Potency of Endophytic Fungi Isolated from Medicinal Plants in Egypt as a Resource for Anticancer Compounds

ABSTRACT

This study aimed to bioprospect endophytic fungi producing anticancer compounds hosted etlmobotanical plant species inhabiting a protected area in Egypt. A total of 58 fungal endophytic species belonging to 32 genera were isolated from 2 medicinal plant species inhabiting Saint Katherine Protectorate. Trichothecium roseum and Stachybotrys chartarum were the most frequently isolated species and were molecularly identified via comparison of their 18S rRNA sequences with those deposited in the GenBank database. Fungi were cultivated on half-strength potato dextrose broth for 15 days at 28°C on a rotaiy shaker at 180 rpm and were then extracted in ethyl acetate (EtOAc). Acute toxicity and brine shrimp lethality bioassays were performed to evaluate the cytotoxicity of the aqueous and EtOAc extracts of the I roseum (GenBank accession number MF399479) and S. chartarum (MF399480) strains. Both taxa exhibited considerable cytotoxic activities against Arteniia saliua, and their effects on MCF-7 breast cancer cell proliferation and apoptosis were investigated in mice. The elevated tumor volumes and cell counts of the tumor-bearing mice decreased significantly after treatment with the I roseum and S. chartarum extracts. A significant decrease in the cell viability of MCF-7 cells was recorded for 550 to 714 mg/kg of the EtOAc extract and for 169 to 178 mg/kg of the aqueous extract of I roseum. Furthermore, the lethal concentration (LC30) value was 2.94 pg/rnL for the EtOAc extract and 1.63 pg/mL for the aqueous extract, as only 50% of the brine stomp were viable after 24 h of treatment at these concentrations. The present study revealed that the secondary metabolites of a native isolate of T roseum (MF399479) hosted by Achillea fragrantissima have a direct inhibitory effect on Ehrlich ascites carcinoma in a mouse model. This type of carcinoma is undifferentiated and originally hyperdiploid and does not exhibit tumor-specific transplantation antigen; moreover, it demonstrates high transplantable capability with no regression and rapid proliferation, exhibiting 100% malignancy, which leads to a shorter life span.

INTRODUCTION

The prospect of fungal endophytes producing active metabolites that may be effective candidates for the treatment of human conditions has attracted the attention of the scientific community in many countries (Abdel-Azeem et al., 2016). Endophytic fungi were recently indicated to produce 51% of previously unknown bioactive substances. The search for new sources of effective natural compounds as novel cancer therapeutic agents is of great importance due to the number of annual cancer deaths worldwide and the high cost and serious side effects of available cancer therapies (Kharwar et al., 2011). The unique environments, substantial diversity, ethnobotanical history, and endemicity of endophyte host plants should be considered during study selection processes (Salem and Abdel-Azeem, 2014). The mountainous region of southern Sinai exhibits greater biodiversity than the rest of Egypt, and 4350 km2 of this area was declared a Protectorate in 1996 (Abdel-Azeem and Salem, 2015). Approximately, 170 plant species that inhabit south Sinai are used traditionally in folk medicine (Fayed and Shaltout, 2004).

This study investigated the capability of endophytic mycobionts hosted by two species of medicinal plants in to produce anticancer metabolites. Furthermore, the potential inhibitory effects of these compounds on Michigan Cancer Foundation-7 (MCF-7) breast cancer cell proliferation and apoptosis in mice were explored.

MATERIALS AND METHODS

SAMPLING OF MEDICINAL PLANTS

Two plant species, Achillea Jragrantissima (Forssk) Sch. Bip and Origanum syriacum L., were collected from 12 sites representing different elevation wadis (1290 m above sea level (m.a.s.l.) up to 2300 m.a.s.l.) in Saint Katherine Protectorate, Sinai, Egypt, following the ethical rules of the protectorates. Samples of aerial parts from each plant species were collected in sterile polyethylene bags, closed using rubber bands, and transferred to the laboratory until plating.

ISOLATION AND IDENTIFICATION OF ENDOPHYTES

The aerial parts of collected plant samples were washed in running water, cut into small pieces, immersed in 75% ethanol for 1 min, dipped in sterile distilled water twice then 0.05 g/mL of a sodium hypochlorite solution for 3-5 min, and rinsed three tunes in sterile distilled water (Abdel-Azeem and Salem, 2012). The surface-sterilized segments were cultured on potato dextrose agar medium (PDA, Difco™) amended with 0.05 g/L rose bengal and 150 mg/L chloramphenicol. Petri dishes were sealed using Parafilm™ and incubated at 28±1°C for 7-21 days. Growing fungi were purified for identification.

6.2.2.1 PHENOTYPIC IDENTIFICATION

Phenotypic identification of endophytic fungal isolates was primarily based on the relevant identification keys for Penicillium (Pitt, 1979), Aspergillus (Klich, 2002), dematiaceous hyphomycetes (Ellis, 1976, 1971), Fusarium (Leslie and Summered, 2006), miscellaneous fungi (Domsch et al., 2007), soil ascomycetes (Guano Safont, 2012), Chaetomium (Doveri, 2013), and Alternaria (Simmons, 2007). The names of fungal taxa have been shortened in accordance to (Kirk and Ansell, 1992), and their systematic arrangement followed Ainsworth and Bisby's Dictionary of the Fungi (Kirk et ah, 2008). All reported taxa were checked against Index Fungorum website database (Kirk, 2017) for name corrections, authorities, and taxonomic assignments.

6.2.22 MOLECULAR IDENTIFICATION

As the most frequent taxa, both Stachybotrys chartarum (Elirenb.) S. Hughes and Trichothecium roseum (Pers.) Link were identified via comparison of their partial 18S rDNA sequences with reference strains data deposited in GenBank using BLAST homology searches on the NCBI website.

Fungal isolates were grown on PDA. DNA extraction, amplification and sequencing of the 18S rDNA were described previously (White et al., 1990; Zaki et al., 2013).

EXTRACTION OF ACTIVE METABOLITES

Selected isolates of 7 roseum and S. chartarum were grown in 2-liter standard flasks containing 500 mL of potato dextrose broth (PDB) on a rotary shaker incubator at 180 rpm/min for 15 days at 28°C. The culture fluid was passed through two layers of filter paper to remove solids, and the metabolites were extracted using ethyl acetate (EtOAc) as the organic solvent. Crude fermentation broths were blended thoroughly and centrifuged at 3000 rpm for 10 min. Supernatants were concentrated to 10% of their original volume via rotary evaporation at 49°C. The concentrated broths were passed through a filtration membrane (d = 0.22-1 m) and reconstituted in 5% dimethylsulfoxide (DMSO, Merck) in ethanol (v/v) to 5 mg/mL prior to toxicity and bioactivity evaluations.

TOXICOLOGICAL BIOASSAYS

BRINE SHRIMP LETHALITY BIOASSAY

Brine shrimp lethality bioassays were used to evaluate the cytotoxicity of the aqueous and ethyl acetate extracts of T roseum and S. chartarum strains according to (Wakawa and Fasihuddin, 2016). A sample of each extract (8 mg) was dissolved in DMSO, and solutions of varying concentrations (800, 400, 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, and 0.78 pg/mL) were obtained via serial dilution using simulated seawater. Each concentration was investigated in triplicate. The solutions were included with premarked vials loaded with 10 live nauplii in 5 mL of simulated sea water. The vials were examined just after one day with a magnification glass, and the amount of surviving nauplii in eveiy vial was measured. The mortality endpoint of this bioassay was described as the lack of controlled onward movement throughout half a minute of observation.3 Vials containing DMSO and the extraction (500 pL) were set as controls. Vincristine sulfate (VS) was adopted as a positive control. Sea salt (Sigma 9883) was applied in activity tests. The complete quantity of shrimp in every container was measured and recorded. The death percentage and lethal concentration (LC) were determined. The LC50 after 24 h was obtained using a plot of the percentage of brine shrimp killed against the logarithm of the extract concentration (toxicant concentration). The best concentration was obtained from the curve data using regression analysis compared to the positive control (VS LC.0 = 0.52 pg/mL). The following regression equation was used: Y = f (X,B) = a + bx according to (Salem and Abdel-Azeem, 2014).

ACUTE TOXICITY BIOASSAY

The acute intraperitoneal toxicity of the fungal extracts was determined by calculation of the lethal dose that kills 50% of annuals (LD.0) using up and down teclmiques according to a previously described method (Bruce, 1985) in 12 albino mice per extract type. Fungal extracts were administered intraperito- neally (i.p.) in graded doses of422.5,550, 714, and 928 mg of extract/kg body weight for the S. chartarum EtOAc extract (SI); 100, 130, 169, and 219.7 mg of extract/kg body weight for the S. chartarum aqueous extract (AQ1); 500, 650, 845, and 1099 mg of extract/kg body weight for the T roseimi EtOAc extract (S2); and 500, 650, 845, 1099, and 1428 mg of extract/kg body weight for the T roseum aqueous extract (AQ2). Mortality rates were recorded within the first 24 h after administration. Doses were selected and adjusted using a constant multiplicative factor of 1.3 for this test. For each successive annual, the dose was adjusted down or up depending on the previous result.

ANIMALS AND ANTICANCER ACTIVITY BIOASSAY

The Michigan Cancer Foundation-7 human breast tumor cell line (MCF-7) was supplied by the Egyptian Institute of Cancer in Cairo, Egypt, and was kept in a female of Swiss albino mice. The in vivo antitumor activity of fermentation broths was tested using a protocol approved by the Scientific Research Ethics Committee of the Veterinary Medicine Faculty at Suez Canal University. Animals were housed as described by standard animal care requirements in groups up to 10 individuals per cage. They maintained under pathogen-free environment on a 12/12 h light/dark system. Seventy mice of Swiss albino were divided into 7 groups (10 animals/group). Each mouse, except those in the negative control group, received an i.p. injection of 0.2 mL of a cell suspension containing 2xi06 Ehrlich ascites carcinoma (EAC) on day 0. Group I mice served as a negative control. Group II mice were left untreated as a positive control. Group III mice were treated with 5-fluorouracil (5-FU) as a standard reference drug. Animal groups IV, V, VI, and VII were subcutaneously injected with 0.1 mL of OS (28 mg/ kg b.wt.), AQS (9 mg/kg b.wt.), ОТ (45 mg/kg b.wt.) and AQT (58 mg/ kg b.wt.), respectively, 24 h after tumor cell inoculation. The abdominal circumferences of the mice were measured after fungal extract injection using a measuring tape. Animals were weighed immediately before tumor inoculation and twice weekly after tumor inoculation. Five mice from each group were sacrificed via cervical dislocation 13 days after injection with the fungal extracts, and changes in the body weight, ascites tumor volume, and viable tumor cell count of the mice were measured and statistically analyzed. Median survival time (MST) and the percent increase in life span (ILS%) for the remaining mice in each group were observed and calculated (Sur and Ganguly, 1994).

BIOCHEMICAL BIOASSAY

Ascetic fluid and serum samples were collected from the annuals treated with fungal extracts and sent directly to the laboratory for analysis. The serum- ascites albumin gradient (SAAG) was used in the differential diagnosis of ascites, particularly with reference to the prediction of portal hypertension, and both fluid and serum samples were required for this analysis. The following examinations were performed: physical parameter history and clinical examination and abdominal (Nyland and Mattoon, 2002) and biochemical examinations (Rudloff, 2005). Blood was immediately transferred to test tubes and maintained at room temperature for 30 min according to the manufacturer's protocol. Blood was centrifuged at 1200 x g for 20 min. Serum assays for albumin, alanine amino transferase (ALT), creatinine, C-reactive protein (CRP), alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) were measured using photometric or enzyme-linked immunosorbent assay (ELISA) methods according to the manufacturers’ instructions.

STATISTICAL ANALYSIS

Data obtained from the endophytic fungal extract experiments were subjected to statistical analyses, including descriptive statistics and graphical presentations using Excel software (Microsoft Office-XP Package 2002). The results of the bioassays are mentioned as the mean value ± standard error of mean. Comparisons were carried out inside treatments with the analysis of variance (ANOVA).). The mean values obtained for the different groups were compared using one-way ANOVA followed by Duncan's multiple ranges (Duncan, 1955), and the least significant difference test was used to determine the significant difference between the means of each parameter. Student's t-test was used for some parameters. The obtained results are expressed as means ± S.E.M.

 
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