Antifungal potential of different parts of Olea europaea and Olea cuspidata growing in Azad Jammu and Kashmir Muhammad

Development of more effective and less toxic antifungal agents is required for the treatment of fungal diseases of plants and human being. The medicinal plants and their various extracts have been used as medicines against infectious diseases and found as potent against crops as well as human pathogenic fungal stains. In this study, the antifungal activity of O. europaea (cultivated olive) leaves, seeds and O. cuspidata (wild olive) leaves, roots bark, stem bark and seeds were evaluated against a range of human and crop pathogenic fungal species by using well diffusion assay. The extracts of investigated parts of O. europaea and O. cuspidata obtained with organic solvents were found to be effective against some of tested fungal strains particularly extracts of O. cuspidata parts proved to be more potent. The ethylacetate extracts of O. cuspidata leaves and seeds were found to be effective against fungal pathogens as compared to O. europaea extracts. Nystatin was also used as positive control and respective solvent as negative control. This study demonstrated that the use of wild olive leaves extracts as medicines may reduce the risk of fungal infections, particularly in situations where lengthy usage of synthetic fungicidal inspire growth of opportunistic contagions. Keyword: Fungicidal; Wild Olive; Cultivated Olive; Organic Solvents; HPLC


Introduction
Extracts of olive leaves has been used in traditional medicine for the treatment of malaria and fever [1].In animals the olive leaf extracts found to be useful particularly in lowering the blood pressure [2] and increases blood flow in coronary arteries, relieves arrhythmia and prevents intestinal muscle spasms [3].Furthermore, olive leaf extract and its phenolic compounds such as oleuropein, tyrosol, hydroxytyrosol, caffeic acid, gallic acid and luteolin have antimicrobial activity against viruses, retroviruses, bacteria, yeasts, fungi, molds and other parasites [4][5][6].The growth of Escherichia coli, Klebsiella pneumonia and Staphylococcus aureus was inhibited by the phenolic compounds isolated from olive fruit [7,8].Gourama and Bullerman [9] evaluated the oleuropein Aspergillus parasiticus's aflatoxin and found that oleuropein used mold growth but inhibited the production of aflatoxins.Markin et al.
[10] also evaluated the antimicrobial potential of olive leaf water extract and found potent against tested yeast and bacterial strains.Moreover, Sousa et al. [11] screen out phenolic compound against a rang of G+ve, G-ve and fungi (Candida albicans and Cryptococcus neoformans) and conferred a prominent activity.The fungi i.e.
Tricophyton mentagrophytes, Microsporum canis and Candida spp.were sensitive from some aldehydes isolated from fruit of olive as conferred good antifungal activity [12].Many of the problems caused by yeasts results from the much higher attention paid to bacteria and moulds which are more significant in terms of public health.Yeasts play a central role in the spoilage of foods and beverages, mainly those with high acidity and reduced water activity [13].Ismail et al. [14] reported that Kloeckera apiculata, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Candida spp.are responsible for the spoilage of foods that have been processed and packaged according to the normal standards of good manufacturing practice.In addition, yeasts are used during biological control following harvest (Candida oleophila, Metschnikowia fructicola, etc.).The objectives of this study were to investigate antifungal activity of organic solvents extracts from wild and cultivated olive parts which has been utilized as traditional folk medicine and to evaluate the potential usage of olive parts extracts as natural preservative.

Materials and Methods
The seeds, leaves, stem and roots bark of wild (Olea cuspidata Wall.Cat.) while seeds and leaves of cultivated (Olea europaea L) were collected from Kotli, Azad Jammu and Kashmir.All parts of wild and cultivated olive were dried carefully under shade then homogenized to powdered material and stored at 4 °C for further analysis.

Extraction of Potential Bioactive Compounds
Extraction is very important first step in the investigation of medicinal plants because it is necessary to extract the desired chemical components from the plant materials for further separation and characterization.Keeping in view the crucial nature of chemical compound extraction procedure, for the present study the homogenized plant parts, already stored at 4 °C were weighed and laid into separate flasks.The 250 grams of each crushed plant portion was macerated separately in 400 ml ethylacetate, acetone, chloroform and methanol for ten days at room temperature (25 ± 2 °C).The solvent extracted material was filtered by using whatmman filter paper in separate sanitized flasks.These extracts of plants portions were dried on rotary evaporator at low temperature (60 °C) and reduced pressure [15].

Culture Medium
For fungal growth PDA (Potato dextrose agar) was used as culture medium.The PDA was mixed and dissolved in distilled water.The solution was sterilized in an autoclave at 121 ºC (with 15 lb/sq-inch pressure) for 15 minutes.The pH of the medium was maintained at 5.4.

Determination of Antifungal Activities
In past research on antifungal activity of medicinal plants has been encountering several problems because of the diversity of the criteria and techniques employed for testing.The lipophilic properties of some extracts such as oil make it very difficult to use an aqueous media in the study of antimicrobial activity.Furthermore, different microorganisms have varying degree of sensitivity to different antimicrobial agents [16].Among the several methods which were employed in the plant research to determine their antimicrobial activities, invitro antifungal activities were assessed by using the well diffusion method [17].Preparation of Inoculum Fungal cultures which were 72 hours old, used as inoculums for test.The slants of PDA were prepared to maintain the fungal culture by following agar slant culture technique [18].The slants of PDA (potato dextrose agar) were prepared in test tubes and were streaked with fungus.Each test tube was labeled with the name of fungus present in it and incubated for 72 hours at 25ºC.

Preparation of Extract Dilution and Suspension
The dried ethyl acetate, acetone, chloroform and methanol extracts were then dissolved in their respective solvents.Test tubes were sterilized in an autoclave at 121ºC for 15 minutes.10 ml of distilled water was taken in each test tube.Then a loop of yeast or fungus was inoculated in distilled water in test tube under aseptic condition.

Preparation of Plates
The petri dishes were sterilized in an oven at 200 ºC for 2 hours.The sterilized petri dishes were also labeled with the yeast and fungal names.The petri dishes were labeled for leaves, seeds, stem bark, roots bark and solvent used.1ml of inoculum was transferred from the test tube into corresponding previously labeled petri dishes, with the help of micropipette.The sterilized potato dextrose agar medium was poured in the petri plates containing fungal suspension then allowed to solidify at room temperature.

Antifungal Assay by Well Diffusion Method
The ethyl acetate, acetone, chloroform and methanol extracts of O. cuspidata leaves, seeds, stem bark, roots bark while O. europaea leaves and seeds were assessed for antifungal activity by using agar well diffusion method [17].The well was made with sterile cork borer of 6.0 mm diameter.The 72 hours old cultures of fungi, grown on potato dextrose agar (PDA) medium were used as inoculum.1 ml of dilution of each fungal strain was transferred into labeled petri plate and then molten PDA was poured in it by pour plate procedure.After solidification of medium, the appropriate wells were made then 10 µl of crude extract laid into these labeled wells.Eight different solid-phase fractions, 100 percent H2O and 5, 10, 15, 20, 30, 40 and 100 percent methanol were also tested in same way that were fractionated form original methanol extracts of leaves by using solid phase extraction procedure.The same volume (10 µl) of nystatin with concentration 100 mg/ml was used as positive control and respective solvent as negative control.The preparative HPLC fraction of 100 percent H2O, 5 and 100 percent methanol were also used to evaluate the antifungal potential of each fraction.All the experiments were carried out in triplicates.All these plates were incubated at 25 ºC for 72 hours.After the incubation period, all the plates were examined for the presence of zones of inhibition as a possession of antifungal activity.The zone of inhibition was taken by measuring the diameter of the zone in millimeters (mm) and was tabulated in tables.

Statistical Analysis
All values were expressed as means ± standard error of means (S.E.M).The data for each microorganism were analyzed by using one way analysis of variance (ANOVA) technique and means were compared by using LSD at 5 percent (0.05) probability level [19].

Results and Discussion
Fruits and oil from cultivated olive trees were broadly used in diet and drugs.The information about chemical constituents with medicinal possessions has been conformed from the widely available fruits, leaves, and oil of O. europaea.It has been revealed through the investigation that the wellbeing effects of olive oil such as postponement of aging must be credited to all its metabolite constituents and not to a single compound.They act through the decrease of the risk factor important to high blood pressure, numerous types of cancer, to the alteration of immunity and inflammatory [20].Leaves 1).O. cuspidata parts extracts prove to be good with antifungal potential while the nystatin an antifungal commercially available medicine was inactive against these pathogens (Fig. 1 A-E).The information presented in literature concerning antimicrobial potential of phenolic compounds particularly obtained from cultivated olive seeds, predominantly oleuropein and hydroxytyrosol [38,39].Investigations of Markin et al. [10] and Pereira et al. [23] about antimicrobial activity of O. europaea leaves revealed the higher minimum inhibitory concentrations for the inhibition of fungal growth than the present investigation.In the present study methanolic extracts of leaves, seeds of cultivated olive and seeds, leaves, roots bark but not stem bark exhibited potent antifungal activity.Further, extracts from wild olive appeared to be particularly potent.The solid phase and HPLC fractions of Olea cuspidata leaves particularly 100 water, 5 and 100 percent methanol fractions exhibited potential antifungal activity against Magnaporthe grisea (Fig. 2, 3).According to Regasini et al. [40] and Helmerhorst et al.
[41], the deaths from opportunistic fungal diseases is above fifty percent and only narrow spectrum antifungal medicines are available, therefore it is a need to isolate new antifungal drugs.On the bases of present investigation it is clear that olive species are best source for the antifungal medicines as they exhibiting excellent inhibitory against human as well as crop pathogens tested particularly wild olive.These results are in agreement with the previous studies [42, 43].Moreover, in current study different extracts of wild and cultivated olives parts were investigated for their potential against eight fungi, which are known to cause reduction in the yield and quality of crops.As all other fungi, they are extremely responsive to environmental pressures and exhibit a capacity to adapt and  Alternaria alternata is an attendant to tomatoes, potatoes, paprika, Botrytis cinerea attacks grapes, berry-fruits, some vegetables too, while Fusarium culmorum causes a degradation of cereal grains.The latter is also known for its production of mycotoxine.Extracts of wild olive parts showed a potential antifungal activity against Aspergillus flavus, Alternaria alternata, Mauginiella scaettae, Saccharomyces cerevisiae and Magnaporthe grisea (Table 1, Fig. 1 A-E).In the present investigations the crude extracts, solid phase and HPLC fractions were used instead of individual phenolic compound due to reasons that the antimicrobial properties of phenolic components are recognized [25, 44] and extracts may be more advantageous as compare to single isolated chemical, since a bioactive single compound can alter its possessions in the presence of other constituents found in extracts [24].The synergistic effects of phytochemicals in vegetables and fruits are responsible for their potent bioactive activities and the benefit of a diet rich in fruits and vegetables is attributed to the complex mixture of phytochemicals present in whole foods

Conclusion
On the bases of present investigation it is concluded that olive species are best source for the antifungal medicines as they exhibiting excellent inhibitory effect against human as well as crop pathogenic fungi tested.In the present study methanolic extracts of leaves, seeds of cultivated olive and seeds, leaves, roots bark but not stem bark exhibited substantial antifungal activity.Further, extracts from wild olive appeared to be particularly potent.This study demonstrate that the use of wild olive leaves extracts as medicines may reduce the risk of fungal infections, particularly in situations where lengthy usage of synthetic fungicidal inspire growth of opportunistic contagions.The use of extracts is suggested to achieve benefits due to the additive and synergistic effects of phytochemicals present in whole extract.

Key:Figure 1 (Figure 1 (Figure 1 (Figure 1 (Figure 1 (Figure- 2 .Figure- 3 .
Figure 1 (A): Antifungal activity of methanol extracts of all parts [45].This explains why no individual antifungal can replace the mixture of natural phytochemicals to attain the health benefits.The Olea cuspidata leaves proved to be highly effective against tested fungi as compared to Olea europaea.The wild olive extracts are efficiently inhibiting or delaying the growth rate of microorganisms.This study is in line with the previous study of Bisignano et al. [38].Antifungal activities observed observed in current study are also in line with the former investigations that reported in literature [10, 33, 34].

Table 1 ,
Fig 1A).The antifungal prospective of methanolic extract of stem bark of O. cuspidata exhibited affective against Magnaporthe grisea (16.30mm) whereas all other fungi verified were unaffected by the extract (