HPLC Phenolic Compounds Analysis and Antifungal Activity of extract's from Cymbopogon citratus (DC) Stapf against Fusarium graminearum and Fusarium oxysporum sp tulipae

Aims: To analyze phenolic compounds and evaluate antifungal activity of methanolic and ethanolic extract's from Cymbopogon citratus against Fusarium graminearum and Fusarium oxysporum sp tulipae. Study Design: Activity directed antifungal activity, total phenolic compounds determination, total antioxidant determination and High Performance Liquid Chromatography (HPLC) phenolic compounds analysis of extract's from Cymbopogon citratus using in vitro methods. Original Research Article Kouassi et al.; JSRR, 15(1): 1-11, 2017; Article no.JSRR.33810 2 Place and Duration of Study: Department of Environment and plant protection, and Laboratory of Chemistry and Biochemistry, University of Agricultural Sciences and Veterinary Medicine (USAMV) between March to July 2014, Laboratory of Biochemistry and Microbiology (Bioactives Natural Substances Unit), Jean Lorougnon Guédé University between September 2014 to January 2015. Methodology: Growth inhibitory effect of ethanolic and methanolic extracts of Cymbopogon citratus were tested in vitro by applying agar slant double dilution method, analyze the profile of phenolic compounds in the two extracts by HPLC and to measure antioxidant activity of the tested extracts through 2, 2’ diphenyl-1-picrylhydrazyl (DPPH) scavenging method. Results: Methanolic and ethanolic extracts of Cymbopogon citratus inhibited the in vitro growth of Fusarium graminearum and Fusarium oxysporum sp tulipae, but the activity of methanolic extract was better. This study revealed the presence of phenolic compounds such as gentisic acid, chlorogenic acid, sinapic acid, protocatechuic acid, caffeic acid, rutin, p-couramic acid, kaempferol and quercetin in the tested extracts. Methanolic extract which was more concentrated in total phenolic, exhibited higher antioxidant activity than ethanolic extract. Conclusion: Cymbopogon citratus is a powerful antioxidant. This plant is not only a natural antifungal but also a potential source of antioxidant and nutraceuticals. Further work in this direction can allow to isolate a novel natural bioactive compound against Fusarium contamination.


INTRODUCTION
Fungal species producing Fusarium mycotoxins (Trichothecenes, Zearalenone and Fumonisins) are regularly implicated in agricultural and horticultural contamination, both during production in the fields and storage [1]. Beyond the considerable economic losses from Fusaria, the presence of mycotoxins they produce and found in food or in the production of tulip bulbs is a real public health problem. Indeed, according to Food and Agriculture Organization of the United Nations (FAO), up to 25% of production in agriculture was contaminated with mycotoxins, and ingestion of mycotoxins is the origin of several disorders including degradation of the nervous system, liver disorders and cancer [2]. To limit such contamination, chemical control using synthetic fungicides is most commonly used with its corollary of adverse effects on the environment [3]. In addition, according to the literature, the filamentous fungi Fusarium spp. are 100% resistant to some synthetic antifungals such as Itraconazole [4]. The use of natural compound, such as phenolic compounds, essential oils and extracts from natural products in the formulation of fungicides in order to reduce the risk of Fusarium contamination and to solve the problem of Fusarium resistance to synthetic antifungals, is an essential component of alternative methods to contribute in the reduction of the use of synthetic antifungals. The struggle based on the use of plant extracts could be an appropriate response, economically and culturally viable for farmers and horticulturists in developing countries. Indeed, many plants have shown antibacterial, antioxidant and antifungal potentialities. And among these plants, figure Cymbopogon citratus (DC) stapf (lemongrass). This plant of gramineae (Poaceae) family is a perennial grass which grows spontaneously in western Africa [5]. Infusions of the leaves are used in traditional medicine as antimicrobial, anti-inflammatory, febrifuge, antiseptic, in skin disorders, rheumatism and cough, headache, malaria [6,7] and sedative [8][9][10]. It was reported that this plant has no toxic effect in human [11]. The essential oil of this plant is used in the food, perfumery, soap, cosmetic, pharmaceutical and insecticidal industries [12,13]. Lemongrass has been used over many years to make caffeine free tea and natural herb drink and this confers its advantage over caffeinated tea products [14]. The essential oil of this plant has been widely studied for its antimicrobial and antioxidant properties whereas the crude extract has been little studied. The aim of this study is to evaluate the antifungal effect of crude extracts obtained from the fine powder of Cymbopogon citratus (DC) Stapf leaves on the in vitro growth of Fusarium graminearum and Fusarium oxysporum sp. tulipae and to analyse their phenolic compounds profile using HPLC.

Collection, Identification and Preservation of the Plant Sample
Cymbopogon citratus (DC) stapf (lemongrass) was obtained from a small garden in Daloa, Region of Haut Sassandra, Côte d'Ivoire in January 2014. Leaves of plant material sample was taken to the National Floristic Center (NFC), Felix Houphouët Boigny University-Abidjan (Côte d'Ivoire), for proper identification and authentication. The identity of the plant to the specimen CNF 11068, was confirmed. The leaves were removed; air dried for a week, powdered, labeled and stored in the air tight container before extraction.

Fungus
Strains of Fusarium graminearum and Fusarium oxysporum sp tulipae plant pathogenic fungi were provided by the Laboratory of Microbiology and plant Biotechnology, University of Agricultural Sciences and Veterinary Medicine (USAMV) Cluj-Napoca. These strains were isolated from corn seeds and Tulipea infected according to recommendations contained in "the Fusarium Laboratory manual" [15].

Preparation of Plant Extracts
Plant extracts were obtained by extraction with SER 148 Velp scientific. A mass of 3 x 10 g of fine powder of the dried leaves of C. citratus (DC) Stapf were introduced into 3 cartridges of the extraction apparatus. Subsequently, these cartridges were introduced separately for 30 minutes into 3 x 100 ml of the boiling extraction solvent contained in 3 extraction beakers according their boiling temperature (ethanol and methanol). After 45 min of refluxing, organic solvent was completely evaporated and the dry extract contained in each extraction beaker was weighed [16]. Extraction yield was determined. The different extracts of C. citratus were coded following: Ethanol extract: CCEt and Methanol extract: CCMet. They were stored in a refrigerator pending further investigations.

Antimicrobial Susceptibility Testing, Determination of Minimum Inhibitory Concentration (MIC) and MFC (Minimum Fungicidal Concentration)
Antifungal susceptibility testing was carried out on PDA (Potato Dextrose Agar) prepared according to manufacturer's instructions. Ethanol and methanol extracts were solubilized in 1% DMSO, then incorporated separately to the medium following method of double dilution tubes on sloping Agar. For each strain per test, eleven (11) tubes were used including nine (09) test tubes containing plant's extract and two (02) control tubes, one without plant's extract, as growth control of fungi and the second without fungi and plant's extract, as a control of sterility of culture medium. Extracts concentrations range in the tubes from 0.39 µg/ml to 100 µg/ml with geometrical connection in order of 1/2. All eleven (11) tubes were autoclaved at 121°C, 1 bar for 15 min and then the tubes were inclined to room temperature of the laboratory to allow their cooling and solidification of the agar. Inoculum was obtained by sampling with a loop diameter of 2 mm with a sterile single colony which was homogenized in 10 ml of distilled water giving a suspension containing 10 6 cells/ml. Then, 1 ml of this suspension was transferred to 9 ml of sterile distilled water giving a suspension 10 -1 . Fungi culture on agar slant previously prepared was made by inoculation of 1000 cells of each strain corresponding to 10 µl standardized inoculum 10 -1 suspension containing 10 5 cells/ml. For each test, the load of inoculum was verified by series of secondary dilutions. Thus, cultures produced were incubated at 25°C for six days (144 hours). After this incubation time, Minimum Inhibitory Concentration (MIC) was determined by the lowest concentration of extract that inhibited visual growth of fungi in an agar dilution susceptibility testing. In addition, for each test, new sterile PDA medium was used for reseeding content of tubes with concentrations superior or equal to MIC values. Then, they were incubated at 25°C for 6 days in the goal to determine Minimum Fungicidal Concentration (MFC) [17]. MFC was determined by the tube with lowest concentration of extract with the growth of one colony. Three replicates for each extract concentration and control against the fungi were used.

Determination of the Total Phenolic Compounds
Total phenolics compounds of crude extract of C. citratus leaves were measured by the Folin & Ciocalteu method and the concentration was calculated using the calibration curve ( Fig. 1) and the results were expressed as mg Gallic acid Equivalents (mgGAE)/g.
Exactly, 200 µl of crude extracts (1 mg/ml) were made up to 3 ml with distilled water, mixed thoroughly with 0.5 ml of Folin-Ciocalteu phenol reagent for 3 min followed by the addition of 2 ml of 7% Sodium carbonate (Na 2 CO 3 ) solution. The mixture was allowed to stand for a further 60 min in the dark, and absorbance was measured at 765 nm on a UV-visible against the reagent blank. To obtain a calibration curve, various concentration of gallic acid solutions were prepared.

Determination of Total Antioxidant Activity by DPPH Radical Scavenging Activity
It's reported that this assay was based on the measurement of the reducing ability of antioxidants toward DPPH (2, 2' diphenyl-1picrylhydrazyl) [18]. Antioxidant assays were based on measurement of the loss of DPPH color at 515 nm after reaction with test compounds, and the reaction was monitored by a spectrophotometer. A measure of total antioxidant capacity would help us understand the functional properties of extracts.
An easier way to present antioxidant activity of extracts would be to reference a common reference standard. (S)-(-)-6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid, also known as Trolox (T), serves such as a common reference standard.
Inhibition percent (I%) was calculated using the formula: where: A B = absorbance of blank solution; A A = absorbance of standard solution (t = 30 min).

Assay protocol for microplates with 24 wells
The DPPH (80 µM) was dissolved in pure ethanol (98%). The radical stock solution was prepared fresh daily. The mixture was shaken vigorously and allowed to stand at room temperature (25°C) in the dark for 10 min. A volume of 250 µl of sample with 1.75 ml radical solution were added to each microplate well. The decrease in absorbance of the resulting solution was monitored at 515 nm for 30 min. The results were corrected for dilution and expressed in mM Trolox per 1g weight. All determinations were performed in triplicate. These analytical methods measure the radical-scavenging activity of antioxidants against free radicals like the 1,1diphenyl-2-picrylhydrazyl (DPPH) radical, the superoxide anion radical (O2), the hydroxyl radical (OH), or the peroxyl radical (ROO). The extracts (0.1 g sample/1 ml solvent) were used.

HPLC Phenolic Compounds Analysis Method
The High Performance Liquid Chromatography (HPLC) analyses were performed with Eclipse XDB C18 column reversed phase (150x4.6 mm) 5 µm particle size from Agilent 1200 system equipped with a quaternary solvent delivery system, coupled to a UV visible detector. A binary gradient composed of solvent A (water/ acetic acid/ acetonitrile 99/0.1/1 v/v/v) and solvent B (acetonitrile/ acetic acid 100/0.1 v/v) was used. Injection volume was adjusted to 20 µl and Chromatogram was recorded at 280 nm. The column flow rate was adjusted to 0.5 ml/min at 30°C with a HPLC solvent gradient as shown in Table 1. Standards were obtained from Sigma Chemical Company (USA). Phenolic acids and flavonoids standards were dissolved in 95% MeOH to make a concentration of 0.10 mg/mL ( Fig. 1 and Table 2).

Preparation of extracts
A mass of 0.2 g sample was extracted using 2 ml methanol with 1% HCl and sonicated for 30 min. The mixture was centrifuged at 3000 rpm for 10 min and filtered through a 0.45 µm nylon filter.

Statistical Analysis
Statistical analysis of total phenol and antioxidant activity was evaluated using the t-test analysis.
The mean was considered statistically significant at p<0.05. The results of all experiments performed were expressed as Mean ± SD of three determinations, the test of significance was applied wherever necessary and values obtained as p<0.05 were considered statistically significant.

Preparation of Extract
Percentages yield of plant extract after 9 cycle extraction by Ethanol and Methanol using SER 148 Velp scientific were calculated to respectively 11.18 ± 0.15% and 15.25 ± 0.20% (Table 3). Yield obtained with Methanol extract (CCMet) was more higher than Ethanol extract (CCEt) because this solvent extracted both same polar, semi polar, non polar compounds and compounds with larger molecular weights. Using SER 148 Velp scientific for extraction allowed to extract maximum of bioactive compounds and prevent the inactivation of phenolic compounds. Percentages of yield should not be considered as a criterion of efficiency but will estimate future returns to scale in production and to select the solvent depending on antifungal activity [19,20].

Susceptibility Testing
Antifungal activity of crude extracts of against Fusarium oxysporum sp have been investigated (Fig. 2)

Total Phenolic Compounds Content and Antioxidant Activity
The total phenolic and antioxidant activity of the crude extracts of Cymbopogon represented in Table 4. Antioxidant activity was recorded in terms of equivalent factor (F) and Inhibition (I) as shown in

Compounds Content
and antioxidant activity of the citratus were . Antioxidant activity was equivalent factor (F) and % Inhibition (I) as shown in Table 4  . Authors showed that the volatile oil from the leaves of Cymbopogon citratus has also an antioxidant property against DPPH [28]. Total phenolic compounds constitute one of the major groups of compounds acting as primary antioxidants or free radical terminators hence it was reasonable to detect their amount in the herbal preparation. Phenolics and flavonoids possess a broad spectrum of chemical and antimicrobial activities including radical scavenging properties. The medicinal effects of plants are often attributed to the antioxidant activity of phytochemical constituents mainly phenolics and flavonoids [29]. It is claimed that phenolic compounds are powerful chain breaking antioxidants. The scavenging activity of phenolic group is due to its hydroxyl group.

Table 4. Total phenolics (GAE) and antioxidant activity (Percentage inhibition (I%) and equivalent factor (F) in mM Trolox)
The antioxidant activities of several natural polyphenol compounds present in vegetables has been reported. Moreover, the polyphenolic compounds have exhibited inhibitory effects on microbial agents and antifungal effects with respect to antioxidant properties was reported by Melyssa et al. [30]. The presence of phenolic compounds identified in the crude's extract of Cymbopogon citratus would probably contribute to their efficiency against Fusarium strains tested.
Exploring secondary metabolites in plant, synthetic antioxidants could be replaced by naturally occurring safer antioxidants, as the synthetics have been suspected of causing or provoking unfavorable side effects, while stronger restrictions are encountered on their application. Several reports have conclusively shown a correlation between the antioxidant activity and amount of total phenolics or total flavonoids [24].

HPLC Phenolic Compounds Analysis Method
HPLC phenolic compounds analysis recorded in Figs. 3, 4 and 5 showed that protocatechuic acid; caffeic acid; rutin; p coumaric acid; ferulic acid; quercetin; kaempherol are common to the two extracts but they were more concentrated in CCMet (Fig. 5). Sinapic acid was specific to CCEt (Fig. 3) while gentisic acid and chlorogenic acid were specific to extract CCMet (Fig. 4).
HPLC phenolics compounds analysis of CCMet and CCEt extract's showed its richness in total phenolic compounds (Phenolics acid and Flavonoïds). Methanol is efficient solvent for extraction of total phenolic compounds from Cymbopogon citratus because in CCMet extract concentrations of phenolics acid (protocatechuic acid, gentisic acid, chlorogenic acid, caffeic acid) and flavonoids (Rutin, Kaempferol and quercetin) were higher. Bassolé et al. [31] showed that the methanol was better to concentrate total phenolic compounds. These compounds have shown to have antioxidant activity [32]. Phenolic compounds are bioactive compounds with antioxidant and anti-inflammatory properties that are found in large quantities in certain plants such as Cymbopogon citratus. They can be simple or complex structures containing at least one aromatic ring in which one or more hydrogens are substituted by a hydroxyl group. This study showed a high level of phenolic compounds in methanolic extract of C. citratus and activity against F. graminearum and F. oxysporum sp tulipae was better. Moreover, previous studies showed that phenolic compounds exhibited antifungal activity against a large number of pathogenic fungi [33,34]. These compounds have a role in various functions, including the protection of plants against ultraviolet and visible light; protection against insects, fungi, viruses, and bacteria; the attraction of animals for pollination; and the action of plant hormones [35].

CONCLUSION
Methanolic and ethanolic extracts of the leaves of cymbopogon citratus possessed both antioxidant and antifungal activity against Fusarium graminearum and oxysporum sp tulipae. Methanolic extract has better antifungal activity. Moreover, extracts tested contained phenolic acids such as protocatechuic acid, gentisic acid, chlorogenic acid, caffeic acid, and flavonoids such as rutin, kaempferol and quercetin. Methanolic extract was more concentrated in total phenolic compounds, and this extract presented the best antioxidant activity. This study would be able to allow the development of natural drugs directed towards fusarium contamination and natural antioxidants for nutraceuticals.

ACKNOWLEDGEMENT
We acknowledge Prof Ioan Gh. Oroian, t of the department of Environmental and Plant Protection (USAMV Romania) encouragement and advices during my stay in the department.

COMPETING INTERESTS
Authors have declared that no competing interests exist. Methanolic and ethanolic extracts of the leaves possessed both antioxidant and antifungal activity against and Fusarium . Methanolic extract has better antifungal activity. Moreover, extracts tested contained phenolic acids such as cid, chlorogenic acid, caffeic acid, and flavonoids such as rutin, kaempferol and quercetin. Methanolic extract was more concentrated in total phenolic compounds, and this extract presented the best antioxidant activity. This study would be able to he development of natural drugs directed contamination and natural