Endophytic fungi associated with Taxus fuana (West Himalayan Yew) of Pakistan: potential bio-resources for cancer chemopreventive agents

Abstract

Context: Endophytic fungi, being a prolific source of bioactive secondary metabolites, are of great interest for natural product discovery.

Objective: Isolation and partial characterization of endophytic fungi inhabiting the leaves and woody parts of Taxus fuana Nan Li & R.R. Mill. (Taxaceae) and evaluation of biological activity.

Materials and methods: Endophytic fungal isolates were identified by molecular analysis of internal transcribed spacer (ITS) regions of 18S rDNA. Extracts of the endophytic fungi cultured on potato dextrose agar and modified medium were evaluated using cancer chemoprevention bioassays [inhibition of TNF-α-induced NFκB, aromatase and inducible nitric oxide synthase (iNOS); induction of quinone reductase 1 (QR1)] and growth inhibition with MCF-7 cells.

Results: Nine of 15 fungal isolates were identified as belonging to Epicoccum, Mucor, Penicillium, Chaetomium, Paraconiothriym, Plectania or Trichoderma. Five of the 15 extracts inhibited NFκB activity (IC₅₀ values ranging between 0.18 and 17 μg/mL) and five inhibited iNOS (IC₅₀ values ranging between 0.32 and 12.9 μg/mL). In the aromatase assay, only two isolates mediated inhibition (IC₅₀ values 12.2 and 10.5 μg/mL). With QR1 induction, three extracts exhibited significant activity (concentrations to double activity values ranging between 0.20 and 5.5 μg/mL), and five extracts inhibited the growth of MCF-7 cells (IC₅₀ values ranging from 0.56 to 17.5 μg/mL). Six active cultures were derived from woody parts of the plant material.

Conclusion: The endophytic fungi studied are capable of producing pharmacologically active natural compounds. In particular, isolates derived from the wood of Taxus fuana should be prioritized for the isolation and characterization of bioactive constituents.

Keywords:

• Aromatase
• endophytes
• growth inhibition
• iNOS
• NFκB
• phylogenetics
• quinone reductase

Introduction

Natural products have long been hailed as candidates for designing drugs against various diseases. Over the years, many researchers have investigated the biological potential of ethno-botanical plants (Haq et al. 2013). Recent investigations have focused on endophytic micro-organisms that reside asymptomatically within the living tissues of higher plants (Joseph & Priya 2011). Endophytes have yielded a broad range of unique structures such as alkaloids, benzopyranones, quinones, flavonoids, phenolic acids and quinines (Pimentel et al. 2010). These secondary metabolites demonstrate various activities and are employed as antibiotics, immunosuppressants, antioxidants, antivirals, antidiabetics or anticancer agents (Gunatilaka 2006). In principle, endophytes provide a cost-effective and renewable source for such valuable natural products.

Paclitaxel (Taxol®) and its derivatives are forerunners of the first major group of anticancer agents produced by endophytes. Taxol interferes with the cell cycle by stabilizing microtubule formation and thus inhibits the proliferation of cancer cells (Cheewarat 2006). The isolation of taxol from the Yew prompted the screening of associated endophytes for the same chemical entity. These efforts turned fruitful with the discovery of a novel taxol-producing endophytic fungus, Taxomyces andreanae, from Taxus brevifolia (Stierle et al. 1993). Subsequently, endophytes have been investigated widely for anticancer agents (Strobel & Daisy 2003; Zhao et al. 2011).

Carcinogenesis is a multistage process involving multiple events including initiation, promotion and progression that ultimately culminate in tumourigenesis (Pezzuto et al. 2005). Therefore, any substance that can halt or reverse these stages is a potential chemopreventive agent (Kinghorn et al. 2004). Terrestrial plants have been widely evaluated for the discovery of cancer chemopreventive agents (Park & Pezzuto 2002; Kondratyuk & Pezzuto 2004). Included among these are plants from the Himalayan region of Pakistan (Haq et al. 2013), where there is a tremendous biodiversity of medicinal plants.

Taxus fuana Nan Li & R.R. Mill (Taxaceae) is an important medicinal plant found in the West Himalayan region of Pakistan. Endophytes associated with Taxus species have been studied for the isolation of potential chemopreventive agents which can target specific pathways in metastasis by inhibiting protein kinases and NFκB (Kondratyuk & Pezzuto 2004; Aly et al. 2010). However, to the best of our knowledge, there is no previous report describing the functional diversity and cancer chemopreventive potential of endophytes associated with Taxus fuana from this region. We have therefore performed such studies with indigenous endo-microflora to establish a cornerstone for isolating lead compounds for drug development.

Materials and methods

Collection of plant sample

Healthy and mature plants of Taxus were selected for sample collection from Nathiagali (District Hazara, Khyber Pakhtunkhwa, Pakistan). Leaves and woody parts (stem and bark) were collected randomly for study. The plant material was brought to the laboratory in sterile bags under cold conditions for further processing. The plant was initially identified at the Department of Plant Sciences, Quaid-i-Azam University, by Prof. Dr. Mir Ajab Khan, as Taxus baccata after comparison with a preserved voucher specimen no. 2978-ZB (accession no. 52035) in the Herbarium of Pakistan. Later, however, it was taxonomically identified as Taxus fuana as described by Shah et al. (2008).

Isolation of endophytes

Isolation of the endophytes was carried out by the surface sterilization method (Petrini 1986). The material was thoroughly washed using distilled water followed by treatment with 70% ethanol for 2 min and 5% sodium hypochlorite for 5 min to accomplish surface sterilization. It was again rinsed with sterile distilled water and finally blotted with sterile blotting paper. The process of sterilization was evaluated by the imprinting method prior to plating (Schulz et al. 1993). Next, the samples were cut into 5–6 pieces (2–6 mm in size) and placed on water agar plates (distilled water, 1.5% agar) and potato dextrose agar (PDA Oxoid) plates supplemented with penicillin G (100 U/mL) and streptomycin (100 μg/mL). Plates were incubated at 28 °C for 2–4 weeks until growth was initiated (Hijri et al. 2002). The hyphal tips emerging from the plant part were selected, purified and maintained on PDA plates for further studies.

Taxonomic identification of fungal isolates

Taxonomic identification of selected endophytic fungi was conducted by analyzing the internal transcribed spacer (ITS) region of the 18S subunit of ribosomal DNA (rDNA) sequence using PCR. DNA was extracted from fungal mycelia by the CTAB method (Paul et al. 2007). A pair of universal primers ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) was used to amplify the target genes. PCR was carried out in a programable MJ Mini^TM gradient thermal cycler (Bio-Rad Laboratories Inc., Berkeley, CA). The reaction mixture (25 μL) contained 5 μL of DNA template, 3 μL of 25 mM MgCl_2, 100 μM of each dNTP, 25 pM of each primer and 1 U of Taq DNA polymerase. All the primers, dNTPs and enzymes were purchased from Fermentas (Fermentas, Thermo Scientific, Waltham, MA). The amplification conditions involved: preheating at 95 °C for 5 min; followed by 35 cycles with a denaturation step at 95 °C for 30 s; annealing step at 55 °C for 1 min; and an extension step at 72 °C for 1 min, followed by final extension at 72 °C for 6 min (Ferrer et al. 2001). The identified sequences were deposited in the NCBI GenBank and accession numbers obtained.

Cultivation of fungi for production of metabolites

Selected isolates were cultivated on potato dextrose agar (PDA) purchased from Oxoid^TM (Oxoid, Basingstoke, UK) and modified taxol medium (TM) according to (Xu et al. 2006). The TM medium contained (g/L): sucrose, 40; phenylalanine, 0.01; peptone, 0.5; yeast extract, 0.8; (NH₄)₂SO₄, 3.0; MgSO₄·7H₂O, 0.5; KH₂PO₄, 2.0; NaCl, 0.6; CH₃COONa, 0.5; C₆H₅COONa, 0.1; agar, 15.0. A mycelial disc of 8 mm diameter, from a freshly grown colony of fungal isolate, was transferred aseptically to the center of the plates and incubated at 25 °C at 150 rpm for 21 d. After incubation, mycelia along with the agar was blended three-times with an equal volume of ethyl acetate and left overnight on a shaker. The organic solvent was pooled by filtration and concentrated to dryness using a rotary evaporator. A stock solution (5 mg/mL) was prepared in dimethyl sulphoxide (DMSO) for biological screening.

Biological evaluation of extracts

Ethyl acetate extracts obtained from 15 fungal strains were evaluated for biological potential using a panel of in vitro assays.

Inhibition of TNF-α-induced NFκB activity

This assay was performed using 293/NFκB-Luc HEK cells (Labvision Corp, Fremont, CA) as reported earlier (Haq et al. 2013). NFκB activity was measured with a luciferase kit (Promega Corporation, Madison, WI) using a LUMIstar Galaxy Luminometer (BMG Labtechnologies, Durham, NC) according to instructions of the manufacturer. Data were calculated as % inhibition relatively to DMSO control. Samples which showed more than 60% inhibition at the test concentration 20 μg/mL were tested for dose-dependence to determine IC₅₀ values (Hoshino et al. 2010). Three NFκB inhibitors were used as positive controls: N-tosyl-l-phenylalanylchloromethyl ketone (TPCK), IC₅₀=3.8 ± 0.6 μM; (E)-3-(4-methylphenylsulfonyl)-2-propenenenitrile (BAY-11), IC₅₀=2.0 ± 0.33 μM; resveratrol, IC₅₀=2.5 ± 0.3 μM. All chemicals were purchased from BD Biosciences, San Jose, CA. Sulphorhodamine B (SRB) cytotoxicity tests were performed in parallel to avoid false-positive results.

Inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine macrophage RAW 264.7 cells

In this assay, potential to inhibit inducible nitric oxide synthase (iNOS) was assessed with lipopolysaccharide (LPS)-activated murine macrophage RAW 264.7 cells as described earlier (Haq et al. 2013). Samples showing more than 70% inhibition at a concentration 20 μg/mL were tested at three-fold serial dilutions to find the IC₅₀ values. NG-Monomethyl-L-arginine, monoacetate salt (L-NMMA) was used as a positive control (IC₅₀=19.7 μM; BD Biosciences, San Jose, CA). Sulphorhodamine B (SRB) cytotoxicity tests were performed in parallel to avoid false positive results.

Aromatase inhibition

Inhibition of aromatase was determined via dealkylation of dibenzylfluorescein as reported previously (Maiti et al. 2007). Fluorescence was measured at 485 nm (excitation) and 530 nm (emission). The experiment was performed in duplicate by using five concentrations of test substance to determine IC₅₀ values. Naringenin (IC₅₀=0.23 μM; BD Biosciences, San Jose, CA) was used as a positive control.

Quinone reductase 1 (QR1)

This assay was performed using Hepa 1c1c7 (murine hepatoma) cells as described earlier (Haq et al. 2013). Quinone reductase activity was measured as a function of the NADPH-dependent menadiol-mediated reduction of 3-(4,5-dimetylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Generation of blue formazan was determined by measuring absorption at 595 nm (Song et al. 1999). Protein content was determined via crystal violet staining of identical plates. Specific activity was defined as nmol of formazan formed per mg protein per min. The induction ratio (IR) was determined at a fixed concentration of 20 μg/mL and defined as the activity obtained in the presence of the test substance divided by the activity in the absence of the test substance. If the IR was approximately 2 or greater, samples were tested in five-fold serial dilutions to find CD values, i.e., the concentration of test sample required to increase activity by 2-fold. 4′-Bromoflavone [concentration required to double activity (CD) = 0.01 μM; BD Biosciences, San Jose, CA] was used as a positive control. A total protein assay using crystal violet stain was performed simultaneously to test the cytotoxic effects of the samples (Haq et al. 2013).

DPPH-free radical scavenging

Free radical scavenging activity was determined using 2,2-diphenyl-1-picryl-hydrazyl (DPPH) as described by Lee et al. (1998). Absorbance was measured at 515 nm using a microplate reader; the highest final concentration of the tested sample was 200 μg/mL. Samples showing more than 70% scavenging activity were further tested to determine IC₅₀ values. Ascorbic acid (IC₅₀=35.6 μM; BD Biosciences, San Jose, CA) and pure DMSO were used as positive and negative controls, respectively. The test was performed in triplicate and IC₅₀ values were calculated. where “Ac” is the absorbance of control and “As” is the absorbance of the test sample.

Growth inhibition with cultured MCF-7 cells

The cytotoxic potential of test substances with MCF-7 cells was determined with sulphorhodamine B (SRB) as described previously (Haq et al. 2013). Cells were seeded in 96-well plates (10⁶ cells/mL), and various concentrations of samples dissolved in 10 μL of 10% DMSO were added to each well. A zero-day control was used in each case. The IC₅₀ values were determined as the concentration of sample required to inhibit the 72 h growth of cells by 50%, relative to a control treated with solvent (0.5% DMSO) only. Paclitaxel (BD Biosciences, San Jose, CA: percentage purity <97%) was used as a positive control. Percent of cell survival was calculated using the formula: (1) where 'OD' is the optical density at 515 nm.

Statistical analysis

The data, in general, are presented as mean ± SEM of triplicate experiments. The least significant differences (LSD) between groups were determined by analysis of variance (ANOVA) and all pairwise comparison tests using statistical software (Statistix 8.1, SPSS Inc., Chicago, IL). *p < 0.05 and **p < 0.001 were set as significance criteria.

Results

A total of 15 endophytic fungi, 10 from wood and five from leaves, were isolated from Taxus fuana (Figures 1 and 2). The preliminary identification of the endophytic fungi was carried out on the basis of macro-morphological features and the in vitro growth pattern on potato dextrose agar. It was further confirmed by molecular analysis of 18S ribosomal DNA and isolates were assigned to different genera. A total of nine isolates were identified at the genus level by comparing percentage homology with closely related genera available at NCBI GenBank (Table 1). Most of the isolates belong to division Ascomycota, with the genus Epicoccum being the most frequent.

Figure 1. Isolation and macro-morphological characteristics of endophytic fungi isolated from the wood of Taxus fuana.

Figure 2. Isolation and macro-morphological characteristics of endophytic fungi isolated from the leaves of Taxus fuana.

Table 1. Percent homology and accession numbers of the ITS region of 18S rDNA nucleotide sequences of endophytic fungi.

Region of isolation	Strain code	GenBank accession number^a	Closest related species	% Similarity	Colony morphology
Wood	NFW3	JX838792.1	Epicoccum nigram EpNi1 [Accession: JQ387578.1]	98%	Redish pink colony
	NFW5	JX845570.1	Tritarichium sp. DFFSCS032 [GenBank: JX156380.1]	94%	Off white flat colony
	NFW6	JX845571.1	Mucorhiemalis MH1 [GenBank: JF303856.1]	94%	Light brown mat with thread like structures
	NFW7	JX838793.1	Epicoccum sp. XAE 132 [GenBank: JN031014.1]	98%	Peach pink colony
	NFW8	KC797170	Chaetomiumsp. ATT039 [GenBank: HQ607810.1]	99%	Dark pink colony
	NFW9	KF597019	Penicillium sp. PX2011a [Accession: JN226993.1]	97%	Greenish colony
Leaf	NFL1	KC812764	Plectaniamilleri [GenBank: GQ377485.1]	94%	White colony with mesh like mat
	NFL2	JX838791.1	Trichodermaasperellum T77 [GenBank: GU176454.1]	96%	Flat, dark green mat
	NL6	KC797169	Paraconiothyrium sp. [GenBank: HQ999974.1]	98%	Flat white colony with black patches

a Sequences corresponding to fungal isolates were recorded as being from Taxus baccata but the source material studied herein was later taxonomically identified as Taxus fuana as described by Shah et al. (2008).

To evaluate the cancer chemopreventive potential of the selected endophytes, a panel of assays was utilized. In the TNF-α-induced NFκB inhibition assay, a total of five samples (NFW1, NFW3, NFW8, NFW9 and NFL1) demonstrated more than 60% inhibition at a concentration of 20 μg/mL, with IC₅₀ values ranging from 0.18 to 17 μg/mL (Table 2). Among the leaf isolates, only one strain, NFL1, showed strong inhibition of 72.0% in TM medium; the IC₅₀ value for the extract was 5.8 μg/mL.

Table 2. Biological activity mediated by extracts derived from endophytic fungi.

		NFκB^b		iNOS^c		Aromatase^d		QR1^e		DPPH^f		SRB^g
Isolate code	PM^a	% Inhibition	IC50 (μg/mL)^h	% Inhibition	IC50 (μg/mL)	% Inhibition	IC50 (μg/mL)	IR	CD^i (μg/mL)	% scavenging	IC50 (μg/mL)	% survival	IC50 (μg/mL)
NFW1	TM^j	99.1 ± 2.0	0.18 ± 0.0	99.7 ± 2.3	0.32 ± 0.1	40.6 ± 1.8	–^k	NT^l	–	5.1 ± 1.0	–	0.00 ± 0.2	0.56 ± 0.1
	PDA^m	99.6 ± 2.2	1.05 ± 0.0	98.3 ± 2.1	0.33 ± 0.0	40.7 ± 1.9	–	NT	–	8.6 ± 2.0	–	16.6 ± 2.8	6.82 ± 0.7
NFW3	TM	66.9 ± 1.7	0.27 ± 0.1	69.7 ± 1.8	7.8 ± 0.8	73.3 ± 1.4	12.2 ± 1.2	2.6 ± 2.0	5.5 ± 0.5	88.8 ± 0.4	11.7 ± 2.0	85.5 ± 2.5	–
	PDA	39.2 ± 1.6	–	−3.70 ± 0.2^n	–	16.6 ± 1.3	–	0.8 ± 0.2	–	NT	–	47.9 ± 3.0	–
NFW4	TM	20.0 ± 1.8	–	5.70 ± 0.0	–	9.34 ± 1.2	–	NT	–	NT	–	NT	–
	PDA	47.6 ± 1.9	–	−7.10 ± 0.1	–	NT	–	0.7 ± 0.1	–	34.4 ± 2.0	–	83.8 ± 4.0	–
NFW5	TM	21.3 ± 2.3	–	6.3 ± 0.5	–	12.6 ± 0.9	–	1.0 ± 0.2	–	3.7 ± 1.0	–	100.0 ± 2.0	–
	PDA	−0.50 ± 0.0	–	NT	–	NT	–	0.10 ± 0.0	–	NT	–	NT	–
NFW6	TM	32.5 ± 1.6	–	52.6 ± 2.5	9.9 ± 1.3	20.8 ± 2.4	–	1.9 ± 0.4	20.4 ± 1.4	90.2 ± 0.1	53.9 ± 8.1	97.0 ± 5.0	–
	PDA	22.8 ± 1.4	–	NT	–	NT	–	NT	–	1.9 ± 1.0	–	NT	–
NFW7	TM	52.5 ± 2.5	–	52.4 ± 2.1	12.9 ± 1.4	20.6 ± 2.3	–	2.5 ± 1.0	0.5 ± 0.1	86.7 ± 0.3	30.8 ± 1.0	87.3 ± 3.5	–
	PDA	5.0 ± 2.3	–	NT	–	NT	–	NT	–	NT	–	NT	–
NFW8	TM	25.6 ± 1.4	–	−5.90 ± 1.0	–	18.1 ± 1.8	–	0.7 ± 0.2	–	4.1 ± 1.3	–	100.0 ± 1.7	–
	PDA	63.8 ± 4.5	17.0 ± 0.2	36.9 ± 2.1	–	22.9 ± 1.8	–	1.8 ± 0.5	–	27.0 ± 1.0	–	30.2 ± 1.3	12.4 ± 0.9
NFW9	TM	41.8 ± 1.2	–	–45.3 ± 2.1	–	22.7 ± 1.4	–	0.9 ± 0.3	–	7.6 ± 2.0	–	83.9 ± 1.7	–
	PDA	61.4 ± 1.3	5.4 ± 0.5	−38.9 ± 0.5	–	76.4 ± 1.6	10.5 ± 1.2	1.3 ± 0.3	–	84.4 ± 0.4	50.1 ± 0.9	46.2 ± 3.5	17.5 ± 1.1
NFW10	TM	32.2 ± 2.3	–	6.0 ± 1.9	–	21.4 ± 1.2	–	1.0 ± 0.2	–	3.5 ± 1.0	–	100.0 ± 2.1	–
	PDA	NT	–	NT	–	NT	–	NT	–	NT	–	NT	–
NFW12	TM	29.0 ± 1.9	–	−14.9 ± 1.0	–	14.1 ± 1.4	–	1.0 ± 0.1	–	4.4 ± 1.4	–	83.6 ± 1.1	–
	PDA	NT	–	NT	–	NT	–	NT	–	NT	–	NT	–
NFL1	TM	72.0 ± 2.5	5.8 ± 1.1	84.2 ± 2.3	4.4 ± 0.5	14.6 ± 1.5		6.9 ± 1.3	0.2 ± 0.1	11.8 ± 2.0		45.7 ± 3.8	11.3 ± 0.8
	PDA	22.6 ± 2.1		0.0 ± 0.5		12.7 ± 1.2		1.1 ± 0.3		15.3 ± 2.0		100.0 ± 1.2
NFL2	TM	21.7 ± 1.2		−12.2 ± 1.7		9.8 ± 1.3		0.8 ± 0.2		8.6 ± 1.6		100.0 ± 1.9
	PDA	11.9 ± 0.9		−12.8 ± 3.1		16.7 ± 1.4		0.6 ± 0.4		9.3 ± 2.0		100.0 ± 3.1
NFL3	TM	NT		NT		NT		NT		NT		NT
	PDA	17.2 ± 1.2		−15.2 ± 2.0		25.0 ± 3.3		1.3 ± 0.2		13.0 ± 2.0		100.0 ± 2.1
NFL5	TM	20.8 ± 1.2		−15.7 ± 2.0		17.6 ± 1.2		0.8 ± 0.4		10.6 ± 1.9		100.0 ± 1.2
	PDA	22.8 ± 3.1		−11.1 ± 1.0		14.6 ± 1.6		1.0 ± 0.1		−1.19 ± 0.0		100.0 ± 1.9
NFL6	TM	36.4 ± 2.1		−6.0 ± 2.1		23.3 ± 3.2		1.5 ± 0.7		13.1 ± 2.0		100.0 ± 1.7
	PDA	26.9 ± 2.1		−25.2 ± 2.3		21.1 ± 1.4		1.1 ± 0.2		9.9 ± 1.9		100.0 ± 1.6

a PM, production media.

b 100% activity observed with TNF-α as activator; % inhibition when tested at 20 μg/mL. Based on the SRB assay, overt toxicity was not observed with these experimental conditions. TPCK was used as a positive control with an IC₅₀ value of 3.8 ± 0.6 μM.

c 100% activity observed with LPS as activator; % inhibition when tested at 20 μg/mL. Based on the SRB assay, overt toxicity was not observed with these experimental conditions. L-NMMA was used as positive control with an IC₅₀ value of 25.1 ± 2.3 μM.

d Percent inhibition was determined at 20 μg/mL. Naringenin was used as a positive control showing an IC₅₀ value of 0.23 ± 0.3 μM.

e The induction ratio (IR) of QR represents the specific enzyme activity of agent-treated cells compared with DMSO-treated control.

f Percent activity for scavenging DPPH free radical. Ascorbic acid was used as positive control with an IC₅₀ value of 3.8 ± 0.6 μM.

g Cytotoxicity using the SRB method with MCF-7 cells in culture; percent inhibition was determined at 20 μg/mL. Taxol was used as positive control with an IC₅₀ value of 0.0029 μM.

h IC₅₀ is the concentration required for 50% inhibition.

i CD is the concentration required to increase activity by 2-fold (μg/mL).

j TM is the modified Taxol medium (Xu et al. 2006).

k Not applicable since % inhibition at 20 μg/mL is less than 50%.

l NT, not tested.

m PDA, potato dextrose agar (Oxoid, UK).

n Negative values indicate growth was greater than the control value.

In the LPS-induced NO inhibition assay, five isolates (NFW1, NFW3, NFW6, NFW7 and NFL1) showed iNOS inhibition greater than 50% at a test concentration 20 μg/mL. The samples showing ≥70% inhibition and ≥70% cell survival at this concentration were selected for dose-dependence to determine IC₅₀ values. Among positive strains, NFW1, NFW3 and NFL1 showed the highest activity with percentage inhibition of 99.6, 69.7 and 84.2% with corresponding IC₅₀ values of 0.32, 7.8 and 4.4 μg/mL, respectively (Table 2).

In the present study, we used the MCF-7 breast cancer cell line to evaluate cytotoxic potential of fungal endophytes. Five samples, i.e., crude extracts of NFW1 (both media), NFW8, NFW9 and NFL1, demonstrated cytotoxicity against MCF-7 cells with percentage survival in the range of 0.00–45.7 and corresponding IC₅₀ values of 0.56–17.5 μg/mL. The crude extract of the wood isolate NFW1 demonstrated strong cytotoxicity [% survival = 0.00 (TM extract) and 16.6% (PDA extract)] with IC₅₀ values of 0.56 and 6.82 μg/mL when grown in TM and PDA media, respectively. One of the leaf isolates, NFL1, also showed cytotoxicity towards the MCF-7 cell line with 45.7% survival in TM media and a corresponding IC₅₀ value of 11.3 μg/mL.

In the aromatase inhibition assay, two isolates, NFW3 and NFW9, demonstrated 73.3 and 76.4% inhibition with corresponding IC₅₀ values 12.2 and 10.5 μg/mL, respectively. Similarly, in the quinone reductase induction assay, it was found that three of the isolates, namely NFW3, NFW7 and NFL1, exhibited significant activity showing induction ratios (IR) of 2.6–6.9 with concentration to double activity (CD) values in the range of 0.2–5.5 μg/mL (Table 2). NFW6 was weakly active with a CD value of 20.4 ± 1.4 μg/mL.

In the DPPH assay for free radical scavenging potential, four samples (NFW3, NFW6, NFW7 and NFW9) showed ≥70% scavenging activity at a test concentration of 200 μg/mL (Table 2). The corresponding IC₅₀ values ranged from 11.7 to 53.9 μg/mL. The most potent activity was observed with NFW3 (an IC₅₀ value of 11.7 μg/mL). Other strains showed low levels of activity, in the range of 3-34% scavenging at a test concentration of 200 μg/mL.

Discussion

Endophytic fungi are considered a valuable source for isolating novel bioactive compounds. In the present study, we isolated 15 endophytic fungi from the medicinal plant Taxus fuana and nine were identified. Most of the isolates belonged to division Ascomycota. Among the wood isolates, NFW3 and NFW7 were identified as the genus Epicoccum. Epicoccum species are often isolated as a major crop endophyte (Martini et al. 2009; Stuart et al. 2010; Fávaro et al. 2012). As reported previously, Epicoccum has the capability of producing tetramic acid derivatives (epiconigine and epicoccamide) and diketopiprazine, and macrolides (Wright et al. 2003; Wangun et al. 2007).

The strain NFW5 was identified as Tritirachium sp. Recently, an endophytic Tritirachium species was also isolated from Quercus pannosa and Rhododendron spp. collected from the Baima Snow Mountain, Southwest China (Li et al. 2012). Macrosphelides macrolide polyketides, exhibiting antitumour activity, have been reported from Tritirachium fungus (Ivanova et al. 2007). However, none of the previous studies report it as an endophyte associated with the Taxus plant species. Consistent with earlier reports from Taxus chinensis (Zhou et al. 2009; Tayung & Jha 2010), we also identified the NFW6 isolate as a strain of Mucor hiemalis. NFW8 was identified as Chaetomium which resides within various plants such as Curcuma wenyujin, Viguiera robusta and Taxus globosa (Momesso et al. 2008; Wang et al. 2010; Soca-Chafre et al. 2011). The genus Chaetomium has great potential to produce bioactive metabolites including chaetoviridins, which exhibit tumour inhibition and antifungal activities (Park et al. 2005; Borges et al. 2011). NFW9 was identified as Penicillium polonicum. Various Penicillium species have been reported to produce anthraquinones, alkaloids, deketopiprazines and steroids possessing antitumour, antioxidant and antimicrobial potential (Nicoletti et al. 2008).

Among the leaf isolates, NFL1 and NFL2 were identified as Plectania and Trichoderma spp., respectively, which have been reported as endophytes from Taxus plants (Liu et al. 2009; Zhao et al. 2010). The Trichoderma genus is also well known for its bioactive metabolites trichothecens, camptothecin (quinoline alkaloid), anthraquinones and lactones (Lu et al. 2012; Pu et al. 2013).

NFL6 was identified as Paraconiothyrium sp., which correlates with the findings of other researchers (Han et al. 2012; Khan et al. 2012). Previous reports indicate that Paraconiothyrium, an endophyte of the marine sponge Ectyplasia perox, can produce meroterpenoids (epoxyphomalins) exhibiting potent in vitro growth inhibitory activity against human tumour cell lines (Mohamed et al. 2009, 2010).

Fungi are considered a rich source of cancer chemopreventive and cytotoxic compounds. Metabolites from endophytes are used to target specific pathways, which consequently inhibit or regulate carcinogenesis and the cell cycle. Based on such a targeted approach, NFκB inhibition is a plausible mechanism for the treatment or prevention of cancer (Schupp et al. 2009; Luqman & Pezzuto 2010). Some fungal metabolites reported for potential to inhibit NFκB include trichodion, tricyclic acid, panepoxydone, hispidin derivatives, chaetoglobosin and mycoepoxydiene (Erkel 2000; Wijeratne et al. 2003; Erkel et al. 2007; Dou et al. 2011; Wu et al. 2011; Wang et al. 2012). In our study, six of the isolates presented more than 60% NFκB inhibition, suggesting their potential to produce cancer chemopreventive compounds.

Similarly, inducible nitric oxide synthase (iNOS) is consistently associated with chronic inflammation and tumourigenesis. During early tumour development, there is up-regulation of iNOS, suggesting its inhibition as a cancer chemopreventive strategy (Park & Pezzuto 2002; Nomelini et al. 2008). In the LPS-induced NO inhibition assay, five isolates showed more than 50% inhibition with IC₅₀ values ranging from 0.32 to 12.9 μg/mL. These results are consistent with the iNOS inhibitory potential of fungal metabolites such as radicicol, sporogen, S14-95, curvularin, azaphilones and lactone derivatives (Jeon et al. 2000; Yao et al. 2003; Quang et al. 2006; Yang et al. 2010; Lee et al. 2011).

Fungal metabolites known to exhibit cytotoxic activity include taxol, norsesquiterpene peroxides, cycloheptapeptides, lactones and chlorinated anthraquinones (Wang & Tang 2011; Chen et al. 2012; Huang et al. 2012). In previous work, one of these strains, NFW1 crude extract, showed overlapping peak distribution at a similar retention time indicating the presence of taxol and related moieties (Jadoon et al. 2015). This was further confirmed in that NFW1 extract showed taxol peaks with molecular ions m/z 854.82 (M + H)⁺, 870.80 (M + NH4)⁺ and 876.82 nm (M + Na)⁺in the LC-ESI-MS spectrum (Jadoon et al. 2015). During the current study, we tested the extracts and could not identify taxol using a Varian 500-MS ion trap mass spectrometer with an electrospray source (ESI). It is likely that fermentation conditions could be optimized to elicit taxol production, and work of this type is ongoing in Pakistan.

In any case, fungal isolates NFW1, NFW8, NFW9 and NFL1 mediated cytotoxicity against MCF-7 cells with percentage survival of 0.00–45.7% and corresponding IC₅₀ values of 0.56–17.5 μg/mL. These values are below the limit established by the National Cancer Institute (IC₅₀ value<20 μg/mL) (Lee & Houghton 2005).

Aromatase is a cytochrome P450 enzyme complex which converts androgens to estrogens (Jongen et al. 2005). Estrogens, being involved in important physiological processes, play a key role in diseases like endometrial and breast cancers (Maiti et al. 2007). Aromatase inhibitors block the production of estrogens and may be effective against estrogen receptor positive breast cancer. Although some agents are already used in the clinic, the search for more efficient aromatase inhibitors continues (Balunas & Kinghorn 2010). Fungal metabolites with azol rings act as inhibitors of different P450 enzymes including aromatase (Zhang et al. 2002). Various other fungal metabolites, such as monomeric xanthones, benzophenone, depsidones and diaryl ethers, also inhibit P450 enzymes (Krick et al. 2007; Chomcheon et al. 2009; Sureram et al. 2012). In this work, isolates NFW3 and NFW9 showed aromatase inhibition of 73.3 and 76.4% with IC₅₀ values of 12.2 and 10.5 μg/mL, respectively. The activity is rather modest but prior to conducting any additional work specificity for aromatase (CYP19) should be assessed, and activity should be reaffirmed in an assay that is not dependent on a fluorescent signal.

One of the strategies for protecting cells from cancer initiation events involves increasing phase II enzymes which can deactivate reactive species. Quinone reductase is an important phase II enzyme and plays an important role in the detoxification of certain reactive chemical species. It is widely distributed in mammalian cells and regarded as a convenient biomarker (Cuendet et al. 2006). Therefore, we studied the effect of endophytes on quinone reductase 1 (QR1) induction using cultured Hepa 1c1c7 (murine hepatoma) cells. Three of the isolates, namely NFW3, NFW7 and NFL1, exhibited significant induction levels, showing induction ratios (IR) of 2.6–6.9 and concentration to double activity (CD) values of 0.20–5.5 μg/mL, respectively. These findings are comparable with the quinone reductase induction potential of two xanthone derivatives from the marine algicolous fungus Monodictys putredinis showing CD values 22.1 and 24.8 μM, respectively (Pontius et al. 2008).

The discovery of novel and safe antioxidants from natural products to combat or prevent diseases is a continuing process. Some compounds suppressing superoxide generation and showing antioxidant activity are associated with cancer chemoprevention (Krick et al. 2007). To determine the free radical scavenging potential of our samples, the DPPH assay was performed. Four fungal extracts, NFW3, 6, 7 and 9, showed significant scavenging activity. The most potent activity was observed with NFW3 (an IC₅₀ value of 11.7 μg/mL). Antioxidant potential has been reported previously with organic extracts of other fungal endophytes such as Phomopsis, Xylaria and Colletotrichum (Joseph & Priya 2011; Tianpanich et al. 2011; Ravindran et al. 2012; Wu et al. 2013). In addition, Nitya et al. (2011) reported DPPH scavenging activities with methanolic extracts of Fusarium, Aspergillus, Penicillium and Mucor, with IC₅₀ values ranging between 200 and 325 μg/mL.

Conclusion

It is well established that endophytes are capable of producing remarkable cancer chemotherapeutic agents (Tayung & Jha 2010; Cragg & Pezzuto 2015). This investigation of biological activities mediated by endophytic fungi isolated from Taxus fuana indicates their diverse potential for the generation of bioactive metabolites. Most of the wood isolates (NFW1, NFW3 and NFW9) displayed significant cancer chemopreventive activity in more than one assay which suggests the production of pharmaceutically relevant biochemical entities, especially when cultured in TM mineral salt medium. This supports the value of these isolates for future studies to isolate and identify active principle candidates for targeted drug development.

Funding information

Funding was provided by the Higher Education Commission of Pakistan under the “International Research Support Initiative Program.”

Acknowledgements

The authors acknowledge Prof. Dr. Mir Ajab Khan and Dr. Mushtaq Ahmad of the Department of Plant Sciences, Quaid-i-Azam University, Islamabad, for their help with plant collection and identification.

Disclosure statement

The authors report that they have no conflicts of interests.