Seeds oil extract of Mesembryanthemum forsskalii from Aljouf, Saudi Arabia: Chemical composition, DPPH radical scavenging and antifungal activities

Hallouma Bilel, Mervat A. Elsherif and Shaima Mohamed Nabil Moustafa 1 Chemistry Department, College of Science, Jouf University, P.O. Box: 2014, Sakaka, Saudi Arabia 2 Chemistry Department, Faculty of Sciences, Gafsa University, Sidi Ahmed Zarroug 2112, Tunisia 3 Food Technology Research Institute, Agriculture Research Center, Giza, Egypt 4 Biology Department, College of Science, Jouf University, P.O. Box: 2014, Sakaka, Saudi Arabia

Mots clés : extrait d'huile de graines / activité antifongique / phytostérols / piégeage de la DPPH / champignons kératinophiles non dermatophytes 1 Introduction Mesembryanthemum forsskalii or in Arabic "Samh" plant is one of the most popular natural plants grown in the region of Al-Jouf, localized in the Eastern North of Saudi Arabia (Showdrei, 1999). Due to the richness of plant in proteins, fats, and carbohydrates , it is used in food preparations . Moreover, this plant has applications in biological and medical fields (Elgasim and Alwesali, 2000;Alqahiz, 2009;Alfaris et al., 2010). Fungi inhabiting human hairs are a common health problem, especially children in poor places. In this aspect, some fungi are considered risk factors because of immunity deficiency in children as well as other nutritional problems (Katona and Katona-Apte, 2008). Tinea capitis is a fungal pathogen that affects human hair and scalp. Kundu et al. (2012) recorded that out of 505 students, 52 were infected with Tinea capitis. Dogo et al. (2016) reported that 45 from 100 children were infected with ringworm, and that the dominance of ringworm infection was 51.4% among girls while the infection among boys was 41.5%. Among ringworm fungal species, it was reported that Trichophyton rubrum (28.8%) and Microsporum canis (22.7%) were predominant dermatophytes and the least common was Trichophyton verrucosum (4.5%) and Trichophyton tonsurans (4.5%). In Nigeria, 55% of school children were infected with fungal skin (Soyinka, 1978), while in India, the infection ranged from 2.9 to 13.9% (Gopinath et al., 1997). Al-Mosawi et al. (1993) found that 5% of children without Tinea capitis had dermatophytes in their scalp. Common ringworm recoded species are Microsporum sp. and Trichophyton sp., but other filamentous skin fungi were isolated from keratin (a fibrous spherical protein) and infected the superficial layer of the skin that excreted hydrolyzed enzymes caused weakened and hair falling (Ali-Shtayeh et al., 2001;East-Innis et al., 2006;Mbata and Nwajagu, 2007;Andrews and Burns, 2008). Keratinolytic fungi are defined as those that can break down keratin, whereas they are the only one that can use naturally related substances from keratin resulting from its destruction or decomposition (Sharma and Choudhary, 2014). According to Rippon (1982), each keratin-analyzed fungus can be considered as a potential pathogen. Not only filamentous dermatophytes have the ability to breakdown keratin, but also, there are several non-dermatophytes keratinophilic fungi and many saprophytic fungi as well (Ulfig et al., 2010).
In this study, seeds oil extract of M. forsskalii was analyzed by gas chromatography coupled with mass spectroscopy GC-MS to give more details about the chemical composition then the antioxidant activity was evaluated by DPPH scavenging. Additionally, series of non-dermatophytes keratinophilic fungi were screened and identified to evaluate the antifungal activity of the tested seeds oil extract.

Chemicals
All chemicals used were of analytical reagent grade: ethanol, purity 99%, dimethyl sulfoxide (DMSO) extra pure. All reagents were purchased from Sigma Aldrich-Fluka.

Collection of plant material and seeds oil extraction
Seeds of M. forsskalii were obtained in April 2019 from desert of Sakaka-Aljouf, according to the following GPS geographical coordinates (latitude: 29.953894, longitude: 40.197044, 29°57' 14.0184" N and 40°11' 49.3584" E). Extract was gained from powder of M. forsskalii seeds by Pyrex ® Soxhlet extractor apparatus. Five grams of the powder were placed in the extraction chamber of the Soxhlet extractor adapted by the condenser, and then 150 mL of ethanol were added in the distillation flask. After refluxing for 8 h, the ethanol was eliminated with a rotary evaporator at 45°C under reduced pressure. Extraction was done in triplicate. Pure seeds oil extract was stored at 4°C in the dark until the beginning of the analysis. Yield of the extracted oil was 0.43 g/5 g w/w (Tab. 1).

Determination of chemical composition by GC-MS
Seeds oil extract from M. forskalii was analyzed using gas chromatography coupled to mass spectrometry Shimadzu GC-MS-QP2010SE single quadrupole apparatus.
The gas chromatograph was equipped with SLB-5MS capillary column (characteristics: L = 30 m, d = 0.25 mm, thickness = 0.25 mm) and FID (flame ionization detection) detector. Injector temperature was fixed at 200°C, oven temperature raised from 45°C to 260°C at 5°C/min, held for 15 min then raised to 360°C at 40°C/min. Detector temperature was set at 365°C. The mass spectrometer was adjusted for an emission current of 10 mA and electron multiplier voltage at 1500 V. Trap temperature was 250°C and mass scanning has been set from 40 to 650 amu. The total analysis time was 80 min, and components were identified based on the comparison of their retention time and mass spectra with those of standards. All determinations were performed in duplicate.

Antioxidant activity: DPPH radical scavenging activity
Antioxidant activity of the seeds oil extract from M. forskalii was determined in vitro using DPPH (2,2-diphenyl-1picrylhydrazyl) radical, according to the method of Blois (1958). In this work, Trolox was used as an internal standard and percentage of inhibition was calculated according to the formula of: Absorbance was measured by spectrophotometer at 517 nm after incubation in the dark within 20 minutes at room temperature (þ/À27°C). Lower absorbance indicates higher free radical scavenging activity.
ABS blank was the absorbance of the control reaction containing all reagents except the tested compound. ABS sample was the absorbance of the test compound.

Antifungal activity 2.5.1 Isolation and identification of fungi
Hair samples were collected from some primary school students in Sakaka city, Aljouf, KSA (5 boys and 5 girls) ranging in age from 5 to 7 years. Samples were transferred to the laboratory for isolation of fungi where potato dextrose agar medium (PDA) supplemented with rose-bengal was used. Examination of the purified growing colonies on the culture medium was carried out using a compound microscope. Fungi were identified according to Barnett and Hunter (1972); Pitt and Hocking (1997).

Percentage and frequency of the isolated fungi
Percentage and frequency of isolated species were calculated according to Krebs (1978). For statistical analysis GraphPad Prism 2.01 and comparison between averages using Least Significant Difference test (LSD) at the level of probability of 0.001 have been used (Ghoodjani, 2019).

Antifungal activity of ethanolic extract of M. forsskalii
A mL of fungal spore suspension (∼10 6 spores) as placed with 1 mL of M. forsskalii oil seeds extract in 15 cm diameter Petri dish contained warm sterilized PDA medium and left until solidification. Five replicates of tested dishes were placed in sterile bags and incubated at 25-27°C for 5-7 days until the appearance of fungal colonies. Control was done using all components with distilled sterilized H 2 O.
3 Results and discussion 3.1 Gas chromatography-mass spectroscopy (GC/MS) GC-MS is an efficient analytical technique for identifying and quantifying components of organic mixture. The brown extract obtained from the seeds of M. forsskalii plant was examined by GC-MS technique and the given analysis had shown a complex composition. Twenty-three components were identified by comparing retention time with those described in literature for the standard compounds. Composition of the extract and their percentage are presented in Tables 2 and 3.
Analysis of chemical composition of seeds oil extract had shown that 68.75% represent total amount of steroid derivatives subdivided into sterols, ketosteroids and stanols (Tabs. 2 and 3). Proportion of unsaturated aliphatic compounds was 12.94%.
Plant steroids are a diverse group of secondary metabolites that can be classified into several groups based on their structures and functions (Sultan and Raza, 2015); they play important pharmacological activities (Gunaherath and Gunatilaka, 2014). It's of importance that plant steroids were analyzed and qualifying. In this study, major steroids are beta-sitosterol (33.05%) which was a phytosterol and 3methoxy-(3-beta,5-alpha)cholestan-6-one (22.14%) which was a ketosteroid derivative, representing 55.19% of the total composition of the extract. In addition, the extract contained two natural tri-terpenoids which were alpha and beta amyrin (isomeric mixture) (Tab. 2), that well-known by their analgesic and anti-inflammatory properties (Pinto et al., 2008).
During the last decade, phytosterols became a center of interest due to their benefits values including reduction of blood cholesterol and prevention to cardiovascular diseases (Woyengo et al., 2009;Othman and Moghadasian, 2011;Alemany et al., 2014;Shuang et al., 2016). According to literature, the three main phytosterols existing in plants extract are stigmasterol, beta-sitosterol and campesterol (Milovanović et al., 2009;Yuang et al., 2018). As shown in Table 2, sterols present in the seeds oil of M. fosskalii were beta-sitosterol (À92.20% of sterol content) and campesterol (À7.80% of sterol content) (Fig. 1).
Due to the presence of appreciable quantity of phytosterols (35.8%) in extract, seeds oil of M. forskalii represent a good choice for patients with high cholesterol and cardiovascular diseases.

Antioxidant activity of the seeds oil extract
Antioxidant activity of the seeds oil extract from M. forskalii was evaluated by DPPH radical scavenging method using Trolox as an internal standard. The obtained results are presented in the next graph (Fig. 2).
Results showed that the extract from seeds of M. forskalii had good antioxidant activity; an increase in the concentration from 0.5 to 5 mg/mL raised the inhibition percentage and reached a maximum value of 69% (Fig. 2). Value of the extract   concentration providing 50% inhibition IC 50 can be calculated directly from the graph plotting inhibition percentage against extract concentration, a lower value of IC 50 indicated a good antioxidant activity. Seeds oil extract of M. forskalii was an effective DPPH radical scavenging agent with an IC 50 value of 3.43 ± 0.19 mg/mL. Anti-oxidant capacity is due to the presence of some chemical compounds responsible for this activity. According to the literature, phenolic compounds were important secondary metabolites present in plants oil (Carpa and Gonzalez, 2001) which were responsible for the stability of unsaturated fatty acids (Siger et al., 2008). Abdel-Farid et al.
(2016) declared that the plant extract of M. forskalii is rich in flavonols, tannins and phenolics, it has been strengthened by Lee et al. (2011), Sutharut and Sudarat (2012) and Abdel-Farid et al. (2014). Good antioxidant activity obtained may be a consequence of its total phenolic compounds present in the seeds oil extract of M. forskalii. Results here support the possibility of using seeds oil extract as a natural antioxidant in different pharmaceutical fields.

Antifungal activity of seeds oil extract 3.3.1 Isolation and identification of fungi
All fungi isolated from the hair of boys and girls were used. It is worth noting that the presence of these fungi is a health hazard due to the ability of them to cause allergies and some skin diseases for humans and animals. Because the hair of the head is close to the respiratory tract in humans, this increases the chance that the fungi germs can enter the lungs of children, causing respiratory-like diseases (Moustafa and Abdelzaher, 2016).
Distribution of fungi from boys and girls showed that a total of 11 species belonging to 7 genera were identified (Tab. 4). Among them, 8 species were isolated from boys' hair samples and 6 species from girls' hair samples. Four species of the genus Aspergillus appeared, 2 species were isolated from boy's hair samples and the other two species were isolated from girl's hair samples. Additionally, two species of the genus Penicillium were isolated from both boy's and girl's hair samples. The predominant isolated species was Aspergillus carneus (32.45%) followed by Penicillium chrysogenium (25.41%). Paecilomyces lilacinus ranked the third species with a frequency of 22.43%, while, the remaining isolates showed lowest frequency ratios ranging from 1.24 to 13.57%.
According to fungal appearance, Penicillium chrysogenium came the first with 17.67% followed by Fusarium oxysporum (12.33%). Aspergillus carneus appeared at 11.50%, which is the third rank, the prevalence of the remaining species was distributed between 5.73 and 10.27%.
Results in Table 4 represent the distribution of species between boys and girls. Penicillium chrysogenium ranked first in frequency of both boys and girls samples with 25.41 and Findings here were consistent with several studies suggesting that males are more susceptible to hair fungi (Fathi and Al-Samarai, 2000). Uneke et al. (2006) pointed out that the short hair of boys compared to girls, facilitated the occurrence of scalp infection as well as the contaminated barbers sharing tools. The present investigation proved the dominance of Aspergillus species in frequency, visibility and in the number of isolated species. Penicillium species represented the second of frequency and visibility, that the dominance of these two species may be due to the nature of their spores widely spread in our surroundings.

Antifungal activity of the seeds oil extract
Several plant extract including ethanol extracts, resins and essential oils were reported to have an antifungal activity. These forms involved simple extraction methods with low production costs (Garcia et al., 2008;Kuster et al., 2009;Gahukar, 2012).
Inhibitory effect of seeds oil extract from M. forskalii was numerous on the isolated fungi and the results are illustrated in Figures 3 and 4.
The present study showed a great inhibitory effect of the seeds oil extract towards P. chysogenum and A. fumigatus which were inhibited by 88%, followed by A. flavus, A. carneus with 85% and the remaining isolated fungi were inhibited from 60 to 75%.
M. forsskalii seeds oil extract showed strong and significant influences on the growth of Alternaria alternata, Aspergillus carneus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Cladosporium cladosporioides, Fusarium oxysporum, Paecilomyces lilacinus, Penicillium chrysogenium, Penicillium oxalicum, and Rhizopus oryzae. Based on these results, seeds oil of M. forsskalii in the form of oil or lotion is recommended for treatments or prevents fungal infection of childrens' hair.

Conclusion
Understanding chemical composition, antioxidant and anti-fungal properties of various extracts are interesting for food, cosmetic and pharmaceutics industries. The study of chemical and biological properties of M. forskalii showed that this extract had many benefits properties. Seeds oil extract of M. forskalii can be used as a natural food preservative to control food spoilage avoiding the use of chemical preservatives due to their good antioxidant activity. Owing to their containing of phytosterols, it can be taken by people having high amount of cholesterol. Additionally, growth of hair fungi can be treated by seeds oil extract of M. forskalii in the form of oil or lotion. In conclusion, our study can be considered as the first detailed document on the in vitro antifungal behavior of seeds oil extract of M. forskalii against a series of nondermatophytes keratinophilic fungi.