infections. Resistance to azoles, including fluconazole, may explain clinical failures [26]. Determining the diameter of the inhibition zones and determination of antifungal parameters (MIC and MFC) were used to assess their sensitivity. In view of the results, we note that the best extraction yields are obtained with the aqueous extracts, but the best antifungal activity is obtained with ethanol extracts.
About A. leiocarpa the diameters of inhibition zones are 18 mm for ETHA the most active extract and 12 mm for EDMA at a concentration of 500 mg/ml. Kubmarawa et al. [27] showed that the ethanol extract of the stem bark of A. leiocarpa has significant inhibitory activity against C. albicans. Similarly Adigun et al. [28] showed that the acid 3,3,4-Tri-o-methylflavellagic glucoside isolated in the stem bark of A. leiocarpa has an antimicrobial effect on Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans. In Togo, studies conducted by Batawila [29] show that the MIC of A leiocarpa for twenty fungal germs vary between 0.25 and 4 mg/ml. Our results show that the MIC range from 0.195 to 12.5 mg/ml and are in agreement with these authors.
As A leiocarpa, S. virosa induces diameters of inhibition zone greater than 10 mm and the MIC range from 1.562 to 25 mg/ml. These results suggest that the plant has an antifungal activity. This is corroborated by the results of Agassounon et al. [30] which have shown that this plant has fungicidal properties.
The both plants have antifungal activity but A. leiocarpa seems to be more active than S. virosa. The roots of A leiocarpa concentrate more active compounds than roots of S. virosa. Therefore, the roots concentrate polar active substances. These results are supported by the work of Moroh [31] which showed that the roots of Morinda morindoides concentrate better the antimicrobial active compounds than other organs of the plant. It should be noted that the antimicrobial activity of medicinal plants varies from one geographic region to another. Then, leaf extracts of A. leiocarpa from Ghana are deemed to have a better antibacterial activity than extracts of leaves of the same plant from Nigeria [32]. The antifungal activity of both plants would be related to their phytomolecules composition. Indeed phytochemical screening of extracts of A. leiocarpa shows the presence of polyterpenes and sterols, polyphenols, flavonoids, catechin tannins and alkaloids. These results are consistent with those of Arbab et al. [33] showing the same chemical constituents in all organs of the plant.
The phytochemical screening of S. virosa extracts shows also polyterpenes and sterols, polyphenols, flavonoids alkaloids and catechin tannins. These compounds have also put in evidence by Ezeonwumelu et al. [34] in extracts of this plant. These extracts are less rich in bioactive compounds than A. leiocarapa. The ethanol extracts were the most active; indicating that they concentrate most of the compounds. Ethanol would be the best extraction solvent of active substances from these plants.
The presence of large chemical groups such as polyterpenes and sterols, polyphenols, flavonoids, catechin tannins and alkaloids in ethanol extracts explains their best activity because these compounds are recognized for their antimicrobial activities [35][36][37]. It may also be explained that the activity of antibiotics in plant extracts against fungi or growth may be due to their mechanism of action, chemical structure or spectrum of activity [38].
Flavonoids are phenolic in nature and they act as cytoplasmic poisons, which inhibit the activity of cytoplasmic enzymes such as aldose reductase, xanthine oxidase, phosphodiesterase and ATPase [39,40]. Tannins coagulate the cell wall proteins resulting in bactericidal activity at higher concentrations, while saponins are surface active agents and they alter the permeability of the cell wall thus facilitating the entry of toxic materials or leakage of vital constituents from the cell [41].

CONCLUSION
The extraction of bioactive substances with solvents of increasing polarity showed that the inhibitory activity of the extracts is due to better polar compounds concentrated in ethanol. The extracts are either fungistatic or fungicidal. These results validate the traditional use of these plants in the treatment of candidiasis.