Pueraria thomsonii Benth-Associated Endophytes: A Multifaceted Exploration of Its Bioactive Molecules

Abstract

Endophytes associated with medicinal plants hold substantial promise as a novel source for therapeutics development. In this study, 11 endophytic fungi and 9 endophytic bacteria were isolated from the roots of Pueraria thomsonii. In the preliminary screening, the ethyl acetate extract of the Apiospora marii FG-Z21 displayed strong inhibitory effects against drug-resistant bacteria Enterococcus faecalis and Methicillin-resistant Staphylococcus aureus (MRSA) with MIC values of 1.563 and 0.098 mg/mL, respectively. Furthermore, this extract exhibited significant inhibitory effects on A549 cells with a GI₅₀ value of 0.036 mg/mL and demonstrated a good antioxidant property with an EC₅₀ value of 1.869 mg/mL. Further investigation of the active ethyl acetate extract of A. marii FG-21 led to the isolation of 4-hydroxybenzoic acid (1), 2-acetamidobenzoic acid (2), benzoic acid (3), dibutyl phthalate (4), 3,4-dimethoxybenzoic acid (5), schizostatin (6), ethyl pyroglutamate (7), and anthranilic acid (8). Notably, compounds 3, 1 and 2 exhibited significant cytotoxicity against A549 cells with GI₅₀ values ranging from 4.97 to 83.08 µg/mL. On the other hand, compounds 4, 5 and 6 demonstrated potent inhibition of E. faecalis growth with low MIC values ranging from 2 to 4 µg/mL. Our study provides a comprehensive exploration of P. thomsonii-derived endophytes and their compounds highlighting their significant bacteriostatic, antioxidative, and cytotoxic activities that may potentially contribute to drug discovery paradigms.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s12088-024-01368-6.

Introduction

Antimicrobial resistance (AMR) has emerged as a critical global health threat in the twenty-first century with alarming projections estimating that will cause 4.95 million deaths annually. By 2050, this number is expected to rise to 10 million people per year [1]. Among the pathogens contributing to AMR-related fatalities, the top six are Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Collectively, these pathogens were responsible for 929,000 deaths attributed to AMR and a staggering 3.57 million deaths associated with AMR in 2019 [2].

In this dire context, multidrug-resistant strains have become a central focus of surgical site infection prevention and antimicrobial resistance initiatives as they are the leading bacterial cause of death in 135 countries worldwide [3]. In fact, infection rates have escalated from 28.4% in 2016 to a troubling 42.6% in 2021 [4]. Additionally, the overuse and misuse of antibiotics have exacerbated the spread of AMR posing a looming threat of bacterial pathogens becoming even deadlier in the future. In light of this, the World Health Organization (WHO) has emphasized the imperative need for novel antibiotic development and the implementation of action plans to combat the relentless spread of AMR [5].

Similarly, there is an urgent need to discover novel compounds with anticancer properties as cancer is the second leading cause of death worldwide. It is responsible for nearly 10 million annual fatalities across the globe and accounts for 1 in 6 deaths globally [6, 7]. The projected economic burden of cancer on a global scale from 2020 to 2050 is a staggering $25.2 trillion in international dollars (at constant 2017 prices). This figure equates to an annual tax equivalent to 0.55% of the world’s gross domestic product. Currently, lung cancer is the leading malignancy in terms of incidence and mortality among cancers globally with an estimated 1.8 million deaths in 2018 (18.4% of total cancer deaths). The annual incidence rate is expected to increase continuously by 67% by 2040 [8]. Tobacco smoking dominates the lung cancer mortality landscape, with a minimum global age-adjusted death rate of 16.71 deaths/100,000. This accounts for 78% of males’ deaths and 53% of females’ deaths from 1990 to 2019. Ambient particulate matter pollution (APMP) and household air pollution (HAP) follow with death rates of 3.85 deaths/100,000 and 2.54 deaths/100,000, respectively [9, 10].

Natural products offer a diverse array of biologically active compounds with specific molecular structures suitable for interactions with biological targets. They have played a vital role in drug discovery contributing to nearly 50% of newly discovered therapeutics from 1981 to 2010, and approximately 75% of anti-infectives [11]. Endophytes, microorganisms residing within plant tissues at various stages of their life cycle represent a rich source of novel natural products that presents an opportunity for drug discovery [12]. Notably, recent research has unveiled that many endophytes produce secondary metabolites including polyketides, nonribosomal peptides, and isoprenoids with antibacterial and antitumor properties [13–20]. Furthermore, endophytes hold potential as antioxidants countering free radicals and contributing to overall health maintenance. This topic has garnered substantial interest among researchers in recent years [21, 22]. Pueraria thomsonii, a perennial medicinal plant has been traditionally used to prevent and treat various chronic ailments making it a valuable resource in herbal medicine [23]. However, it remained unexplored in terms of associated endophytes and their potential antibacterial, antioxidant, and cytotoxic properties. Therefore, this investigation sheds light on the untapped potential of P. thomsonii endophytes for drug discovery and therapeutic applications.

Materials and Methods

Plant Material and Isolation of Endophytes

Roots of P. thomsonii (Fig. S1) were obtained from the Chinese Medicinal Botanical Garden in Western Hubei Province, China (109° 45′ 24′′ E, 30° 10′ 51′′ N) which is an important region for P. thomsonii production. Briefly, the healthy plant roots were washed with sterile water. Under aseptic conditions, the plant materials were surface-sterilized by immersing them in 5 mL of 70% ethanol for 2 min, followed by 5-min soak in 5 mL of 4% sodium hypochlorite. They were then rinsed twice with 5 mL of sterile water to remove any traces of sodium hypochlorite and ethanol. The dried roots were finely powdered and inoculated onto two different media: Potato Dextrose Agar (PDA) supplemented with kanamycin at a final concentration of 50 µg/mL and Luria–Bertani (LB) medium containing ketoconazole at a final concentration of 25 µg/mL. The PDA plates were incubated at 25 °C for 7 days with daily monitoring for visible fungal growth. Fungal colonies were sub-cultured onto fresh PDA media to ensure purity. The LB plates were incubated at 37 °C for 2 days until bacterial colonies became visible and then bacterial strains were transferred to newly prepared LB plates for purification. The pure isolates were identified through phenotypic and genotypic analysis and they were maintained on appropriate agar and stored at 4 °C for subsequent studies [24].

Phenotypic and Genotypic Characterization of Endophytes

The endophytes were morphologically characterized using previously established methods with minor modifications [25]. In brief, the radial growth rate of fungal isolates was determined by measuring the slope of the linear regression of colony radius over time using the equation: r(t) = a + V × t, where ‘r(t)’ represents the colony radius in millimeters, ‘a’ is the linear regression constant, ‘V’ is the radial growth rate in millimeters per day, and ‘t’ is the cultivation time. Subsequently, cell morphology was examined under a microscope focusing on spore chain arrangements, hyphae, and mycelium structure. For bacterial isolates, Gram staining was performed using the Gram Stain Kit from Haibo Biotechnology Co., Ltd., Qingdao. Identification of the isolates was based on the analysis of highly conserved regions specifically the internally transcribed spacers (ITS) for fungal isolates and the 16S rRNA for bacterial isolates. These conserved regions were amplified using ITS 1 and ITS 4 primers for fungi, and 27F and 1492R primers for bacteria and the PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd., for sequencing. The resulting DNA sequences were compared to reference sequences in the GenBank database and assigned unique accession numbers. Fungal and bacterial phylogenetic trees were constructed using the Neighbor-Joining method in MEGA-X 10.2.2 (https://www.megasoftware.net) based on the similarity of the ITS regions and 16S gene regions of rDNA. Phylogenetic analysis included closely related strains listed in Table S2.

Small-Scale Fermentation and Extraction of Endophytic Crude Extracts

The well-grown fungal and bacterial endophytes were inoculated into 250 mL conical flasks containing 50 mL of production medium such as potato dextrose broth (PDB) and Luria–Bertani broth (LB) for secondary metabolites production, respectively. The inoculated fungal flasks were kept on a rotary shaker for 14 days at 28 ± 2 °C and the bacterial flasks incubated at 37 ± 2 °C for 7 days [1, 26]. After incubation, the fermented broth along with cells were soaked with equal volume of petroleum ether followed by ethyl acetate. The mixtures were then subjected to ultra-sonication for 30 min before extraction. The fermented broth and mycelium were then freeze-dried at − 60 ± 2 °C and 80 Pa for 12 h to obtain the freeze-dried powders. The processed fermented broth and mycelium were soaked with an 10 × volume of methyl alcohol (v/m mL/g) at room temperature and ultra-sonicated for 30 min and then dried using a rotary evaporator under reduced pressure.

Test Microbes

The antibiotic activity of the endophytes was assessed using Gram-positive bacteria such as Bacillus subtilis ATCC 19659, Bacillus thuringiensis ATCC 10792, Enterococcus faecalis ATCC 29212, Micrococcus luteus ATCC 4698, Staphylococcus aureus ATCC 29213, Methicillin-resistant Staphylococcus aureus ATCC 43300 (MRSA), Methicillin-resistant Staphylococcus epidermidis ATCC 12228 (MRSE); Gram-negative bacteria such as Escherichia coli ATCC 25922, Klebsiella pneumonia ATCC 13883, Pseudomonas aeruginosa ATCC 9027 and Vibrio alginolyticus ATCC 17749. All the test bacteria were obtained from the Key Laboratory of Tropical Marine Biological Resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences. All the test strains were grown at 37 °C in Mueller–Hinton agar (OXOID, USA).

Antibacterial Assay

The antibacterial activity of the crude extracts from the endophytes was evaluated using a modified filter paper disc method [15]. Briefly, the sterile filter paper discs were impregnated with 20 μL of crude extract and placed on pre-coated bacterial plates (1 × 10^4–6 CFU/mL) of Mueller–Hinton agar. The positive control consisted of kanamycin at a concentration of 50 µg/mL, while the negative control consisted of DMSO. The plates were initially incubated at 4 °C for 12 h to allow the extract to diffuse into the medium and then incubated at 37 °C for 24 h. After the incubation period, the inhibition zones were measured and expressed in millimeters. The average inhibition zone was determined based on 5 replicates.

Minimum Inhibitory Concentrations (MIC) and Minimum Bactericidal Concentrations (MBC)

MIC and MBC of the potent endophyte extract were determined using a broth microdilution assay in 96-well plates following the method of Li et al. [1]. Briefly, a two-fold dilution of the extract was prepared with double-strength Mueller–Hinton broth (MHB) achieving concentrations ranging from 25 to 0.097 mg/mL for extracts and 100 to 1.0 μg/mL for compounds. After treatment the microtiter plate was incubated at 37 °C for 24 h, and microbial growth was assessed using a color change from yellow to purple after adding iodonitrotetrazolium chloride. To determine MBC, 50 μL diluted and tested microbial suspension (approximately 1 × 10^4–6 CFU mL⁻¹) was streaked onto MHB solid medium and incubated at 37 °C for 24 h in a constant temperature incubator. After the incubation period, the MBC was defined as the lowest concentration of endophyte extracts that killed > 99.9% of the initial bacterial population which viable bacteria were less than five on MHB solid medium.

Cell Line Maintenance and Cytotoxicity Assay

Human lung adenocarcinoma A549 cells (A549) were cultured in RPMI-1640 medium supplemented with 10% FBS, along with 1% antibiotic and antimycotic solution containing 1000 U/mL penicillin and 10 mg/mL streptomycin sulfate. These cells were maintained in a CO₂ incubator at 37 °C, with a controlled atmosphere of 5% CO₂ and 95% air at 90% relative humidity.

To evaluate their cytotoxicity, the extracts and compounds were tested on the on aforementioned cancer cell lines. These cells were seeded into a 96-well microtiter plate at a density of 5 × 10⁴ cells/well and treated with different concentrations of extracts and compounds, ranging from 5 to 0.01 mg/mL and 100 to 1.0 μg/mL, respectively. DMSO and cisplatin were included as the solvent and positive controls, respectively. Following 24 h of treatment, the cells underwent an additional 2 h incubation at 37 °C with a 10% Quanti-Blue solution (v/v). Cell viability was assessed by measuring OD values at 544 nm and 590 nm using a Spectronic Genesys 5 spectrophotometer [1].

Compound Isolation

To obtain sufficient crude extracts for compound isolation and structural elucidation, the active isolate FG-Z21 was mass produced using PDB at 28 °C with continuous shaking at a rate of 200 rpm for 14 days. After incubation, the cultures were soaked with an equal volume of ethyl acetate at room temperature and the resulting filtrate was subjected to drying using a rotary evaporator under reduced pressure to obtain black gum (6.40 g). The obtained extract was further fractionated using a silica gel chromatographic column eluting with solvents of increasing polarity of chloroform and methanol (CHCl₃/MeOH). This process yielded a total of 6 fractions (Fr–A1–Fr–A6), with similar fractions being combined based on their Thin-Layer Chromatography (TLC) profiles. Fr–A2 (425.78 mg) was subjected to further fractionation over a silica gel column chromatography to obtain 8 sub-fractions (Fr–B1–Fr–B8). Fr–B1 was subsequently purified through semi-preparative High-Performance Liquid Chromatography (HPLC) resulted in the isolation of compound 2 (6.40 mg) and compound 7 (8.30 mg). Similarly, Fr–B3 was purified by semi-preparative HPLC, leading to the isolation of compound 5 (10.70 mg) and compound 6 (6.80 mg). Fr–B4 was purified over semi-preparative HPLC and resulted in the isolation of compound 1 (4.80 mg). Based on the TLC profile, Fr–A4 (206.27 mg) was combined and further fractionated using reversed-phase silica gel column chromatography and yielded compound 3 (5.40 mg), compound 4 (11.60 mg), and compound 8 (8.70 mg).

Statistical Analysis

The statistical analysis results were presented as means ± standard deviations. To compare data from different groups, a one-way ANOVA was conducted using SPSS 19.0 software (http://www.spss.com). Subsequently, multiple comparison analyses were performed using the LSD test to assess variance homogeneity between the groups. Differences were deemed significant at a threshold of P < 0.05.

Results

Isolation and Identification of Pueraria thomsonii Associated Microbes

A total of 20 endophytic microorganisms were isolated and purified from P. thomsonii roots consisting of eleven endophytic fungi and nine endophytic bacteria. The isolates were characterized and identified based on the combination of morphological features and conserved molecular regions (Table 1 and Fig. 1). Furthermore, the evolutionary relatedness of the isolates clustered within various lineages including Trichocomaceae, Saccharomycetaceae, Didymosphaeriaceae, Schizophyllaceae, Tuberculariaceae, Phanerochaetaceae, Bionectriaceae, Diaporthaceae, Moniliaceae and Polyporaceace which were identified and named as Penicillium glabrum FG-Z4, Debaryomyces hansenii FG-Z5, Didymella macrostoma FG-Z6, Schizophyllum sp. FG-Z9, Fusarium oxysporum FG-Z12, Porostereum crissum FG-Z15, Bionectria ochroleuca FG-Z20, Apiospora marii FG-Z21, Clonostachys rosea FG-Z35, Phoma herbarum FG-Z36 and Trametes sp. FG-Z4 (Fig. 2A). Similarly, the phylogenetic analysis of the nine endophytic bacteria indicated their placement within the lineages of Enterobacteriaceae, Pseudomonadaceae, Staphylococcaceae, and Bacillaceae. These bacteria were identified and named as Enterobacter asburiae FG-X1, Rahnella aquatilis FG-X2, Pseudomonas jessenii FG-X4, Pseudomonas fragi FG-X8, Lelliottia sp. FG-X18, Pseudomonas oryzihabitans FG-X20, Pseudomonas rhodesiae FG-X35, Staphylococcus argenteus FG-X48 and Paenibacillus shenyangensis FG-X50 (Fig. 2B).

Table 1.

Morphological features of microbes associated with P. thomsonii

Isolates	Macroscopic observation on potato dextrose agar	Microscopic observation	Radial growth rate (mm/d)
FG-Z4	Velvet and sulcate colonies with green color	Highly branched and septate hyphae	0.984
FG-Z5	Grayish white and shiny colonies with soft and smooth surface	Cells were oval, conical or reniform	2.40
FG-Z6	Colonies were rosy-whitish to peach-colored and neat at margin	Conidia were regular-spherical, smooth and hyaline	4.04
FG-Z9	Colonies were round, flat, velvet-like and the edges were like snowflakes	Thin to dense hyphae with diaphragm and granular salience	12.64
FG-Z12	Pink colored colonies with fluffy aerial mycelium, irregular and margin	Mycelia were delicate, felt-like and funiculous	4.90
FG-Z15	Colonies were hyaline to light white, velvety texture and growth pattern was round with irregular margin	Clamp connections of mycelium were clearly visible	17.80
FG-Z20	Colonies were cottony, white, dense and irregular margins	Mycelium had many branches with small elliptical and ovate spores on the branchlets	4.31
FG-Z21	Colonies circular form, concentrically spreading with sparse aerial mycelium, margin entire	Mycelia hyaline to white colored	12.36
FG-Z35	Powdery yellowish-white colonies	Conidia formed on both verticillate and penicillate conidiophoresn and was globose to subglobose, smooth, hyaline	4.33
FG-Z36	The colonies were downy to woolly with a regular margin, greyish surface, with brown diffusible pigment	The hyphae were filamentous septate and hyaline to brown	2.93
FG-Z41	Colony had regular margin, white and penetrated the agar	Hyphae were branched, long hyphal segments and simple septate with scattered single clamp connections	2.13

	Macroscopic observation on Luria–Bertani Agar	Observation	Gram stain
FG-X1	Single colonies appeared oval, milky white, and smooth, with bright and slightly raised surfaces, and with moist neat edges	Thallus were rhabditiform	G−
FG-X2	Colonies were pale yellow to cream-colored, circular, convex with entire margin	Cells were short rods	G−
FG-X4	Colonies were smooth circular, yellow and slightly raised surfaces	Cells were asporogeneous and rod shaped motile	G−
FG-X8	Colonies appeared circle, slightly convex and pale yellow	Cells were coccus and shiny	G−
FG-X18	Colonies appeared round, slightly convex, opaque, smooth and beige	Cells were straight rod-shaped	G−
FG-X20	Colonies were circular, smooth-margined and yellow	Cells were rod-shaped	G−
FG-X35	Colonies were smooth, circular and milky white	Cells were motile, oval-shaped or rod shaped	G−
FG-X48	Colonies were round, even, regular, low convex, smooth, opaque, orange pigmented	Cells occurred in pairs, tetrads or clusters	G−
FG-X50	Colonies were light white to yellow, convex and circular, viscous, smooth	Cells were rod-shaped and non-motile	G−

Fig. 1.

Morphological features of P. thomsonii associated endophytes; colony of 20 isolates on Potato dextrose agar (PDA) and Luria–Bertani Agar, a1–a2 Macroscopic and microscopic images of Apiospora marii FG-Z21, b1–b2 Clonostachys rosea FG-Z35, c1–c2 Didymella macrostoma FG-Z6, d1–d2 Schizophyllum sp. FG-Z9, e1–e2 Fusarium oxysporum FG-Z12, f1–f2 Porostereum crissum FG-Z15, g1–g2 Bionectria ochroleuca FG-Z20, h1–h2 Penicillium glabrum FG-Z4, i1–i2 Debaryomyces hansenii FG-Z5, j1–j2 Phoma herbarum FG-Z36, k1–k2 Trametes sp. FG-Z41, l1–l2 Lelliottia sp. FG-X18, m1–m2 Pseudomonas rhodesiae FG-X35, n1–n2 Paenibacillus shenyangensis FG-X50, o1–o2 Pseudomonas oryzihabitans FG-X20, p1–p2 Enterobacter asburiae FG-X1, q1–q2 Staphlococcus argenteus FG-X48, r1-r2 Pseudomonas fragi FG-X8, s1s1–s2  s1-s2 Rahnella aquatilis FG-X2, t1–t2 Pseudomonas jessenii FG-X4

Fig. 2.

Evolutionary relatedness of endophytes obtained from tender roots of P. thomsonii and bootstrap values are shown at the branches (1000 replicates), A phytogenic trees of endophytic fungal isolates, B phytogenic trees of endophytic bacterial isolates

Biological Activities of the Endophytes

Antibacterial Activity

The preliminary screening indicated significant antibacterial activity of the endophytic extracts. About 85% of the ethyl acetate extracts showed broad spectrum of antibacterial activities against the tested seven Gram-positive bacteria and four Gram-negative bacteria followed by methanol and petroleum ether extracts (Fig. 3 and Table S1). Among them the ethyl acetate extract of A. marii FG-Z21 had a highly sensitive inhibitory effect on drug-resistant bacteria E. faecalis and MRSA with the highest zones inhibition of 23.76 ± 0.04 and 19.61 ± 0.16 mm, respectively (Fig. S2). Followed by the methanol extract of C. rosea FG-Z35 with inhibition zone of 16.66 ± 0.07 mm against E. faecalis and 17.21 ± 0.4 mm against MRSA. In addition, the methanol extract of S. argenteus FG-X48 and the ethyl acetate extract of F. oxysporum FG-Z12 displayed promising antibacterial activity against drug-resistant bacteria P. aeruginosa with inhibition zones of 18.15 ± 0.28 and 16.79 ± 0.71 mm, respectively. Meanwhile, the methanol extract of C. rosea FG-Z35 showed significant antibacterial activity against the Gram negative bacterial pathogens A. bammannii with inhibition zones of 18.98 ± 0.75 mm.

Fig. 3.

Parallel plot showing the in vitro antibacterial activity of endophytic extract on bacterial pathogens (M-methanol, E-ethyl acetate, P-petroleum ether)

Determination of MIC and MBC

Based on preliminary antibacterial activity of the active endophyte extracts were tested against selected pathogenic bacterial strains to determine their MICs and MBCs. The resulting MIC and MBC of the endophyte extracts against tested pathogenic bacteria are depicted in Table 2. Ethyl acetate extracts from 11 endophytes showed strong antibacterial activity against the tested strains with MIC values of 0.082 to 12.50 mg/mL followed by methanol extract of 7 endophytes ranging from 0.098 to 25.00 mg/mL and petroleum ether extract of 3 endophytes 0.391 to 6.250 mg/mL. Ethanol extracts of A. marii FG-Z21 and D. macrostoma FG-Z6 showed broad-spectrum antibacterial activity against 11 tested strains with MIC values ranging 0.391–7.125 mg/mL and 0.195–6.250 mg/mL, respectively and their MBC values were 0.195 to 14.25 mg/mL and 0.781 to 6.250 mg/mL. Furthermore, the ethyl acetate extract of D. macrostoma FG-Z6, F. oxysporum FG-Z12, P. crissum FG-Z15 and A. marii FG-Z21 showed good antibacterial activity against drug-resistant bacteria E. faecalis, MRSA, MRSE and A. bammannii with MIC of 0.098 to 7.125 mg/mL and MBC of 1.563 to 25.00 mg/mL, respectively. Remarkably, the A. marii FG-Z21 at a lower concentration of MIC (0.098–1.563 mg/mL) efficiently restricted the growth of E. faecalis, MRSA and MRSE in comparison with positive control kanamycin.

Table 2.

Minimal inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of the endophytic extracts

Endophytes	Extract	Minimum inhibitory and minimum bactericidal concentrations (mg/mL)
		Gram positive bacteria														Gram negative bacteria
		Bs		Bt		Ef		Ml		Sa		MRSA		MRSE		Kp		Pa		Ab		Va
		MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC
FG-Z6	EtOAc	1.563	3.125	0.195	0.781	1.563	3.125	3.125	6.250	0.781	1.563	1.563	6.250	1.563	3.125	0.781	1.563	0.781	3.125	1.563	3.125	3.125	6.250
FG-Z9		–	–	0.781	3.125	–	–	0.781	6.250	–	–	0.781	3.125	–	–	0.391	12.50	0.781	12.50	–	–	–	–
FG-Z12		6.250	25.00	0.781	25.00	3.125	12.50	0.195	25.00	0.781	12.50	0.391	25.00	–	–	0.781	25.00	0.049	1.563	–	–	1.563	50.00
FG-Z15		–	–	0.781	3.125	1.563	12.50	6.250	12.50	–	–	0.328	1.313	1.563	1.563	–	–	–	–	–	–	–	–
FG-Z20		–	–	0.082	2.625	–	–	–	–	2.625	2.263	7.125	7.125	–	–	–	–	0.082	1.313	2.625	2.625	–	–
FG-Z21		1.563	3.125	7.125	7.125	1.563	1.563	0.195	0.781	7.125	14.25	0.098	0.195	1.563	1.563	0.781	3.125	0.391	0.781	7.125	7.125	0.391	0.781
FG-Z35		12.50	12.50	0.098	3.125	–	–	–	–	–	–	–	–	–	–	–	–	0.098	3.125	–	–	–	–
FG-Z36		–	–	0.195	0.781	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
FG-Z41		–	–	–	–	1.563	3.125	0.195	0.781	3.125	3.125	–	–	–	–	0.781	6.250	0.195	0.781	–	–	1.563	1.563
FG-Z5	MeOH	–	–	0.781	12.50	0.781	12.50	–	–	0.781	12.50	–	–	–	–	–	–	0.781	6.250	1.563	6.250	6.250	1.563
FG-Z6		–	–	–	–	3.125	12.50	–	–	3.125	25.00	–	–	–	–	0.781	1.563	3.125	12.50	1.563	3.125	1.563	3.125
FG-Z12		3.125	3.125	–	–	3.125	3.125	0.098	0.195	–	–	1.563	3.125	–	–	3.125	3.125	6.250	6.250	–	–	–	–
FG-Z35		–	–	–	–	–	–	–	–	12.50	12.50	–	–	–	–	1.563	6.250	–	–	3.125	25.00	–	–
FG-X35		–	–	–	–	–	–	–	–	–	–	1.563	6.250	–	–	–	–	–	–	–	–	–	–
FG-X48		–	–	0.391	–	12.50	25.00	–	–	–	–	–	–	–	–	–	–	25.00	25.00	–	–	–	–
FG-X50		–	–	0.781	–	–	–	–	–	–	–	–	–	3.125	–	–	–	–	–	–	–	–	–
FG-Z4	PE	3.125	3.125	–	–	–	–	0.391	3.125	–	–	1.563	3.125	–	–	–	–	1.563	12.50	–	–	–	–
FG-Z9		–	–	–	–	3.125	6.250	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
FG-Z12		–	–	–	–	1.563	3.125	0.391	–	–	–	–	–	–	25.00	6.250	6.250	3.125	3.125	–	–	6.250	6.250
Kan *		0.025	0.025	0.125	19.5	–	–	12.5	19.5	25	25	–	–	15.625	19.5	25	25	12.5	19.5	6.25	9.8	25	25

Bs-B, subtilis; Bt-B, thuringiensis; Ef-E, faecalis; Ml-M, luteus; Sa-S, aureus; Ab-A, bammannii; Kp-K, pneumonia; Ec-E, coli; Pa-P, aeruginosa; Va-V, alginolyticus, Positive control

*Kanamycin (40 µg/mL)

– indicates no antibacterial activity

DPPH Scavenging Activity

The radical scavenging activity of all the 60 extracts viz., petroleum ether, ethyl acetate and methanol from the endophytes were depicted in Fig. 4. A reduction in the EC₅₀ value meant a higher level of scavenging activity were significantly different with EC₅₀ ranging from 0.085 to 11.984 mg/mL obtained by the plot of inhibition percentage and samples concentrations. As shown in Fig. 4 the ethyl acetate extract of P. glabrum FG-Z4 displayed the most notably antioxidant activity followed by B. ochroleuca FG-Z20 and Lelliottia sp. FG-X18 with the EC₅₀ value of 0.085 mg/mL, 0.320 mg/mL and 0.846 mg/mL, respectively. In addition, the ethyl acetate extract of A. marii FG-Z21 also showed moderate antioxidant activity with EC₅₀ of 1.869 mg/mL. The results were compared with that of well-known antioxidant standard drug ascorbic acid.

Fig. 4.

Parallel plot showing the antioxidant activity of endophytic extracts

Cytotoxic Potential Against Human Lung Adenocarcinoma A549 Cell Line

The sequential extracts of all the endophytes were evaluated for cytotoxic activity on Human lung adenocarcinoma A549 cell line. As shown in Fig. 5 and Fig. S3, ethyl acetate extracts from 12 endophytes exhibited significant abilities in inhibiting A549 cell line with GI₅₀ values ranging from 0.036 to 0.732 mg/mL followed by petroleum ether extracts from 8 endophytes were 0.059 to1.004 mg/mL and methanol extracts from 7 endophytes were 0.260 to 0.527 mg/mL. Among them, ethyl acetate extracts of A. mari FG-Z21 showed the strong cytotoxic against tested cell lines with the GI₅₀ value of 0.036 mg/mL while S. argenteus FG-X48 also showed good cytotoxicity with GI₅₀ value of 0.039 mg/mL. In addition, petroleum ether extracts of F. oxsporum FG-Z12 showed moderate cytotoxic activity with GI₅₀ value of 0.059 mg/mL. The results were compared with that of well-known anticancer standard drug cisplatin.

Fig. 5.

Parallel plot showing the cytotoxic effects of endophytic extract

Isolation and Identification Bioactive Compounds from A. marii FG-Z21

The remarkable antibacterial, antioxidant and cytotoxic activities of A. mari FG-Z21 further encouraged us to investigate the bioactive constituents that led to the isolation of eight compounds and the structures were identified on the basis of physiochemical and detailed spectroscopic analyses (Fig. 6). 4-Hydroxybenzoic acid (1) was obtained as a white powder and the molecular formula was established as C₇H₆O₃ by positive HREIMS m/z 138.0312 [H + M]⁺ (calcd. for C₇H₆O₃) (Fig. S6). ¹H-NMR (600 MHz, DMSO-d₆) δ ppm: 12.43 (1H, s, –COOH), 10.22 (1H, s, –OH), 7. 78 (2H, d, J = 8.7 Hz, H-2, 6), 6.82 (2H, d, J = 8.7 Hz, H-3, 5); ¹³C-NMR (125 MHz, DMSO-d₆) δ ppm: 167.20 (C-7), 161. 63 (C-4), 131. 56 (C-2, 6), 121. 37 (C-1), 115. 15 (C-3, 5) (Fig. S7–S8). Further, the identity of the compound was also confirmed by comparison with the spectroscopic data with those reported in the literature [51]. 2-Acetamidobenzoic acid (2) was obtained as a white powder and the molecular formula was established as C₉H₉NO₃ by positive HREIMS m/z 179.0577 [H + M]⁺ (calcd. for C₉H₉NO₃) (Fig. S9). ¹H-NMR (500 MHz, DMSO-d₆) δ ppm: 12.73 (1H, s, –OH), 8.44 (1H, d, J = 8 Hz, H-3), 7.97 (1H, d, J = 7.5 Hz, H-6), 7.39 (1H, t, J = 7.5 Hz, H-4), 7.01 (1H, t, J = 7.5 Hz, H-5), 2.08 (3H, s, –CH₃); 13C-NMR (126 MHz, DMSO) δ ppm: 169.95 (–COOH), 167.99 (–COCH3), 140.83 (C-2), 131.70 (C-4), 131.12 (C-6), 121.71 (C-5), 118.82 (C-3), 25.11 (C-9) (Fig. S10–S11). The identity of the compound was confirmed by comparing the spectroscopic data reported by Ramírez et al. [47]. Benzoic acid (3) was obtained as an acicular crystal and the molecular formula was established as C₇H₆O₂ by positive HREIMS m/z 121.0290 [M–H]⁻ (calcd. for C₇H₆O₂) (Fig. S12). ¹H-NMR (600 MHz, DMSO-d₆) δ ppm: 7.95 (2H, d, J = 7.7 Hz, H-3, 7), 7.61 (1H, t, J = 7.4 Hz, H-5), 7.49 (2H, t, J = 7.6 Hz, H-4, 6); ¹³C-NMR (151 MHz, DMSO) δ ppm: 167.44 (C-1), 132.79 (C-5), 131.02 (C-2), 129.28 (C-3, 7), 128.56 (C-4, 6) (Fig. S13–S14). The structure of the compound was further confirmed by comparing the spectroscopic data reported by Ali et al. [48]. Dibutyl phthalate (4) was obtained as light yellow oil and the molecular formula was established as C₁₆H₂₂O₄ by positive HRESIMS m/z 301.1416 [M + H]⁺ (calcd. for C₁₆H₂₂O₄) (Fig. S15). ¹H-NMR (600 MHz, DMSO-d₆) δ ppm: 7.71 (2H,dd, J = 5.58, 3.36 Hz, H-3, 6), 7.66 (2H, dd, J = 5.64, 3.3 Hz, H-4, H-5), 4.22 (4H, t, J = 6.6 Hz, H-8, H-13), 1.64 (4 H, m, H-9, H-14), 1.37 (4 H, m, H-10, H-15), 0.91 (6 H, t, J = 7.4 Hz, H-11, H-16); ¹³C-NMR (151 MHz, DMSO) δ ppm: 166.98 (C-7, C-12), 131.72 (C-1, C-2), 131.54 (C-3, C-6), 128.68 (C-4, C-5), 65.04 (C-8, C-13), 30.02 (C-9, C-14), 18.68 (C-10, C-15), 13.57 (C-11, C-16) (Fig. S16–S17). The structure of the compound was further confirmed by comparing the spectroscopic data reported by Egorov et al. [49]. 3,4-Dimethoxybenzoic Acid (5) was obtained as a white crystal and the molecular formula was established as C₉H₁₀O₄ by positive HREI-MS m/z:182.0573 [M]⁺ (calcd. for C₉H₁₀O₄). ¹H-NMR (600 MHz, CD₃OD) δ ppm: 7.67 (1H, dd, J = 8.4, 2.0 Hz, H-1), 7.56 (1H, d, J = 2.0 Hz, H-5), 7.01 (1H, d, J = 8.4 Hz, H-2), 3.86 (3H, s, 3-OCH₃), 3.89 (3H, s,4-OCH₃); ¹³C NMR (151 MHz, CD₃OD) δ ppm:124.23 (C-1), 113.55 (C-2), 150.08 (C-3), 154.69 (C-4), 111.82 (C-5), 125.06 (C-6), 169.78 (C-7), 56.40 (3-OCH₃), 56.44 (4-OCH₃). Further, the identity of the compound was also confirmed by comparison with the spectroscopic data with those reported in the literature [50] (Fig. S18–S20). Schizostatin (6) was obtained as a white powder and the molecular formula was established as C₂₀H₃₀O₄ by positive HRESI-MS m/z:333.2068 [M–H]⁻ (calcd. for C₂₀H₃₀O₄). ¹H-NMR (600 MHz, CDCl₃) δ ppm: 6.89 (1H, s, H-2), 2.85 (2H, t, J = 7.5 Hz, H-4), 2.22 (2H, q, J = 7.5 Hz, H-5), 5.18 (H, t, J = 7.0 Hz, H-6), 2.00–1.94 (4H, m, H-8, H-9), 5.10 (2H, dt, J = 7.0, 3.6 Hz, H-10, H-14), 2.09–2.03 (4H, m, H-12, H-13), 1.59 (6H, s, H-16, H-20), 1.67 (6H, s, H-18), 1.61 (3H, s, H-19); ¹³C NMR (151 MHz, CDCl₃) δ ppm: 170.32 (C-1), 127.57 (C-2), 149.07 (C-3), 28.05 (C-4), 27.71 (C-5), 122.63 (C-6), 137.09 (C-7), 39.85 (C-8), 26.89 (C-9), 124.53 (C-10), 135.21 (C-11), 39.80 (C-12), 26.72 (C-13), 124.25 (C-14), 131.45 (C-15), 25.84 (C-16), 171.67 (C-17), 16.06 (C-18), 16.12 (C-19), 17.82 (C-20). Further, the identity of the compound was also confirmed by comparison with the spectroscopic data with those reported in the literature [51] (Fig. S21–S23). Ethyl Pyroglutamate (7) was obtained as a colorless oil and the molecular formula was established as C₇H₁₁NO₃ by positive HREI-MS m/z: 157.0733 [M]⁺ (calcd. for C₇H₁₁NO₃). ¹H-NMR (800 MHz, CD₃OD) δ ppm: 2.39–2.28 (2H, m, H-3), 2.51–2.45 (H, m, H-4a), 2.17–2.12 (H, m, H-4b), 4.28 (H, dd, J = 4.5, 9.0 Hz, H-5), 4.21 (2H, q, J = 7.1 Hz, H-7), 1.28 (3H, t, J = 7.1 Hz, H-8); ¹³C NMR (201 MHz, CD₃OD) δ ppm: 30.31 (C-3), 25.87 (C-4), 57.13 (C-5), 174.03 (C-6), 62.53 (C-7), 14.42 (C-8). Further, the identity of the compound was also confirmed by comparison with the spectroscopic data with those reported in the literature [52] (Fig. S24–S26). Anthranilic Acid (8) was obtained as a white powder and the molecular formula was established as C₇H₇NO₂ by positive HREI-MS m/z:137.0471 [M]⁻ (calcd. for C₇H₇NO₂). ¹H-NMR (800 MHz, CD₃OD) δ ppm: 6.72 (1H, d, J = 8.3 Hz, H-2), 7.21 (1H, dd, J = 8.3, 1.4 Hz, H-3), 6.56 (1H, dd, J = 8.0, 7.5 Hz, H-4), 7.80 (1H, d, J = 8.0 Hz, H-5); ¹³C NMR (201 MHz, CD3OD) δ ppm:152.72 (C-1), 116.58 (C-2), 134.82 (C-3), 117.73 (C-4), 132.66 (C-5), 112.28 (C-6), 172.04 (C-7). Further, the identity of the compound was also confirmed by comparison with the spectroscopic data with those reported in the literature [53] (Fig. S27–S29).

Fig. 6.

Chemical structures of the compounds (1–8) isolated from A. marii FG-Z21

Antibacterial Activities Compounds (1–8)

With respect to the significant antibacterial activity evidenced in the preliminary screening, the isolated compounds were tested for their antibacterial potential. All eight compounds demonstrated varying levels of antibacterial activity against the drug-resistant pathogens E. faecalis and MRSA. Notably, dibutyl phthalate (4) displayed significant antibacterial efficacy against E. faecalis with a zone of inhibition measuring 24.73 ± 0.55 mm. Following closely were 3,4-dimethoxybenzoic acid (5) with 18.63 ± 0.52 mm, schizostatin (6) with 15.08 ± 0.28 mm, anthranilic acid (8) with 14.97 ± 0.35 mm, 2-acetamidobenzoic acid (2) with 13.63 ± 0.17 mm, and ethyl pyroglutamate (7) with 11.19 ± 0.82 mm. These results were compared with the activity of kanamycin on MRSA, where no activity was observed. On the other hand, compounds 4-hydroxybenzoic acid (1), 2-Acetamidobenzoic acid (2), benzoic acid (3), 3,4-Dimethoxybenzoic acid (5), schizostatin (6), and ethyl Pyroglutamate (7) exhibited moderate activity against MRSA displaying zones of inhibition ranging from 9.22 ± 0.05 to 11.27 ± 0.13 mm (Table 3, Fig. S5). Further assessments were conducted on the compounds with significant antibacterial activity to determine their MIC and MBC against the aforementioned drug-resistant bacteria pathogens. As anticipated, compound 4 efficiently inhibited the growth of E. faecalis with MIC and MBC values of 2.00 and 4.00 µg/mL, respectively. Similarly, compounds 5 and 6 displayed potent activity with low MIC values of 4.00 µg/mL and MBC values of 8.00 µg/mL. On the other hand, compounds 3 and 7 exhibited moderate activity against MRSA both showing identical MIC and MBC values of 8.00 µg/mL. Additionally, compound 6 demonstrated weak antibacterial activity against MRSA with an MIC value of 8.00 µg/mL and an MBC value of 16.00 µg/mL.

Table 3.

Antibacterial and cytotoxic activity of compounds 1–8 from extracts of A. marii FG-Z21

	Anti-bacteria assay						Cytotoxicity
	E. faecalis			MRSA			Cell line A549
	Zone of inhibition (mm)	MIC (µg/mL)	MBC (µg/mL)	Zone of inhibition  (mm)	MIC (µg/mL)	MBC (µg/mL)	IG50 (µg/mL)
1	–	–	–	9.22 ± 0.05	16.00	–	8.63
2	13.63 ± 0.17	8.00	16.00	10.27 ± 0.30	8.00	32.00	10.46
3	–	–	–	11.15 ± 0.38	8.00	8.00	4.97
4	24.73 ± 0.55	2.00	4.00	–	–	–	83.08
5	18.63 ± 0.52	4.00	8.00	10.21 ± 0.02	16.00	32.00	–
6	15.08 ± 0.28	4.00	8.00	10.90 ± 0.06	8.00	16.00	–
7	11.19 ± 0.82	4.00	16.00	11.27 ± 0.13	8.00	8.00	–
8	14.97 ± 0.35	8.00	16.00	–	–	–	–
Cisplatin	–	–	–	–	–	–	4.34
kan	–	–	–	–	–	–	–

– indicates no antibacterial activity/ cytotoxic activity

Cytotoxic Effects of Compounds (1–8)

The isolated compounds 4-hydroxybenzoic acid (1), 2-acetamidobenzoic acid (2), benzoic acid (3), dibutyl phthalate (4), 3,4-dimethoxybenzoic acid (5), schizostatin (6), ethyl pyroglutamate (7), and anthranilic acid (8) showed cytotoxic effects in a dose-dependent manner against the human non-small cell lung carcinoma cells. Interestingly, compound 3 demonstrated a substantial impact by inducing cell death in the A549 cell line ranging from 19.02 ± 2.37 to 95.22 ± 2.00% with the GI₅₀ value of 4.97 μg/mL. Following this, compounds 1, 2 and 4 displayed a notable effect resulting in cell death ranging from 21.43 ± 0.25 to 96.57 ± 2.37%, 22.74 ± 0.08 to 96.54 ± 0.07% and 71.15 ± 1.09 to 97.32 ± 1.72% with the GI₅₀ values of 8.63, 10.46 and 83.08 µg/mL, respectively. On the other hand, compounds 5, 6, 7 and 8 did not exhibit any cytotoxic effect on A549 cells and the results were compared with that of well-known anticancer standard drug cisplatin (Table 3, Fig. S4).

Discussion

Natural products are valuable because of their therapeutic qualities and people have long utilized medicinal plants for therapeutic as well as culinary purposes. In this line, endophytes have been investigated as a special resource for the discovery of novel compounds that reside in plant tissues during part or all of their life cycle and function to defend the host against infections. Scientifically, endophytes from medicinal plants are capable of producing secondary metabolites that resemble or match those of the host and have been found to possess significant antibacterial, antioxidant, and anticancer properties. In this instance, P.ueraria thomsonii an endemic medicinal plant yielded a total of 20 culturable fungal and bacterial endophytes which provided insight into the microbial diversity of this plant. The combined morphological characterization and molecular analyses facilitated the classification of these isolates into distinct lineages revealing a taxonomically diverse consortium encompassing Trichocomaceae, Saccharomycetaceae, Didymosphaeriaceae, Schizophyllaceae, Tuberculariaceae, Phanerochaetaceae, Bionectriaceae, Diaporthaceae, Moniliaceae, and Polyporaceac for fungi [26–35], and Enterobacteriaceae, Pseudomonadaceae, Staphylococcaceae, and Bacillaceae for bacteria [6, 36–42]. The study further explored the biological activities of these endophytes unveiling notable antibacterial, antioxidant, and cytotoxic potentials.

The ethyl acetate extracts of A. marii FG-Z21 exhibited significant antibacterial activity against both Gram-positive and Gram-negative bacteria showing particularly efficacy against drug-resistant bacteria such as E. faecalis and MRSA. The diversity in antibacterial activities across different extracts underscores the potential of these endophytes to produce bioactive compounds with broad-spectrum activities. Antioxidant activity evaluated through DPPH scavenging assays revealed the ethyl acetate extract of P. glabrum FG-Z4 as the most potent antioxidant emphasizing its potential in mitigating oxidative stress. The ethyl acetate extracts showed significant cytotoxicity when tested against the human lung adenocarcinoma A549 cell line, with A. mari FG-Z21 showing very potent inhibitory effects. These findings underscore the potential of endophytes associated with P. thomsonii as a source of bioactive compounds with diverse biological activities.

Overall, it is noteworthy that the endophytic fungi A. marii FG-Z21 displayed significant activity against A549 cell line with GI₅₀ value of 36.03 µg/mL, promising antibacterial activity against 11 drug-resistant bacterial pathogens and moderate antioxidant activity with EC₅₀ of 1.869 µg/mL. However, few literatures documented on secondary metabolites and their biological activity of A. marii. Consequently, additional chemical investigation into the ethyl acetate extract of A. marii FG-Z21 led to the isolation of 8 active monomer compounds that have potent biological activities.

The structures of compounds 1–8 are elucidated as 4-hydroxybenzoic acid, 2-acetamidobenzoic acid, benzoic acid, dibutyl phthalate, 3,4-dimethoxybenzoic acid, schizostatin, ethyl pyroglutamate and anthranilic acid based on their comprehensive spectroscopic data and by comparison of their NMR data with the reports of Ge et al., Ramírez et al., Ali et al., Egorov et al., Rustamova et al., Tanimoto et al., Cao et al., Park et al. [43–50]. During our ongoing search for potent compounds from aforementioned endophytic fungi A. marii FG-Z21, compounds 1–8 were evaluated for their cytotoxicity and antibacterial activities. As a result, benzoic acid (3) showed significant cytotoxicity against A549 cell lines, followed by 4-hydroxybenzoic acid (1) and 2-acetamidobenzoic acid (2). Surprisingly, our discovery of remarkable cytotoxic activity of benzoic acid against A549 provided a new idea for the development of anticancer drugs and functional foods which are widely used in food as a kind of preservatives and no effect on health within the limited dosage of 1.0 g/kg [51]. These results are supported by the earlier reports of Abd-Ellatif et al. [52] in which 2-acetamidobenzoic acid (2) was proved to visualize the potentially cytotoxic activity against the human colon Caco-2 cancer cell line. Another study reported by Sannino et al. [53] further demonstrate that 4-hydroxybenzoic acid (1) inhibited A549 cancer cell proliferation via activating at the gene and protein levels (caspase-1, IL1β, and IL18), a specific cell death signaling pathway named pyroptosis. We found that dibutyl phthalate (4) showed weak cytotoxicity against cells A549. Similarly, Yan et al. [54] reported that dibutyl phthalate (4) induces cytotoxicity of the human glioma cell line U251 through activation of nuclear factor-jB. It should be noted that the cytotoxicity of these compounds’ benzoic acid derivatives 4-hydroxybenzoic acid (1), benzoic acid (3) and dibutyl phthalate (4) are reported for the first time through this current research. Therefore, the cytotoxicity by these compounds in the study was regarded as an important step in developing potent anticancer regimes.

Moreover, the isolated compounds showed strong to weak antibacterial against Gram positive multi-drug resistant bacterial pathogens E. faecalis and MRSA at very low MIC and MBC values. The previous report of Cho et al. [55] who showed that 20 of the Gram-positive and some Gram-negative bacteria were sensitive to 4-hydroxybenzoic acid (1) at IC₅₀ concentrations of 100–170 µg/mL; 2-acetamidobenzoic acid (2) showed strong to weak antibacterial against the pathogens tested viz., C. albicans, P. aeruginosa, B. subtilis, E. coli [52]; As a kind of antibacterial and antifungal preservative, benzoic acid (3) was widely used in foods and feeds [51]; antimicrobial agents dibutyl phthalate (4) have been reported to be cathepsin B inhibitor inducing microbial death [56]; 3,4-dimethoxybenzoic acid (5) inhibited the growth of the two different bacterial stains K. pneumoniae and S. aureus [57]; schizostatin (6) could be used as a biochemical pesticide exhibited antimicrobial activities against plant pathogenic fungi Rhizoctonia solani, Diaporthe sp., Botrytis cinerea, and Alternaria solani and bacterial pathogens such as B. subtilis and S. aureus [58]. Anthranilic acid (8) exogenously also exhibited antibacterial activity against Legionella pneumophila, the pathogenic agent of Legionnaires' disease [59]. Due to the insufficient yield of compounds, the antioxidant assay results were not included in the final report. Moreover, preliminary antioxidant assays indicated that A. mari demonstrated only modest activity which did not warrant further detailed analysis. This moderate activity, combined with the limited compound availability led to the decision to exclude these results from the study.

Conclusion

In summary, 11 endophytic fungi and 9 bacteria were isolated from Pueraria thomsonii displayed a broad-spectrum of antibacterial activity along with significant cytotoxic, and antioxidant activities in preliminary screening. Among the endophytic extracts tested, the ethyl acetate extract of A. marii FG-Z21 exhibited a significant DPPH radical scavenging activity and antibacterial activity with extremely very low MICs and MBCs values as well as significant inhibitory effect on A549 cells with low GI₅₀ value. Subsequent chemical investigations led to the isolation of bioactive compounds such as 4-hydroxybenzoic acid, 2-acetamidobenzoic acid, benzoic acid, dibutyl phthalate, 3,4-dimethoxybenzoic acid, schizostatin, ethyl pyroglutamate and anthranilic acid. These compounds exhibited strong antibacterial and cytotoxicity properties which advances our knowledge of the endophytes' potential as therapeutic agents and forms a strong basis for additional pharmacological research to treat various cancer and emerging bacterial infections.

Supplementary Information

Below is the link to the electronic supplementary material.

Funding

This research was funded by Youth Fund of Institute of Chinese Herbal Medicines, grant number, 2024NKYJJ37; Study on anticancer activity and mechanism of vine tea, grant number, XYJ2023000138; Study on key technology of fermentation tea cake processing of vine tea grant number, XYJ2023000049; Using selenium-rich Chinese medicinal materials to develop whitening and liver protection active products in Enshi, grant number XYJ2023000044.

Declarations

Conflict of interest

The authors declare no conflict of interest.