Antifungal Activity of  the Extracts of Two Medicinal Plants from South Western Algeria: Ephedra alata and Bubonium graveolens

Medicinal plants and plant derived medicine are widely used in traditional cultures all over the word and they are becoming increasingly popular in modern society as naturel alternatives or supplements to synthetic chemicals (Van et al., 2018) Nowadays, a large number of medicinal plants have very important biological properties that find many applications in various fields, The use of plants for medical purposes is experiencing growing success; namely in medicine, pharmacy, cosmetology and agriculture (Badiaga, 2011; Vaou et al., 2021; Bendifallah, 2023). The use of chemicals is currently the most widely used technique for controlling harmful molds, due to their efficacy, as well as their easy and convenient applications. However, their use remains a very delicate task because of their very high cost and their harmful side effects on consumers. It is estimated that over 30% of the world’s population has ever had a fungal infection (Achimón et al., 2022), These various difficulties aroused our interest in the search for other fungitoxic substances of natural origin which could constitute an alternative solution to chemicals. (Balakumar et al., 2014; Liu et al., 2014).
 
South Algeria with its rich floral resources and ethnobotanical history is an ideal place of new pharmacological compounds (Berreghioua and Cheriti, 2018). The use of plants by local populations in the treatment of infectious diseases, such as those caused by fungi have been recorded by several studies (Violante et al., 2012). The therapeutic value of these plants is due to their secondary metabolites, especially phenolic compounds. Phenolic compounds (mainly flavonoids, phenolic acids and tannins) constitute a wealth widely exploited by the agro-food, cosmetics and pharmaceutical industries. The extraction of active principles from these metabolites is a very important step in their isolation, as well as in their identification (Pacôme et al., 2018). The exploration of natural products remains very interesting given the great diversity of antifungal compounds identified in certain plant extracts (Angaman et al., 2018). Various fungi including Aspergillus niger and Penicillium sp are involved in these infections.                                                                                                                                                     
       
The genus Asteriscus (family Asteraceae) includes eight species. Asteriscus graveolens (Forssk.) Less. (syn. Bubonium graveolens, Odontospermum graveolens or Nauplius graveolens), is an endemic aromatic plant mainly distributed in southwestern Algeria. in Sahara folk medicine, this species is used as stomachic, treating fever, gastrointestinal tracts, cephalic pains, bronchitis, and as anti-inflammatory (Said et al., 2017; Znini et al., 2012) and also previous studies revealed that the extracts from this species have an antifungal effect against Fusarium oxysporum (Boulenouar et al., 2012), antioxidant (Haddouchi et al., 2016), antimicrobial activity (Ramdane et al., 2017).
       
Ephedra alata belongs to the family Ephedraceae which is one of the most widely distributed range plants in Algeria. Ephedra is a genus of gymnosperm belonging to the family of Ephedraceae (D’Auria et al., 2012) Ephedra is a medicinal plant that has been used in traditional medicine to treat a variety of ailments,  is one of the oldest range and medicinal herbs in the world. The decoction of Ephedra alata aerial parts has been reported in folk medicine to relieve nasal decongestion, in the treatment of asthma, digestive system disorders and as a general respiratory decongestant and antifungal properties (Alqarawi et al., 2014). The hot broth of the dried green stems is used in traditional medicine, as hot tea, after abortion and to treat bacterial and fungal infections a variety of specific ingredients have been found in Ephedra herb, mainly including alkaloids, flavonoids, tannins, polysaccharides, organic acids, volatile oils, and many other active compounds (Shuang-Man et al., 2020). As part of the research by our laboratory on the biological activity and phytochemistry of medicinal plants from south western Algeria, it seemed important to us to explore the nature of natural antifungal substances in traditional Saharan medicine. The use of bioactive natural compounds offers a first-choice alternative for plants with antifungal activity in the control of pathogens of several products.
       
The effect of plant extracts on fungi, for this our study of medicinal plants and Saharan endemic species, by the use of different extraction techniques were carried out on the aerial part of these plants tested against fungi.
       
The present study was aimed at evaluating the crude extracts of Ephedra alata and Bubonium graveolens with different solvent for antifungal potential. The antifungal activity was determined on two fungi namely, Penicillium sp and Aspergillus niger.
 

Description of the study area
 
The current exploratory research was conducted in the South Western Algeria Bechar. Bechar district (31°37' N latitude, 2°13' W longitude), is located in the south west of Algeria (Fig 1) covering an area of 161.400 km². The arid climate spreads to the region go through long periods of heat from May to mars, where the temperature reaches sometimes 40°C to 45°C maximum and very low annual rainfall rate (16.9 mm). The flora of Algerian is estimated at more 3000 species belonging to several botanical families. The arid regions constitute an important reservoir of medicinal plants used by people who know the Sahara not only identify the plant but also its cycle of development, its habitat and its use (Sekkoum et al., 2011).
 

 
Plant material
 
The plants were collected during the month of February 2022 in the city of Bechar, exactly about 15 to 30 km north of town (near the Lahmer region). The plant parts were dried in the open air, protected from light and moisture for three weeks and then stored until use.
 
Fungal material
 
The fungal (Penicillium sp, Aspergillus niger) used to evaluate the antifungal properties of plants. They belong to the fungus collection of Biology Laboratory of Tahri Mohamed Bechar University. They are cultivated on nutritive medium PDA (potato dextrose agar) during seven days in the darkness and at room temperature 25°C. To prepare spore suspension, the fungi grown spores suspended in 0.85% (w/v) sodium chloride to prepare the homogeneous spore suspension (Danmek et al., 2014; Gad et al., 2022).
 
Extraction method
 
In a 1000 ml flask surmounted by a reflux condenser, 150 g of the plant are placed in the presence of 300 ml of different solvents (Methanol, Ethyl acetate and Distilled water). Extraction under reflux for 2 hours, then filter the evaporated solutions to dryness using a rotavapor.
 
Antifungal activity
 
Different crude extracts (Methanolic, ethyl acetate and aqueous) of the plants were dissolved in dimethyl sulfoxide (DMSO) with the concentration of 1mg/ml being evaluated for biological potential (Jacinto et al., 2011).
       
The antifungal activity was determined by Agar tube dilution Method. Two strains of fungus were used such as Aspergillus niger and Penicillium sp and sample was prepared by dissolving 1 mg/ml in DMSO while 25 ml of media was poured in each Petri plate. Media were allowed to solidify and spores of fungus were applied in the center of the plate (Nisar et al., 2010). The crude extract was dissolved in DMSO (1mg / ml). Sterile Sabouraud’s dextrose agar medium (5 ml) was placed in a test tube.
 
Reading the results
 
The results were read after seven days of incubation at 25°C by the measurement of the diameter of the zone of fungi growth. In parallel, we determined the diameter of the fungal strains in the absence of the extract plant. The antifungal effect is determined by measuring the percentage diametral growth inhibition using the following formula:
   
DC: Diameter growth and test (mm).
DT: Diameter growth in control (mm).
 
Statistical analysis
 
All experiments were spotted three times. Ms Excel 2007 was used to express the values as the mean±deviation.

The following results represent the effect of extracts from the Ephedra alata and Bubonium graveolens plants on the two fungal strains Aspergillus niger and Penicillium sp. These results, it is necessary to carry out antifungal tests by the direct contact method in solid medium from the extracts (aqueous, methanolic and ethyl acetate) for the two plants, to examine on two fungal strains generally (Aspergillus niger, Penicillium sp) (Table 1, 2).
 

 

       
The results obtained showed that this extraits had significant antifungal activity against all the molds tested. The inhibition of penicillium sp growth was observed at a concentration of 25 mg/ml and 50 mg/ml for methanol and ethyl acetate extracts of Bubonium graveolens 99.7% and 90.29% for extrait ethyl acetate of Ephedra alata (Table 3).
 

       
The minimum inhibitory and fungicidal concentrations of Ephedra alata were in the order of 25 and 50 mg/ml, respectively 40.53% and 41.21% of methanol and ethyl acetate extracts. Indeed, this inhibitory activity is more marked on Aspergillus niger by inhibition its growth from C=25 mg/ml while the methanolic extract for Bubonium graveolens. While Bubonium graveolens was the most resistant strain with a C= 25 mg/ml and C=50 mg/ml (Table 4). However, Penicillium sp was shown to be the most sensitive, it was completely inhibited from the 25 mg/ml concentration of Bubonium graveolens of methanol and ethyl acetate extracts. Thus, the concentration of 25 mg/ml of Ephedra alata was sufficient to stop the growth of penicillium sp. Whereas, the strain that showed some remarkable resistance which resisted up to extraits concentration 25 mg/ml (Table 4).
 

       
The results obtained with the different concentrations of Ephedra alata show that the inhibitory activity is important of different species tested. The methanol extract of Bubonium graveolens is very active against Penicillium sp and Aspergillus niger with a 100% inhibition rate at the concentration of 25 mg/ml. At the same concentration, inhibition is less significant the aqueous extract. However, these fungal agents are completely inhibited under the action other medicinal plants.
       
This can be explained by the effect of secondary metabolites contained in the plant tested.
       
Therefore, the activity of a natural plant is in direct relationship and in correlation with its chemical composition. Several studies have shown the antifungal activity of this extract by studying the antifungal activity of Bubonium graveolens. The study of the antifungal activity of the two sesquiterpenes of Asteriscus graveolens subsp odorus showed better efficacy on the three fungi tested. The results obtained that the essential oil of Asteriscus graveolens sub sp. odorus may be a new potential source of natural antifungal activity against Penicillium digitatum, Penicillium expansum (Alilou et al., 2014).
 
Ephedra has long been considered an important source of the alkaloid ephedrine, a vasoconstrictor drug once widely used in the treatment of asthma and as a general respiratory decongestant. 60 Many alkaloids, lignans and phenolic compounds have been isolated from Ephedra alata (Mandaville, 2013). The Ephedra genus is known for its alkaloids, hence, there are fewer studies about other phytocomponents of its species members, Ephedra contains flavonoids (leucodelphinidin, leucopelargonine, leucoanthocyanidin, lucenine, vicenin-1 and vicenin-2), tannins, benzylmethylamine. Tannins, mainly proanthocyanidines, are constituents of many Ephedra  species (Ibragic and Sofić, 2015 ; Guasch et al., 2012).
       
Biological control through the use of natural alternatives gave a lot of interest in this moment. Many Researchers noted that the possibility of using the extract from plants as an effective natural alternative. A study performed on the essential oils of three plants which Laurus nobilis one of them for 17 fungal species that are: Aspergillus niger, A. ochraceus, A. versicolor, A. flavus, A. terreus, Alternaria alternata, Aureobasidium pullulans, Penicillium ochrochloron. The results obtained showed that this plant that is rich in 1,8-Cineole has moderate power over the mushrooms tested (Goudjil et al., 2016).
       
 
The work by Ebi and Kamalu (2001) showed the activity antifungal extracts dichloromethane, methanolic, ethanolic from Mitracarpus scaber (Rubiaceae), on Aspergillus flavus and C. albicans. They indicated the presence of alkaloids and saponins in these extracts and attributed the activity extracts with saponins which would interact with sterols, proteins and phospholipids fungal cell membranes. Therefore, more in-depth studies can be undertaken for the development of natural products based on extracts to exploit its antifungal properties in the prevention, and treatment of certain fungal infections, and to combat molds resistant to conventional antifungals. The fungal resistance is a major problem in plant protection. There remain few effective antifungal agents against some resistant products. Scientists are looking for new products of natural origin as secondary metabolites of medicinal plants and having an antifungal activity (Hajji et al., 2016).
       
Our results suggest that extracts from this plants Bubonium graveolens and Ephedra alata could be used as an alternative source of antifungal agents against fungal infections. Further, more in-depth research is more than necessary to discover the true bioactive molecules responsible for antifungal activities.

Medicinal plants represent an important source of bioactive natural substances and compounds. The Crude extracts  of the specie Bubonium graveolens and Ephedra alinda showed antifungal activity against investigated by disk diffusion method. The evaluation of the antifungal activity of the methanolic and ethyl acetate extracts have a higher activity against all the molds tested compared to the aqueous extract.
       
The phytochemical and pharmacological results encourage the application of this medicinal specie for further evaluations of other possible bioactivities and detection of active pure compounds as constituents of drugs.It is necessary to develop the phytochemical and pharmacological aspects of local medicinal plants as a potential source of new active compounds.

The Authors are grateful to the DGRSDT (Algeria) for supporting.