ASSESSMENT OF AQUEOUS AND ETHANOLIC EXTRACTS ANTIFUNGAL ACTIVITY FROM STEM BARKS OF ENANTIA POLYCARPA (ANNONACEAE) ON CANDIDA ALBICANS, ASPERGILLUS FLAVUS, ASPERGILLUS NIGER AND CRYPTOCOCCUS NEOFORMANS

OUATTARA Sitapha 1 , KPOROU Kouassi Elisée 1,2 , YAO Kouakou 2 , KRA Adou Koffi Mathieu 1 , GUESSAN Jean David 1 and DJAMAN Allico Joseph 1,3 . 1. Biochemical Pharmacodynamics laboratory, University Felix Houphouët Boigny (Ivory Coast). 2. Biochemistry-Microbiology laboratory, BIONAS Team, University Jean Lorougnon Guédé (Ivory Coast). 3. Biochemistry Medical and Fundamental, Pasteur institute of Cocody (Ivory Coast). ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


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The neurominingeal cryptococcosis is the second most frequent opportunist infection and the most dangerous systemic mycosis during the VIH infection (Luma and Temfackn, 2013; Millogo and al., 2004). Whereas the impact of the illness decreased in the western countries with the triple therapy, the neurominingeal cryptococcosis remained a reason principal of meningitis in sub-Saharan Africa.
According to Shivaswamy and Neelambike (2014), 60 to 80% of patients positive VIH develop a candidosis with a death rate of 10 to 20%. The candidosis, the neurominingeal cryptococcosis and the aspergillosis are frequent opportunist infections during the AIDS compromising the handling of the people living with VIH (PLVIH).
The antifungal multiresistance to the germs responsible for these infections like Candida albicans, Cryptococcus neoformans, Aspergillus flavus, Aspergillus niger etc. impose the scientific community to develop new therapeutic approaches from the natural substance of plant origin. Besides, the cost raised of the antifungal, the long period of treatment and the poverty are many factors that force the population to move toward traditional medicine by healing plants. These  Among these plants Enantia polycarpa a plant species reputed in traditional environment for its antimicrobic, antiparasitic, anti-inflammatory properties.
The present work is a contribution to the valorization of Ivory Coast healing plants.
The general objective is to value in vitro aqueous and ethanolic extracts antifungal activity from stem barks of Enantia polycarpa (Annonaceae). To reach this general objective, some following specific objectives have been defined: 1. Produce aqueous and ethanolic extracts from stem barks of Enantia polycarpa (Annonaceae) 2. Determine the antifungal parameters: the Inhibitory Minimal Concentration (IMC) that is the smallest concentration from which no colony is visible to the naked eye, the Fungicidal Minimal Concentration (FMC) that gives 99, 99% of inhibition compared to the tube witness of control growth (100%) and the Concentration for 50% of inhibition (CI 50 ) that is determined graphically and corresponds to 50% of inhibition compared to the tube witness of control growth.

Material and Method:-
Plant material:-It is constituted of the powder of stem barks of Enantia polycarpa Engl. & Diels. (Annonaceae). The plant material has been harvested by a herbalist and a herbarium has been achieved for its identification at the Floristic National Center (FNC) of the university Felix Houphouët Boigny of Cocody. It has been identified at the specimen n°11561. After the identification, Enantia polycarpa barks have been harvested, washed, cut and dried during one week under cover to the sun. After the drying, the barks have been ground finely in an electric grinder. The gotten powder served at the preparation of extracts for the assessment in vitro of antifungal activities.

Microbial material:-
The antifungal activities assessment has been made on Aspergillus flavus, Aspergillus niger, Candida albicans and Cryptococcus neoformans. These germs were provided by the mycology laboratory from Medical Sciences of Felix Houphouët Boigny's university (Abidjan, Ivory Coast). These germs have been isolated at patients in consultation at the service of the infectious illnesses of from Treichville's university hospital.
Methods:-Aqueous (Xaq) and 70% ethanolic (Xet) extracts preparation:-This preparation has been achieved according to the method described by authors Ouattara, (2004); Ackah and al., (2008a). To get aqueous and 70% ethanolic total extracts, Enantia polycarpa's powder has been extracted using 100g of the powder in 1L distilled water in a blender of mark Life's Superb (LS-317) or 1L of 70% ethanol during 650 three cycles of three minutes each at the ambient temperature. The gotten homogenate is first wrung in a white cloth square, filtered five times then successively on the absorbent cotton wool and on the filter paper Whatman 3mm. Extraction solvents water and 70% ethanol were evaporated respectively with a steam room controlled at 60°C during 72 hours and 48 hours. The gotten dry evaporate has been codified Xaq for the aqueous total extract and Xet for the 70% ethanolic extract.
Antifungal activity's assessment:-Antifungal tests were carried out on culture medium Sabouraud (BioRAD /Réf: 64494; Batch: 6J2218). Vegetable extract incorporation to agar was made according double dilution method of tilted tubes. 12 test tubes were used including 10 test tubes containing vegetable extract and 2 pilot tubes. Among these two tubes, one without vegetable extract was used as witness of germs control growth while the other without germs and extract was used as witness of culture medium sterility control. Extract concentrations range in the tubes go from 800 to 1.56 µg/mL with geometrical connection of reason ½. All the tubes were pressure-sealed (121°C during 15 min), then tilted with small base at room temperature to allow their cooling and solidification of the agar. Germs culture on agar slant On C. albicans, the ethanolic extract activity curve has a relatively strong slope that the aqueous extract. The antifungal parameter values have been determined for the aqueous extract at IMC=50 mg/mL, FMC = 100 mg/mL and IC 50 = 12,55 mg/mL whereas for the ethanolic extract at IMC = 3,125 mg/mL, FMC = 3,125 mg/mL, and IC 50 = 1,80 mg/mL.
On C. neoformans, the ethanolic extract activity curve has a relatively strong slope that the aqueous extract. The antifungal parameter values have been determined for the aqueous extract at IMC=200 mg/mL, FMC = 400 mg/mL and IC 50 = 55 mg/mL whereas for the ethanolic extract at IMC = 3,125 mg/mL, FMC = 6,25 mg/mL, and IC 50 = 2,40 mg/mL.

Discussion:-
The antifungal test was about the yeasts mushrooms (Candida albicans and Cryptococcus neoformans) and filamentous mushrooms (Aspergillus niger and Aspergillus flavus) on the culture medium Sabouraud. The control cultures (100% of survival) presented an aspect of germs growth very dense on the different streaks witnesses. It means that this agar is a culture medium enable for the germ growth. The results analysis showed that all tested germs were sensitive to the different extracts from Enantia polycarpa. Indeed, there was a progressive reduction of the number of colonies as the concentrations of the various extracts from E. polycarpa increased in the experimental Ethanolic extract (%).
The antifungal parameter values of the Table I revealed that: 1. The aqueous extract had a better activity on C. albicans than the other mushrooms because the IMC value is the weakest. Aspergillus flavus seems to be the least sensitive with IMC = 400 mg/mL most elevated. 2. The ethanolic extract is more active on C. albicans (IMC = 3,125 mg/mL). The others strains sensibility is identical. For this extract, the others strains had IMC and FMC identical values, while taking into account the IC 50 value, it came out again that A. niger is less sensitive than ethanolic extract because the IC50 is the most elevated. Considering the two extract activities on each of the mushrooms, it appears that the ethanolic extract (Xet) would be the most effective with lowest antifungal parameter values. The activity difference of Xaq and Xet could explain by the nature of the molecules contained in these extracts. Indeed, there is a capacity difference of solubilization and extraction of the solvents to the secondary metabolites. We could deduce that the antifungal compounds contained in E. polycarpa are more soluble in the ethanol than in the water. The 70% ethanol concentrate better the active principles.
The germs susceptibility to the extracts revealed the plant antifungal character. The decreasing shape of the activities curves showed that the 2 extracts have acted according to a relation amount-effect Besides, it comes out that C. albicans was the most sensitive mushroom because the antifungal parameter values were the weakest. On the basis of activity report (FMC-Xaq / FMC-Xet), ethanolic extract was respectively 32, 32, 64 and 128 times more active than the aqueous extract on C. albicans, A. niger, C. neoformans and A. flavus. On each target strain, the antifungal parameter values (IMC and FMC) for the aqueous extract were different; so aqueous extract was fungistatic on these germs. On the other hand ethanolic extract was fungicidal on C. albicans, A. flavus and C. neoformans (IMC=FMC) and fungistatic on A. niger.
On the basis of the FMC value gotten, the activity of Xet on C. albicans is identical to the one gotten by authors  (2010) works and also on the basis of the FMC reports, the activity hydroethanolic extract from Terminalia superba was 2 times more active than the activity ethanolic extract from Enantia polycarpa (Ambé and al., 2016). Besides, the activity hydroethanolic extract from Terminalia mantaly was 8 times more active than the activity ethanolic extract from Enantia polycarpa (Yapi  and al., 2010).
For the same germ Candida albicans with the same plant ethanolic extract from E. polycarpa gotten by Ambé and al. in 2016 had an activity 2 more important times than the Xet extract. This extract activity's difference could be due to the difference of place of species harvest, or harvest period, even the temperature and drying length.

Conclusion and Perspectives:-
This survey permitted to identify the Enantia polycarpa antifungal potential. The Enantia polycarpa aqueous and ethanolic extracts of Enantia polycarpa had antifungal activity more or less accentuated on Candida albicans, Cryptococcus neoformans, Aspergillus niger and Aspergillus flavus in vitro growth at 48 hours of incubation. The extracts acted according to a relation amount-effect and the inhibitions have been gotten with different extracts at concentrations from 100 to 800 mg/mL for the aqueous extract and 3,125 to 12,5 mg/mL for the ethanolic extract. The ethanolic extract was more active than the aqueous extract on each of the studied strains. The aqueous extract shown a fungistatic effect on the four germs whereas the ethanolic extract a fungicidal action on C. albicans, A. flavus and C. neoformans and a fungistatic on A. niger. This study revealed that 70% hydroethanolic solvent permits to concentrate better the active compounds of E. polycarpa. In perspectives, we consider: