Antimicrobial and L-Asparaginase Activities of Endophytic Fungi Isolated from Datura Innoxia and Hyoscyamus Muticus Medicinal Plants

European Journal ISSN 2449-8955 Research Article of Biological Research

Antimicrobial and L-asparaginase activities of endophytic fungi isolated from Datura innoxia and Hyoscyamus muticus medicinal plants

Ahmed H. M. El-Said 1,2 , Yassmin M. Shebany 1,2 , Mohamed A. Hussein 1*, Eman G. A. El-Dawy 1

1 Botany Department, Faculty of Science, South Valley University, Qena, Egypt 2 Biology Department, Faculty of Science, Taif University, Saudi Arabia *Corresponding author: Mohamed A. Hussein; E-mail: [email protected]

Received: 21 March 2016; Revised submission: 08 June 2016; Accepted: 20 June 2016 Copyright: © The Author(s) 2016. European Journal of Biological Research © T.M.Karpi ński 2016. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial 4.0 International License, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. DOI: http://dx.doi.org/10.5281/zenodo.56056

ABSTRACT Keywords: Datura innoxia ; Hyoscyamus muticus ; Endophytic fungi; Antimicrobial activity; L-aspara- Thirty-six species and two varieties belonging to 16 ginase. genera of fungal endophytes were isolated from the leaves of Datura innoxia and Hyoscyamus muticus 1. INTRODUCTION plants. The most prevailing fungi were: Asper- gillus fumigatus , A. niger , A. terreus var. africanus , Endophytic fungi colonize healthy living Cladosporium cucumerinum, C. oxysporum , Penicil- plant tissues without causing visible negative lium aurantiogriseum and P. chrysogenum . Endo- symptoms [1]. Endophytic fungi are known to be phytic fungi from D. innoxia and H. muticus plants associated with medicinal plants and proved to be were tested for antibacterial and antifungal activities an important source of various secondary metabo- from which 68.98 and 78.26% respectively showed lites and bioactive compounds valuable for the antibacterial against at least one of the tested pharmaceutical industry [2-4]. Datura innoxia and microbe, but didn’t had effect on tested fungal Hyoscyamus muticus belonging to Solanaceae isolates. Aspergillus niger SVUAn1 was in the top family, medicinally and economically these plants in producing L-asparaginase among tested isolates are important as it contains widely used tropane obtained from two plants. Maximum production of alkaloids, scopolamine, hyoscyamine and atropine this enzyme obtained after 4 days of incubation with [5]. Many investigations have been carried out on culture medium containing glucose as a carbon the endophytic mycobiota associated with various source. This study indicated that the endophytic types of medicinal plants by several researches fungi from D. innoxia and H. muticus plant another [6-8]. Many problems associated with using antibio- potential source of bioactive antimicrobial and tics as antimicrobial agent including antibiotic anticancer agents. resistance, host hypersensitivity, host immune- suppression and allergic reactions. Therefore, there is a need to develop alternative antimicrobial drugs

European Journal of Biological Research 2016; 6 (3): 135-144

136 | El-Said et al. Antimicrobial and L-asparaginase activities of endophytic fungi for the treatment of infectious diseases from fungi rinsed gently in running water to remove dust and [9]. Fungi are a good source of antimicrobial debris. Then, leaves were cut into 1 cm in diameter compounds used for medicine [10]. Numerous with mid rib. The surface sterilization was done investigations have been carried out on the by sequel immersion in 75% ethanol for 1 min antimicrobial activity of endophytic fungi associated followed by sodium hypochlorite (5% available with various types of medicinal plants by several chlorine) for 2 min and treated with 75% ethanol researches [11-13]. for 1 min. Later the segments were rinsed three L-Asparaginase (EC 3.5.1.1) is a tetrameric times with sterile distilled water and dried between protein [14] belonging to amidase group that sterile filter paper. Finally, four segments were ctalyses the hydrolytic deamination of asparagine inoculated on GPY plate amended with chloram- to yield aspartic acid and ammonium ion [15]. phenicol. The plates incubated at 28±2˚C for 2-3 L-asparaginase (EC 3.5.1.1), a medically important weeks then the developing fungi were counted and enzyme possesses abroad spectrum of anticancer identified morphologically, based on macro- and activity [16]. Using in treatment of different forms microscopic characters [26-30]. of cancer including acute lymphoblastic leukiemia [17]. L-asparaginase is produce by many micro- 2.3. Crude extracts from fungi organisms including fungal species [18]. Several endophytic fungi appear to be a good source of this Firstly, the endophytic fungi isolates were therapeutic enzyme including Alternaria tangelonis, grown in GPY medium at 28±2˚C for 3-5 days. Cladosporium cladosporioides, Curvularia akaii After that, 10 mm discs of the growth culture were and Fusarium subglotinans [19] and Aspergillus introduced into 250 ml Erlenmeyer flasks containing terreus [20]. Optimization of growth parameters 50 ml of GPY broth and incubated at 28±2˚C on a increases the yield of enzyme activity; several rotary shaker at 160 rpm with normal daily light and workers have revealed that, incubation periods dark periods for 10 days. Then, the culture broth and carbon sources affecting the production of was filtrated through Whatman filter paper and the L- asparaginase by fungi [21-23]. filtrate was extracted with chloroform (1:1 v/v) This study is aimed to isolate endophytic under constant shaking. The organic phase was fungi from Datura innoxia and Hyoscyamus muticus concentrated under reduced pressure using a rotary plants, identify them, and detect their antimicrobial evaporator at 45˚C and, finally, the concentrated potential and L-asparginase activity. extract was stored in a vacuum desiccator until constant weight [31]. 2. MATERIAL AND METHODS 2.4. Antimicrobial assay 2.1. Collection of plant samples The antimicrobial activity test was carried out Twenty samples from each plant ( Datura by disk diffusion method [32] against the following innoxia and Hyoscyamus muticus ) were chosen to bacteria ( Enterobacter aerogenes , Enterococcus isolate endophytic fungi, which collected from faecalis , Escherichia coli , Klebsiella pneumonia , desert habitat in Qena governorate, Egypt. Each Pseudomonas aeruginosa , Salmonella typhi , Salmo- sample was put in a sterile polyethylene bag [24]. nella typhimurium , Shigella flexneri and Staphy- Samples were transported in the same day to lococcus aureus ) and fungi ( Alternaria alternata , laboratory and were kept at 5˚C for mycological A. citri , Aspergillus niger , A. flavus , Cochliobolus analysis. spicifer , Stemphylium vesicarium and Ulocladium botrytis ). The crude extracts of endophytic fungi 2.2. Determination of Endophytic Fungi (0.001 g) dissolved with 1000 μl of dimethyl- sulfoxide (DMSO). For antibacterial test, sterile Isolation of endophytic fungi from plant parts 7 mm paper disks were impregnated with 10 μl of was done according to the method described by these extracts and placed on the Petri dishes surface Rossman et al. [25]. Firstly, the plant leaves were containing Luria Bertani agar medium [33] previou-

European Journal of Biological Research 2016; 6 (3): 135-144

137 | El-Said et al. Antimicrobial and L-asparaginase activities of endophytic fungi sly spread with bacterial suspension. Subsequently, culture filtrate was used as crude enzyme to estimate the Petri were incubated at 37±2˚C and the diameter enzyme activity. of the inhibition zones was measured after 24 hr. For antifungal test, the fungal species were 2.5.2.2 Effect of carbon sources employed with GPY agar medium and the plates were incubated at 28±2˚C up to 5-7 days [34]. The basal medium of modified Czapek Dox’s Chloroamphenicol and Nystatin used as positive liquid media with pH 6.2 was supplemented with control for the bacterial and fungal isolates, 0.2% of one of the following carbon sources: respectively. carboxymethylcellulose, fructose, maltose, starch, sucrose and yeast extract, in addition to glucose as 2.5. L-asparaginase activity of fungal isolates control. After inoculation cultures were incubated at 30 ˚C for 96 h and the cultures were filtered, 2.5.1. Screening of fungal isolates for L-aspara- centrifuged and the filtrate was used for the ginase production detection of L-asparaginase activity according to method described by Imada et al. [37]. Based on isolation results Aspergillus niger was the most prevalent species. Six isolates from 3. RESULTS A. niger isolated from Datura innoxia and Hyoscya- mus muticus were chosen randomly and screened 3.1. Endophytic mycobiota of Datura innoxia and for their abilities to produce L-asparaginase as Hyoscyamus muticus plants described by Gulati et al. [35]. Modified Czapek Dox’s medium [36] contained (g/L) glucose 2.0, Thirty six and 2 varieties belonging to 16

L-asparagine 10.0, KH 2PO 4 1.5, KCl 0.5, MgSO 4. genera were collected from 40 plant samples. The

7H 2O 0.5, CuNO 3. 3H 2O 0.03, ZnSO 4.7H 2O 0.05, most prevalent genera on D. innoxia plant were

FeSO 4.7H 2O 0.03, agar 18, initial pH 6.2 supple- Aspergillus and Penicillium isolated from 65% and mented with 0.09% phenol red as indicator was used 50% of the samples comprising 25.92% and 24.69 to detect L-asparaginase activity by tested isoltes. of total fungi, respectively. From these genera, The plates were inoculated with the 6 selected the most prevalent species were A. terreus var. fungal isolates and incubated at 30°C for 48 h. The africanus , P. aurantiogriseum and P. chrysogenum , developing pink zones around the fungal colonies they recovered from 25, 25 and 35% of the samples which indicated L-asparaginase production were comprising 6.17, 8.64 and 12.34% of total fungi, measured. respectively. Chaetomium and Cladosporium were the third frequent genera recovered from 30% of 2.5.2. Factors affecting L-asparaginase production the samples contributing 12.34% and 16.04 of total fungi respectively (Table 1). Aspergillus and The effect of incubation periods and carbon Cladosporium were the most common genera on sources on L-asparaginase production by Asper- H. muticus plant were recovered from 90% and 80% gillus niger SVUAn1 were studied; since isolate of the sample matching 45.61% and 27.20% of was found to be highly active in L-asparaginase total fungi, respectively. From previous genera production. A. fumigatus , A. niger , C. cucumerinum and C. oxysporum were the most prevalent species they 2.5.2.1. Effect of incubation periods recovered from 25, 25, 35 and 40% of total samples comprising 9.65, 28.94, 5.38 and 9.65% of total The test isolate A. niger SVUAn1 was grown fungi, respectively. Penicillium was the third on the basal medium modified Czapek Dox’s liquid frequent genus recovered (35%) of the samples media with pH 6.2. The flasks were incubated at matching 11.40% fungi (Table 1). These species 30°C at different incubation periods (24-144 h). were isolated with different numbers and Uninoculated media served as controls. The cultures frequencies from various plants in many places of filterated through Whatman No. 1 filter paper. The the world by several works [6, 38-40].

European Journal of Biological Research 2016; 6 (3): 135-144

138 | El-Said et al. Antimicrobial and L-asparaginase activities of endophytic fungi

Table 1. Total counts (TC, calculated per 240 leaf segments), percentage of fungal counts (%TC, calculated per total fungi) and frequency of fungal species (%F, calculated per 20 samples) of various fungal genera and species recovered from leaves of Datura innoxia and Hyoscyamus muticus . Datura innoxia Hyoscyamus muticus Genera and species TC %TC %F TC %TC %F Alternaria alternata (Fries) Keissler 5 4.38 25 Aspergillus 21 25.92 65 52 45.61 90 A. carneus (V. Tiegh.) Blochwitz 1 1.23 5 A. flavus Link 1 1.23 5 2 1.75 10 A. fumigatus Fresenius 1 1.23 5 11 9.65 35 A. niger Van Teighem 8 9.87 35 33 28.94 80 A. ochraceous Wilhelm 1 1.23 5 A. terreus var. africanus Fennell and Raper 5 6.17 25 5 5.38 20 A. ustus (Bainier) Thom and Church 2 2.46 5 A. versicolor (Vuill.) Tiraboschi 2 2.46 10 1 0.87 5 Chaetomium 10 12.34 30 2 1.75 10 C. atrobrunneum Ames 9 11.11 30 C. globosporum Rikh and Mukerji 1 0.87 5 C. globosum Kunze 1 0.87 5 C. hexagonosporum Carter and Malloch 1 1.23 5 Cladosporium 13 16.04 30 31 27.20 80 C. chlorocephalum (Fres.) Mason and M. B. Ellis 1 0.87 5 C. cladosporioides (Fres.) de Vries 1 1.23 5 C. cucumerinum Ellis and Arth 4 4.93 10 5 5.38 25 C. oxysporum Berk. and Curt 2 2.46 10 11 9.65 40 C. sphaerospermum Penzig 5 5.38 10 C. spongiosum Berk. and Curtis 3 3.70 10 3 2.63 10 C. uredinicola Speg 3 3.70 10 6 5.26 20 Cochliobolus spicifer Nelson 4 4.93 10 1 0.87 5 Cordella clarkia M.B. Ellis 1 0.87 5 Emericella nidulans var . lata (Thom and Raper) 1 1.23 5 Subram Eurotium chevalieri Mangin 3 3.70 15 1 0.87 5 Mucor hiemalis Wehmer 1 1.23 5 Penicillium 20 24.69 50 13 11.40 35 P. aurantiogriseum Dierckx 7 8.64 25 3 2.63 15 P. chrysogenum Thom 10 12.34 35 7 6.14 15 P. spinulosum Thom 1 1.23 5 P. duclauxii Delacroix 1 0.87 5 P. funicolusum Thom 1 0.87 5 P. rubrum Stoll 1 0.87 5 P. steckii Zaleski 1 1.23 5 P. waksmanii Zaleski 1 1.23 5 Periconia atropurpurea (Bek. and Curt.) Litvinov 1 1.23 5 2 1.75 10 Phialophora fastigiata (Lagerb., Lundberg and 1 1.23 5 Melin) Conant Scopulariopsis brevicaulis (Sacc.) Bainier 1 1.23 5 Stemphyllium botryosum Sacc. 1 1.23 5 Sterile Mycelia 3 3.70 15 6 5.26 25 Ulocladium alternaria (Cooke) Simmons 1 1.23 5 Total account 81 114 No. of genera 13 9 No. of species 27+2 var. 22+1 var.

European Journal of Biological Research 2016; 6 (3): 135-144

139 | El-Said et al. Antimicrobial and L-asparaginase activities of endophytic fungi

Dos Santos et al. [41] reported that, among inhibition zone from 8-16 mm. A. fumigatus and the fungi isolated from Melia azedarach , the A. versicolor isolated from D. innoxia showed endophytic fungi belonging to Aspergillus and strongest inhibition against Enterobacter aerogenes Penicillium were predominant; on the other hand, growth (Table 2 and 3). Among 144 endophytic Selvi and Balagengatharathilagam [42] found that isolates, A. fumigatus isolates were the most Cladosporium sp. was commonly present in most effective fungi against Staphylococcus aureus and of the tested medicinal plants in Virudhunagar Klebsiella pneumoniae [46]. The antimicrobial District. Also, Ramesha and Srinivas [43] isolated activity of Aspergillus fumigatus against bacteria endophytic fungi from different parts of Plumeria and fungi was previously reported [47]. Fumifungin acuminata and Plumeria obtusifolia and identified and synerazol, new antibiotics, were recovered from them morphologically from which Alternaria sp., the culture broth of A. fumigatus [48, 49]. Another Aspergillus sp., Chaetomium sp., Cladosporium sp., antibiotic, fumagillin, is produced by certain strains Cochliobolus sp., Curvularia sp., Mycelia Sterilia, of A. fumigatus and because it was reported as an Fusarium sp. and Penicillium sp. angiogenesis inhibitor [50]. Two aroyl uridine derivatives kipukasins H and I from A. versicolor 3.2. Antimicrobial effects of endophytic fungal strain ATCC 9577 exhibited antibacterial activity extracts isolated from Datura innoxia and against Staphylococcus epidermidis [51]. Many Hyoscyamus muticus other compounds including anthraquinone derivatives, averantin, averufin, methyl-averantin Generally, fungal endophytes isolated from nidurufin, sterigmatocystin and versiconol extracted D. innoxia and H. muticus did not have any from cultures broth of A. versicolor exhibited antifungal effect against tested fungi. In total, antibacterial activities [52-54]. Thus, it was clear 68.96% and 78.26% of the endophytic fungal that, chloroform extracts of endophytic fungi isolates from D. innoxia and H. muticus exhibited obtained from the same plant showed different antibacterial activities, respectively (Table 2 and 3). antibacterial activity (Table 2 and 3). These diffe- This result were in agreement with obtained by rences in susceptibility could be attributed to the Mahdi et al. [44] they found fungal extract showed type of isolates and nature and level of the inhibition for bacterial growth but were not antimicrobial agents present in their extracts as active against tested fungi. Ramesha and Srinivas well as their mode of action on different test [35] screened the potential of endophytic fungi microorganisms [55]. from Plumeria obtusifolia and P. acuminate for antimicrobial activity and they found that, 66.6% 3.3. L-asparaginase activities of endophytic and 58.8% recovered from P. obtusifolia and Aspergillus niger isolates P. acuminate plants, respectively, exhibited activity against tested organism. 70 % of fungal endophytes Six isolates of Aspergillus niger were from Celastrus paniculatus plant displayed screened using Czapek Dox’s medium supple- antimicrobial activities against tested bacteria and mented with phenol red for L-asparaginase fungi [45]. production. All tested isolates were positive for Endophytic fungi showed different antibac- extracellular L-asparaginase production but with terial activities against tested bacteria, the most different degrees (Table 4). One isolate representing potent fungi from D. innoxia plant were: Asper- 16.6 % of total isolates showed high L-asparaginase gillus fumigatus , Cochliobolus spicifer , Phialophora activity and these were SVUAn1 which recovered fastigiata and Stemphylium botryosum exhibited from D. innoxia plant. The remaining other five antimicrobial activity ranged from 77.7-88.8 % with isolates showed moderate L-asparaginase activities inhibition zone from 8-23 mm. Aspergillus flavus , with pink zones ranged from 20-32.5 mm. Several A. versicolor , Cordella clarkia , Eurotium chevalieri , investigators have shown that the different Penicillium aurantiogriseum and P. funicolusum fungal isolates exhibit variable capabilities in the were the most potent fungi from H. muticus plant L- asparaginase production [56-59]. Patro et al. [60] with antimicrobial activity from 77.7-88.8 and found, among 66 fungal isolates screened for

European Journal of Biological Research 2016; 6 (3): 135-144

140 | El-Said et al. Antimicrobial and L-asparaginase activities of endophytic fungi

L-asparaginase production on glucose-asparagine broth medium seven isolates showed higher enzyme activity. Twelve fungal strains isolated from wheat flour including A. niger exhibited high L-asparaginase activity [61]. High enzyme producer Aspergillus niger (SVUAn1) was chosen to study the effects of incubation time and carbon source on enzyme activity. Maximum production of L-asparaginase by A. niger (SVUAn1) isolate was achieved at 96 h (4 days) with incorporation of glucose as carbon Figure 1. Production of L-asparaginase by A. niger source in the culture medium (Figs.1, 2). These (SVUAn1) at different time intervals. finding are almost in agreement with those results reported by several workers with different fungal species [22, 62-64].

Table 2. Antibacterial effects (in mm diameter) of selected fungal endophytes from Datura innoxia against different nine pathogenic bacteria. Pathogenic bacteria Antibacterial Fungal endophytes A B C D E F G H I activity (%) Aspergillus flavus - - - - - 9 - - 10 22.2 A. fumigatus 23 19.5 10 - - 8.5 15 17.5 23 77.7 A. niger 10 8 7.5 16 - - 11 8 - 66.6 A. versicolor 40 - - - - 9 - - 11 33.3 A. ochraceous 9 ------8.5 - 22.2 A. ustus ------12 11.1 Chaetomium atrobrunneum ------11.5 11.1 C. hexagonosporum 10 - - - -. - - - - 11.1 Cladosporium spongiosum 10.5 9 10 - - 9 10.5 10 10 77.7 C. uredinicola ------11 11.1 Cochliobolus spicifer 10 8 11 13.5 8 9.5 11 8 - 88.8 Emericella nidulans var . lata 9 ------11.1 Mucor hiemalis ------8 - 11.1 Penicillium aurantiogriseum ------8 - 11.1 P. chrysogenum 11 - - - -. - - - - 11.1 P. steckii - 10 10 - - 9 8.5 - 9.5 55.5 P. waksmanii - - - - 9 - - - - 11.1 Phialophora fastigiata 9.5 8 - 10 8 9 11 8 - 77.7 Stemphylium botryosum 14.5 11 10 - - 8 9 9 10.5 77.7 Ulocladium alternariae - - - 11.5 - - - - 12.5 22.2 Chloramphenicol (10 mg -1) 37 35 12.5 32 20 32 32 34 32 A: Enterobacter aerogenes (ATCC13048), B: Enterococcus faecalis (ATCC29212), C: Escherichia coli (ATCC25922), D: Klebsiella pneumonia (ATCC13888), E: Pseudomonas aeruginosa (ATCC278223), F: Salmonella Typhi (ATCC19430) , G: Salmonella typhimurium (ATCC14028), H: Shigella flexneri (ATCC12022) and I: Staphylococcus aureus (ATCC43300) .

European Journal of Biological Research 2016; 6 (3): 135-144

141 | El-Said et al. Antimicrobial and L-asparaginase activities of endophytic fungi

Table 3. Antibacterial effects (in mm diameter) of selected fungal endophytes from Hyoscyamus muticus against different nine pathogenic bacteria. Pathogenic bacteria Antibacterial Fungal endophytes A B C D E F G H I activity (%) Alternaria alternata ------9.5 11.1 Aspergillus flavus 9 - 16 12 9 - 11 9 12 77.7 A. niger 10 - - -. - 8 - 8 - 33.3 A. terreus var. africanus - - - 8.5 - - - - 8.5 22.2 A. versicolor 8 - 15 10.5 8.5 - 8 9 10.5 77.7 Chaetomium globosporum ------20 11.1 C. globosum 8 -. - - 8 - 9 - 8 44.4 Cladosporium chlorocephalum 9 - 13.5 13.5 9 - 8 - 13.5 66.6 C. oxysporum 8 - - - 8 - 8 8 - 44.4 C. sphaerospermum 9 - 9 - - - - 8 - 33.3 C.uredinicola 8 - 13 13.5 8 - 8 - 13.5 66.6 Cochliobolus spicifer 8 -. - - 8 - - 8 - 33.3 Cordella clarkii 8 8 8 8 8 - 8 8 8 88.8 Eurotium chevalieri - 8 9 9 8 9 8 9 8 88.8 Penicillium aurantiogriseum - 8 9 11 8 8 9 8 8 88.8 P. chrysogenum 12 - 10 - - 8 8.5 8 - 55.5 P. funicolusum 8 8 8 9 8 - 9 9 8 88.8 P. rubrum - -. -. 8.5 - - - - - 11.1 Sterile mycelium ------. - - 9 11.1 Chloramphenicol (10 mg -1) 37 35 12.5 32 20 32 32 34 32 A: Enterobacter aerogenes (ATCC13048), B: Enterococcus faecalis (ATCC29212), C: Escherichia coli (ATCC25922), D: Klebsiella pneumonia (ATCC13888), E: Pseudomonas aeruginosa (ATCC278223), F: Salmonella Typhi (ATCC19430) , G: Salmonella typhimurium (ATCC14028), H: Shigella flexneri (ATCC12022) and I: Staphylococcus aureus (ATCC43300) .

Table 4. Degree of L-asparaginase activities (calculated as average diameter of pink color zone around the colony mm) of the tested Aspergillus niger isolates. A. niger Activity Source of isolation isolates remarks SVUAn1 Datura innoxia 67.5 H SVUAn2 Datura innoxia 25 M SVUAn3 Datura innoxia 30 M SVUAn4 Hyoscyamus muticus 32.5 M SVUAn5 Hyoscyamus muticus 25 M Figure 2. The effect of carbon sources on the production SVUAn6 Hyoscyamus muticus 20 M of L-asparaginase by A. niger (SVUAn1).

Activity remarks: high activity, H = 35-70 mm; moderate activity, M = 20-34 mm and weak activity, W = less than 20 mm. Zia et al. [65] reported that, the maximum production of L-asparaginase by Aspergillus niger was achieved after 4 days of incubation and with glucose as carbon source. The shorter incubation time reduces the chance of L-asparaginase decom-

European Journal of Biological Research 2016; 6 (3): 135-144

142 | El-Said et al. Antimicrobial and L-asparaginase activities of endophytic fungi position by proteolytic enzymes while, the prolon- 7. Abdou R. Bioactive metabolites from the endophyte ged incubation time led to a decrease in L-asparagi- Botryospheria obtuse of the medicinal plant Bidens nase secretion and this may be due to the exhaustion pilosa . Int J Pharmacy Pharm Sci. 2013; 5(3): 579- of some medium constituents or the production of 584. inhibitory compounds [66]. 8. Devi NN, Prabakaran JJ. Bioactive metabolites from an endophytic fungus Penicillium sp. isolated from Centella asiatica . Curr Res Environ Appl Mycol. 4. CONCLUSIONS 2014; 4(1): 34-43.

From above results, it can conclude that 9. Supriya GNR, Audipudi AV. Screening for antimicrobial activities of endophytic fungi isolated medicinal plants are a reservoir for many endo- from ripened fruit of Capsicum frutescence L. World phytic fungi which consider a good source for J Pharm Sci. 2015; 3(2): 258-262. antimicrobial and anticancer compounds. The 10. Newman DJ, Cragg MG. Natural products as source maximum production of L-asparaginase by Asper- of new drugs over the last 25 years. J Nat Prod. gillus niger SVUAn1 was achieved at shorter 2007; 70: 461-477. incubation time with glucose as carbon source. 11. Kumala S, Izzati H. Isolation IPG3-1 and IPG3-3, endophytic fungi from Delima ( Punica granatum AUTHORS’ CONTRIBUTION Linn.) twigs and in vitro assessment of their antimicrobial activity. Int Res J Pharmacy. 2013; YMS: Designed and collect the samples, EGAE: 4(6): 49-53. laboratory work and recorded the experimental data. 12. Kumar S, Aharwal RP, Kumar S, Sandhu SS. MAH: participate in collections of samples, fungal Isolation and detection of anti-bacterial activity of identification and wrote the research and editing. endophytic fungi from Bombex cebia and Argemone AHME; Supervision, revision. The final manuscript Mexicana. J Chem Pharm Res. 2014; 6(11): 95-100. has been read and approved by all authors. 13. Raju DC, Victoria TD. Isolation, characterization and antibacterial activeity of endophytic fungi from TRANSPARENCY DECLARATION Calophyllum inophyllum L. Der Pharma Chemica. The authors declare no conflicts of interest. 2015; 7(7): 250-254. 14. David SG. The molecular perspective: L-aspara- REFERENCES ginase. Oncologist. 2005; 10: 238-239. 1. Hyde KD, Soytong K. The fungal endophyte 15. Ghasemi Y, Ebrahimminezhad A, Amini SR, Zarrini dilemma. Fungal Divers. 2008; 33: 163-173. G. Ghoshoon MB, Raee MJ, et al. An optimized 2. Zhang HW, Song YC, Tan RX. Biology and medium for screening of L-asparaginase production chemistry of endophytes. Natural Prod Rep. 2006; by Escherichia coli . Am J Biochem Biotechnol. 23: 753-771. 2008; 4(4): 422-424. 3. Krishnamurthy YL, Naik SB, Jayaram S. Fungal 16. McCredie KB, Wang Ho DH, Freireich EJ. L-aspara- communities in herbaceous medicinal plants from the ginase for the treatment of cancer. A Cancer J Clin. Malnad Region, Southern India. Microbes Environ. 2008; 23(4): 220-227. 2008; 23(1): 24-28. 17. Jain R, Zaidi KU, Verma Y, Saxena P. L-aspara- 4. Khan R, Shahzad S, Choudhary I, Khan SA, Ahmad ginase: A promising enzyme for treatment of acute A. Communities of endophytic fungi in medicinal lymphoblastic leukiemia. J Sci Res. 2012; 5(1): 29- plant Withania somnifera . Pak J Bot. 2010; 42(2): 35. 1281-1287. 18. Yadav CN, Sarkar S. Production of L-asparagi- 5. Niber BT, Helenius J, Varis AL. Toxicity of plant nase by Fusarium oxysporum using submerged extracts to three storage beetles (Coleoptera). J Appl fermentation. Int J Pharm Sci Invention. 2014; 3(6): Entomol. 1992; 113(2): 202-208. 32-40. 6. Bharathidasan R, Panneerselvam A. Biodiversity of 19. Masumi S, Mirzaei S, Kalvandi R, Zafari D. the endophytic fungi isolated from Avicennia marina Asparaginase and amylase activity of thyme in Ramanathapuram District, Karankadu. World J Sci endophytic fungi. J Crop Prot. 2014; 3: 655-662. Technol. 2011; 1(9): 1-5. 20. Kalyanasundaram I, Nagamuthu J, Srinivasan B, Pachayappan A, Muthukumarasamy S. Production,

European Journal of Biological Research 2016; 6 (3): 135-144

143 | El-Said et al. Antimicrobial and L-asparaginase activities of endophytic fungi

purification and characterisation of extracellular 34. Maria GL, Sridhar KR, Raviraja NS. Antimicrobial L-asparaginase from salt marsh fungal endophytes. and enzyme activity of mangrove endophytic fungi World Journal Pharm Pharmac Sci. 2015; 4(3): 663- of southwest coast of India. J Agric Technol. 2005: 677. 67-80. 21. Thirunavukkarasu N, Suryanarayanan TS, Murali 35. Gulati R. Saxena RK, Gupta R. A rapid plate assay TS, Ravishankar JP, Gummadi SN. L-asparaginase for screening L-asparaginase producing micro- from marine derived fungal endophytes of seaweeds. organisms. Lett Appl Microbiol. 1997; 24: 23-26. Mycosphere. 2011; 2(2): 147-155. 36. Saxena RK, Sinha U. L-asparaginase and gluta- 22. Rani SA, Sundaram L, Vasantha PB. Isolation and minase activities in culture filtrates of Aspergillus screening of L-asparaginase producing fungi from nidulans . Curr Sci. 1981; 50: 218-219. soil samples. Int J Pharmacy Pharm Sci. 2011; 4: 37. Imada A, Igarasi S, Nakahama K, Isono M. 279-282. Asparaginase and glutaminase activities of micro- 23. Lincoln L, More SS. Isolation and production of organisms. J General Microbiol. 1973; 76: 85-99. clinical and food grade L-asparaginase enzyme from 38. El-Morsy EM. Fungi isolated from the endo- fungi. J Pharmacognosy Phytochem. 2014; 3(3): rhizosphere of halophytic plants from the Red Sea 177-183. Coast of Egypt. Fungal Divers. 2000; 5: 43-54. 24. Bills GF. Isolation and analysis of endophytic fungal 39. Abdel-Motaal FF, El-Zayat Y, Kosaka Y, El-Sayed communities from woody plants. In: Redlin SC, M A, Nassar MSM, Ito SI. Antifungal activity of Carris LM, eds. Endophytic fungi in grasses and endophytic fungi isolated from Egyptian henbane woody plants: systematics, ecology and evolution. (Hyoscyamus muticus L.). Pak J Bot. 2010; 42(4): APS Press, St. Paul, 1996. 2883-2894. 25. Rossman AY, Tulloss RE, O’Dell TE, Thorn RG. 40. Singh SK. Endophytic fungi from Jatropha curcus : Protocols for an all taxa biodiversity inventory of A preliminary study. J Pharm Sci Innov. 2013; 2(2): fungi in a Costa Rican conservation area. Parkway 26-29. Publications, Inc., Boone, North Carolina, 1998. 41. Dos-Santos RMG, Rodrigues-Fo E, Rocha WC, 26. Ames LA. A monograph of the chaetomiaceae. Teixeira MFS. Endophytic fungi from Melia Wheldon and Wasley L.T.D. New York, 1969. azedarach . World J Microbiol Biotechnol. 2003; 19: 27. Ellis MB. Dematiaceous hyphomycetes. Common- 767-770. wealth Mycological Institute, Kew, England, 1971. 42. Selvi KB, Balagengatharathilagam P. Endophytic 28. Raper KB, Fennell DJ. The genus Aspergillus . fungi from medicinal plants of Virudhunagar district Williams & Wilkins, Baltimore, USA, 1965. for antimicrobial activity. Int J Sci Nature. 2014; 29. Raper KB, Thom C. A manual of the Penicillium . 5(1): 147-155. Williams & Wilkins, Baltimore, USA, 1949. 43. Ramesha A, Srinivas C. Antimicrobial activity and 30. Domsch KH, Gms W, Anderson TH. Compendium phytochemical analysis of crude extracts of of soil fungi. Acad Press, London, 1980. endophytic fungi isolated from Plumeria acuminata L. and Plumeria obtusifolia L. Eur J Exp Biol. 2014; 31. Silva MRO, Almeida AC, Arruda FVF, Gusmao N. 4(2): 35-43. Endophytic fungi from Brazillian mangrove plant Laguncularia racemosa (L.) Gaertn. (Combre- 44. Mahdi T, Mohamed I, Yagi S. Endophytic fungal taceae): their antimicrobial potential. In: Science communities associated with ethno-medicinal plants against microbial pathogens: communicating current from Sudan and their antimicrobial and antioxidant research and technological advances. Mendez-Vilas prospective. J Forest Prod Industr. 2014; 3(6): 248- A, ed. 2011: 1260-1266. 256. 32. Bauer AW, Kirby WMM, Sherris JC, Turck M. 45. Powar PV, Patil KS. Screening of endophytic fungi Antibiotics susceptibility test by a standardized isolated from Celastrus paniculatus for antimicrobial single disc method. Am J Clin Pathol. 1966; 45: 493- potential. World J Pharmacy Pharm Sci. 2015; 4(7): 496. 717-722. 33. Tripathi NK, Shrivastva A, Biswal KC, Rao PVL. 46. Gherbawy YA, Elhariry HM. Endophytic fungi Optimization of culture medium for production of associated with high-altitude Juniperus trees and recombinant dengue protein in Escherichia coli . their antimicrobial activities. Plant Biosystems. Indust Biotechnol. 2009; 5(3): 179-183. 2016; 150(1): 131-140.

European Journal of Biological Research 2016; 6 (3): 135-144

144 | El-Said et al. Antimicrobial and L-asparaginase activities of endophytic fungi

47. Shaaban M, Nasr H, Hassan AZ, Asker MS. 57. Siddalingeshwara KG, Lingappa K. Production and Bioactive secondary metabolites from endophytic characterization of L-asparaginase - a tumour Aspergillus fumigatus : structural elucidation and inhibitor. Int J PharmTech Res. 2011; 3(1): 314-319. bioactivity studies. Rev Latinoam Quím. 2013; 58. Audipudi AV, Supriya GNR, Pallavi R, Mani PG. 41(1): 50-60. Characterization of L-asparaginase producing 48. Mukhopadhyay T, Roy K, Coutinho L, Rupp RH, endophytic fungi isolated from ripened fruit of Ganguli BN. Fumifungin, a new antifungal antibiotic Capsicum frutescence . Int J Pharm Develop Technol. from Aspergillus fumigatus Fresenius 1863. J 2014; 4(1): 52-57. Antibiot. 1987; 40: 1050-1052. 59. El-Hefnawy MAA, Attia M, El-Hofy ME, Ali MAS. 49. Ando O, Satake H, Nakajima M, Sato A, Nakamura Optimization Production of L asparaginase by locally T, Kinoshita T. Synerazol, a new antifungal isolated filamentous fungi from Egypt. Curr Sci Int. antibiotic. J Antibiot. 1991; 44: 382-389. 2015: 4(3): 330-341. 50. Han SK, Choi ANS, Hong RK, Moon SK, Chun HS, 60. Patro KKR, Satpathy S, Gupta N. Evaluation of Lee SJ. Design and synthesis of highly potent some fungi for L-asparaginase production. Indian J fumagillin analogues from homology modeling for a Fund Appl Life Sci. 2011; 1(4): 219-221. human metAP-2. Bioorg Med Chem Lett. 2000; 10: 61. Alhussaini MS. Mycobiota of wheat flour and 39-43. detection of α- amylase and L-asparaginase enzymes. 51. Xu L, Meng W, Cao C, Wang J, Shan W, Wang Q. Life Sci J. 2013; 10(1): 360-371. Antibacterial and antifungal compounds from marine 62. Tippani R, Sivadevuni G. Nutritional factors fungi. Marine Drugs. 2015; 13: 3479-3513. effecting the production of L asparaginase by the 52. Lee YM, Li H, Hong J, Cho HY, Bae KS, Kim MA, Fusarium sp. Afr J Biotechnol. 2012; 11(15): 3692- et al. Bioactive Metabolites from the Sponge- 3696. Derived Fungus Aspergillus versicolor . Arch Pharm 63. Vijay B, Raju KJ. Production of L-asparaginase by Res. 2010; 33(2): 231-235. Aspergillus terreus MTCC 1782 under solid state 53. Zhang Y, Li XM, Wang BG. Anthraquinone fermentation using pearl millet and finger millet as derivatives produced by marine-derived fungus mixed substrate. J Chem Biol Phys Sci. 2014; 5(1): Aspergillus versicolor EN-7. Biosci Biotechnol 366-377. Biochem. 2012; 76: 1774-1776. 64. Aparna C, Raju KJ. Optimization of process 54. Song FH, Ren B, Chen CX, Yu K, Liu XR, Zhang parameters for L-asparaginase production by YH, et al. Three new sterigmatocystin analogues Aspergillus terreus MTCC1782 under solid state from marine-derived fungus Aspergillus versicolor fermentation using mixed substrate. Int J Res Eng MF359. Appl Microbiol Biotechnol. 2014; 98: 3753- Technol. 2015; 4(5): 354-360. 3758. 65. Zia MA, Bashir R, Ahmed I, Iftikhar T. Production 55. Barbour EK, Sharif MA, Sagherian VK, Habre AN, of L-asparaginase from Aspergillus niger using agro Talhouk RS, Talhouk SN. 2004. Screening of wastes by-products in submerged fermentation selected indigenous plants of Lebanon for process. J Teknologi. 2013; 62(2): 47-51. antimicrobial activity. J Ethnopharmacol. 2004; 93: 66. Thakur M, Lincoln L, Niyonzima FN, More SS. 1-7. Isolation, purification and characterization of fungal 56. Sidalingeshwara KG, Lingapa K. Key fermentation extracellular L-asparaginase from Mucor hiemalis . J factors for the synthesis of L-asparaginase - an anti Biocat Biotrans. 2014; 2(2): 1-9. tumour agent through ssf methodology. Int J Pharm Sci. 2010; 1(1): 103-112.

European Journal of Biological Research 2016; 6 (3): 135-144