Bioactive Secondary Metabolite from Endophytic Aspergillus Tubenginses ASH4 Isolated from Hyoscyamus muticus: Antimicrobial, Antibiofilm, Antioxidant and Anticancer Activity

Scientists are tackling different diseases in our society due to rehabilitated environment and lifestyle. Several researchers are working on these emerging diseases to understand and cure them using different chemical and natural formulations. However, many areas are untouched due to poor knowledge and techniques.1,2 Researchers are trying to isolate new bioactive compounds from new species of fungal endophytes for medicinal, agricultural and industrial applications as reported by Anisworth et al. (1973); Smith et al. (2008); Garg et al. (2011); Fahmy et al. (2017); Hamed et al. (2018); Nurunnabi et al. (2020).3-8


INTRODUCTION
Scientists are tackling different diseases in our society due to rehabilitated environment and lifestyle. Several researchers are working on these emerging diseases to understand and cure them using different chemical and natural formulations. However, many areas are untouched due to poor knowledge and techniques. 1 [3][4][5][6][7][8] Endophytic fungi have a symbiotic relationship between fungi and their host plants. 6 Symbiosis refers to the living together of several species by which symbiont and host are beneficiaries. [9][10][11] The secondary endophytic fungal metabolites have a various biological activity. The multitude of secondary metabolites derived from fungal endophytes has established therapeutic applications such as anticancer, antimicrobials, antitumor and antidiabetics, and cholesterol inhibitors and antioxidant agents. 7,12-14 Wang et al. (2007) showed that, endophytic fungal metabolites can also be used effectively in weed control. For example, Cladosporium sp. created the Brefeldin A. that suppresses the maturity of weed pollen tubes of Picea meyeri. 15 Khan et al. (2012) declared that, Paraconiothyrium sp. generates ascotoxin which influences seed germination of Echinochloa crus-galli and Lactuca sativa. 16 Fungal endophytes possess defensive mechanisms and greatly affect plant community growth. 17 Therefore, the aim of this study was to investigate the bioactive metabolites of endophytic Aspergillus tubenginses with biological evaluation of purified secondary metabolites.
in SDW, immersed in 2% sodium hypochlorite for 1 min. followed by rinsing with SDW triply. The sterilized plant parts were allowed to dry in laminar flow, and a healthy leaf was cut into small pieces of 1 cm 2 and placed on a PDA plate. To check the efficiency of surface sterilization procedure, 4-5 water drops from the final rinse were inoculated on a PDA medium, held for approximately 5-6 days to investigate the growth of any endophytic fungi. The most potent obtained fungal endophyte was identified using 18S rRNA analysis, Macrogen Company, South Korea.

Genetic identification of endophytic fungus
Here, 18S rRNA analysis was performed on the endophytic fungus by biosynthesizing the major compound. In the laboratory, fungal DNA was extracted using the Qiagen DNeasy Mini Kit protocol. The extracted DNA was subjected to polymerase chain reaction using (5′-TCCGTAGGTGAACCTGCG-3′)/ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) universal primers. In Macrogen Companies, Seoul, South Korea. Amplified DNA was subjected to DNA sequencing and the gained DNA sequence was parallel to the DNA sequence presented at NCBI GenBank *. The resulting gene sequence was submitted to the NCBI GenBank database and an accession number was attained. http://www.ncbi.nlm.nih.gov.blast

Production of bioactive compounds
Rice in the solid state was used as a fermentation media, 100g rice was soaked in 100mL distilled sea water (Mediterranean Sea), and autoclaved, and 1ml of endophytic fungus was introduced to each medium flask. The inoculated flasks were incubated under static conditions at 30°C for 15 days. The extraction was performed using ethyl acetate, and then the organic phase was gathered, stored, and dried at 37°C. [19][20][21] Antimicrobial activity The antimicrobial activity of the obtained crude extracts was performed using agar diffusion method according to Abdel-Aziz et al. (2019). 22 Each fungal crude extract was dissolved in MeOH at 500 µg/ml and Aliquots of 50 μl were loaded on disks (Whattman No. 1 filter paper, 5 mm). The inhibition zone diameter was measured against various of pathogenic microorganisms comprising: Gram+ve bacteria; (Staphylococcus aureus ATCC6538-P) and Bacillus subtilis. Gram-ve bacteria (Pseudomonas aeruginosa ATCC27853) and E. coli. and Yeasts (Candida albicans ATCC10231). On the other hand, the antibacterial activity of the pure compound isolated from selected fungus was measured using Antimicrobial assay and MIC were performed as described by Ingebrigtsen et al. (2016). 23 All test microbes were obtained from the Culture Collection Center (Microbial Chemistry Department and National Research Center, NRC), Egypt.

Antibiofilm activity
MTP assays were conducted to test antibiofilm activity of the fungal crude extracts and pure compound. The test was performed using 96 well-flat bottom polystyrene titer plates and four clinical microbes were also used (P. aeruginosa, S. aureus, E. coli, and B. subtilis) according to Christensen et al. (1985) 24  . 25 The optical density was determined at 570 nm using a spectrostar nanomicroplate reader (BMG LABTECH GmbH, Allmendgrun, Germany).

Determination of total antioxidant capacity (TAC) and total phenolic content (TPC)
The antioxidant activity of the ethyl acetate crude extracts along with pure compound was determined by phosphor molybdenum assay according to Ghareeb et al. (2014Ghareeb et al. ( & 2016 26,27 and Saad et al. (2017) 28 . The antioxidant activity was conducted as the number of ascorbic acid equivalent (AAE), in triplicate. The total phenolic content of fungal endophytic ethyl acetate extract was also estimated using phosphomolybdenum assay according to Shoeb et al. (2014). 29 Extraction, purification and structure elucidation Purification started by fractionation using liquid-liquid partitioning according to the Kupchan original protocol. 30 The extraction procedures mainly depend on the polarity of interest compounds (which can be determined by thin layer chromatography with varying polarity eluents or by analytical HPLC. The most potent fraction was further purified using Size-exclusion chromatography technique via using Sephadex LH-20. The separation was based on molecular weight. The purified compound was subjected to NMR spectroscopy for structure elucidation as well as Thermo Scientific LTQ Orbitrap XL Mass Spectrometer for molecular formula identification.

Anticancer activity
Four different cell lines were used, human lung fibroblast normal cell line (WI-38) , colorectal carcinoma colon cancer (HCT-116), breast cancer of the mammary gland (MCF-7), and hepatocellular cancer (Hep-G2). The cell lines were obtained from American Type Culture Collection (ATCC), through Biological Products and Vaccine Holding Company (VACSERA), Cairo, Egypt. In reference, doxorubicin has been used as a traditional anticancer drug.
Using the MTT test, the abovementioned cell lines were employed to assess the inhibitory effects on cell growth of pure compounds extracted from endophytic fungus. The colorimetric assay depends on the conversion of yellow tetrazolium bromide (MTT) in viable cells into a violet formazan derivative by mitochondrial succinate dehydrogenase in viable cells. The cell lines were cultivated using 10 % fetal bovine serum in the RPMI-1640 medium. Antibiotics added in a 5% CO 2 incubator were 100 units/ml of penicillin and 100μg / ml of streptomycin at 37°C. The cell lines were seeded for 48 h under 5 percent CO 2 in a 96-well plate at a density of 1.0 × 10 4 cells/well at 37°C. The cells were treated with different concentrations of compounds after incubation for 24 h. A 20 µl of MTT solution at 5 mg/ml was used in the drug treatment, added and incubated for 4 h, and 100µl of DMSO was added to dissolve the purple formazan formed in each well. The colorimetric assay was recorded at 570 nm using a plate reader (EXL 800, USA). The relative cell viability in percentage was calculated as (A570 of treated samples/A570 of untreated sample) x 100. 32,33

Sample collection and isolation of endophytes
The plant sample was gathered from Wadi-Elnatrun Valley, Egypt. The plant was identified based on the morphological features as Hyoscyamus muticus L. Ten endophytic fungi were isolated from Hyoscyamus muticus L, coded and kept at microbial culture collection, Microbial Chemistry Department, Dokki, Giza, Egypt.

Evaluation of the antimicrobial activities of fungal endophytes
The crude extracts from the isolated 10 endophytic fungi were prepared by cultivation of the 10 fungal isolates on rice medium followed by ethyl acetate extraction. The antimicrobial activity of the crude extracts was evaluated (in vitro) against several test pathogenic microorganisms comprising 2 Gram -ve, 2 Gram +ve bacteria and one yeast. Results showed that, nearly 70% of the endophytic fungi isolated from H. muticus L exhibited antibacterial activities. The most promising endophytic fungi from H. muticus L plant were AF3, 15F6, 15F8, 15F13 and 15F14 with inhibition zone 17-21, 15-17, 14.5-19.2, 14.5-17, and 7-14 mm, respectively, as recorded in Table 1 Observably, different endophytic fungal extracts from the same plant display various antimicrobial activities Table (1). These sensitivity differences could be attributed to the isolate species, level, nature, and mode of action of antimicrobial agents existing in their extracts and on various trial microorganisms as reported by Barbour et al. (2004) 40 . In this study, the most potent endophytic isolates (AF3, 15F6, 15F8, 15F13, and 15F14) were selected for other screening methods and investigations.

Antioxidant capacity of endophytic fungal extract using phosphomolybdenum assay
Phosphomolybdenum assay is based on Mo+ 4 reduction to Mo +5 through interaction with the tested sample and sequent formation of a green color [phosphate = Mo +5 ] complex with maximum absorption of 695 nm in the acidic medium. 41,42 . Results showed that, TAC values for the tested crude endophyte extracts ranged from 111.66 -880.66 mg AAE/g dry extract. The results are in the order: 15F13 > 15F8 > AF3 > 15F6 > 15F14 as presented in Table 2. Rudgers et al. (2007) stated that plant phenolic compounds influenced the community of endophytes. 43 Therefore, the higher the content of active compounds in host plant, the more richness with endophytic microorganisms. Previous reports revealed that endophyte extracts and their derived pure isolates exhibit noticeable antioxidant potentials. 44,45. Antibiofilm activity of endophytic extract of fungal isolates against some pathogenic microorganisms The evaporated ethyl acetate extract was diluted with methanol for MTP test that was performed according to Christensen et al. (1985). 24 From the extracts, a concentration (100μg/mL) was selected for biofilm inhibition assay against tested pathogens and their virulence factor in terms of biofilm formation. The test was performed in a 69-well microtiter plate. The crude extract from fungal endophyte AF3 showed better inhibition percentage in (100 μg/mL) with 50.06% against Bacillus subtilis, 37.68% against E coli, 28.44% against Pseudomonas aeruginosa and 60.8% against Staphylococcus aureus Figure 1. The results showed that the crude compound of AF3 was the most active against biofilm colonization and cell adherence

Molecular identification the most active endophytic fungal isolate
The 18S rRNA gene sequence was used to identify and oppose other identified sequences available in the GeneBank database using BLAST to indicate the score similaritie sand calculate the statistically significant differences of matches (http://www.blast.ncbi.nlm.nih.gov/Blast). The results established a very close similarity with Aspergillus tubenginses using the 18S rRNA gene sequence with 100 % homology of the isolate AF3. The phylogenetic analysis and tree were composed using the neighboring method ( Figure 2) by MEGA 7 program according to Kumar et al. (2016). 49 Based on the analysis of the DNA sequence and morphological characteristics of the AF3 isolate (Supplementary 1), the isolated strain was identified as Aspergillus tubenginses ASH4 and deposited in GenBank with the accession no. MN784618.1.

Extraction and Bio-guided fractionation and purification
Large scale fermentation of the most potent endophytic fungus Aspergillus tubenginses ASH4 was carried out on rice medium, incubated for 15 days, extracted by ethyl acetate and evaporated using a rotary evaporator at 40°C. Purification was started by fractionation using liquid-liquid partitioning according to the method of Kupchan original protocol. 30 Five aqueous fractions: methanol, n-hexane, n-butanol, dichloromethane (DCM) and water were performed as shown in Figure 3. Antimicrobial screening indicated that only dichloromethane (DCM) followed by n-hexane extracts for Aspergillus tubenginses ASH4 exhibited antimicrobial activity against the tested pathogenic microorganisms.
Conversely, antioxidant evaluation of the five fractions, showed that butanol and methanol extracts of Aspergillus tubenginses ASH4 exhibited high antioxidant activity. Also, the results revealed various total phenolic concentrations of ethyl acetate fractions in endophytic fungal extracts as manifest in Table 3. The estimation varied from 48.0 -134.62 mg gallic acid equivalent (GAE)/g of dry weight. The highest concentration of phenols was observed in extract of ASH4 BuOH (134.62 mg GAE/g extract). Considering findings of other studies, the polyphenolic-rich extracts could loss an electron through their redox reaction with a molybdotungstate reagent (Folin Ciocalteu's reagent). This electron transfer reaction induces a blue color, which can be quantified simply by spectrophotometry at 765 nm. 42 Also, based on the antimicrobial screening (Table 4), the fraction dichloromethane (DCM) was active, and therefore was selected for further processing using different chromatographic techniques. Compound (1) was identified as anofinic acid (2,2-dimethyl-2H-1-benzopyran-6-carboxylic acid).
Anofinic acid (Figure 3) was isolated as a yellow fine crystal from the DCM fraction using Sephadex LH-20 subcolumn eluted with a gradient mix elution system using aqueous methanol. It showed a molecular formula (C 12 H 12 O 3 ) and molecular weight of 204.22 (Supplementary 1). In 1 H-NMR spectrum, it showed characteristic signals in the aromatic region between δ H 6.95 to δ H 7.45 ppm, resonating methyl protons appeared at δ H 1.5 ppm. Two olefinic protons were resonated at δ H 6.33 and 5.77 ppm. The hydroxyl proton of the carboxylic group was resonated at δ H 11.69 ppm. 31 Evaluation of bioactivity of pure compound antibacterial activity Data illustrated in Figure 5 represent the antimicrobial activity of the pure compound from Aspergillus tubenginses ASH4 against a set of microorganisms comprising Gram-ve bacteria and Gram+ve bacteria (P. aeruginosa, S. aureus, B. subtilis and E. coli). The results revealed that     anofinic acid exhibited antibacterial activity against all tested bacteria.
Recently, the introduction of new pathogens, such as SARS, H1N1 and different forms of influenza, has become a significant public health threat. Most of these emerged diseases are occasioned by microorganisms and occasional microbes become increasingly drug resistant with time. 50 To overcome such infectious diseases, novel microorganism and plant bioactive compounds may stand by the best substitutional provenance of potential medicines. 51 Since, plant is the prime source of bioactive compounds, endophytes may nevertheless compete a vital role in the quest for novel compounds of biological activity. 52 Antibiofilm activity of pure compound Using microtiter biofilm plate assay, the biofilm inhibition activity of the pure compound of Aspergillus tubenginses ASH4 was measured against four clinical pathogenic bacteria (P. aeruginosa, S. aureus, E. coli and B. subtilis), and the results were compared with untreated biofilms (control) for each pathogen (Table 5).

TAC
The results presented in Table 6 show that the TAC of pure anofinic acid is 409.92 mg AAE/g compound. Ghareeb et al. (2019b) 45       against examined cell lines cancer of the colorectal colon (HCT-116), breast cancer of the mammary gland (MCF-7), and (Hep-G2).
Extraction of natural bioactive compounds and screening them for pharmacological advantages provide a path for identifying of drug nominee as reported by Salvador-Reyes and Luesch (2015) 53 and Hamed et al. (2020) 25 . Many endophytes have been described and documented to generate new compounds are successful in anticancer inspection. 54,55 In another study, endophyte isolate Aspergillus sp. was led to manufacture utmost yield of 100 μg/g of mycelia dry weight. 56 Also, Budhiraja et al. (2013) isolated Aspergillus sp. from Gloriosa superba. 57 A new compound colchatetralene was isolated along with three known compounds namely ergosterol, 4-hydroxy-phthalic acid-dimethyl ester and 5-(hydroxymethyl) furan-2 carbaldehyde which was tested for cytotoxic effect on seven cell lines. Colchatetralene was found to be influential against

CONCLUSION
In this study, 10 endophytic fungal strains were isolated from Hyoscyamus muticus plant, biological screening including antibacterial, antibiofilm, and antioxidant activities of 10 fungal ethyl acetate extracts, showed that, Aspergillus tubenginses ASH4 exhibited maximum antibacterial activity against tested pathogenic strains with inhibition zone of 21 mm, and the TAC values for the tested crude extract of Aspergillus tubenginses ASH4 was 753.33 mg AAE/g dry extract. Moreover, the isolated pure compound anofinic acid inhibited biofilm formation up to 69.51%. Moreover, the antioxidant activity of anofinic acid was 409.92 mg AAE/g. The IC 50 values of anofinic acid against some carcinoma cells such as HCT-116, Hep-G2 and MCF-7 were 728.14, 31.65, and 22.61 μg/well, respectively, described as very strong activities.