Novel secondary metabolites from endophytic fungi: synthesis and biological properties

Abstract

Plant-associated endophytic fungi are rich sources of novel bioactive and structurally diverse secondary metabolites and other natural products. Newly synthesized secondary metabolites of endophytes in terrestrial plants and their biological properties are presented and reviewed herein. The review highlights natural products from plant-associated fungal endophytes that demonstrate significant inhibition against pathogenic strains, human cancer cell lines and other biological targets. We have summarized 221 recently isolated, structurally diverse and novel secondary metabolites. Secondary metabolites that failed to demonstrate activity in the bioassays are not included. The review is intended to assist scientists in the fields of phytochemistry, organic chemistry, and pharmacology.

Graphic abstract

Introduction

Endophytic fungi are microorganisms that inhabit the internal tissues of the plants without causing apparent disease symptoms (Nisa et al. 2015). Differences between pathogenic and endophytic microorganisms are sometimes difficult to define, especially when both present similar genetic signatures. Many fungal species comprise individual subspecies having both endophytic and pathogenic properties (Schouten 2019a, b). However, most pathogenic microorganisms produce toxins harmful to the host plant, while secondary metabolites produced by endophytic strains—usually antimicrobial agents—protect their host plants by blocking or inhibiting the appropriate pathogenic microorganisms (Schouten 2019a; Ludwig-Müller 2019). Endophytic microorganisms may also control growth and development of host plants (Neilson et al. 2013). Importantly, endophytic microorganisms living inside their host can often be cultured and grown after isolation from their host plants.

The isolation of endophytic fungi is normally carried out using surface sterilization, vacuuming (particularly for wood plants) and other accepted methods (Martinez-Klimova et al. 2017). Among these, most common approach is the screening of surface-sterilized plant tissue placed on growth media suitable for fungi (Schouten 2019b); antibacterial agents may be added to prevent proliferation of endophytic bacteria. Identification of endophytic microorganisms can be performed by morphological and molecular phylogenetics (rDNA, 16S rDNA sequences) techniques (Ko Ko et al. 2011; Rustamova et al. 2020).

One approach to efficient production of secondary metabolites is represented by fermentation, a microorganism-driven process that enzymatically converts substrates into bioactive compounds (Parvez et al. 2006). Endophytic strains sometimes produce novel pharmacologically active and structurally diverse secondary metabolites not present in their host’s tissues. In this way endophytes can live independently and produce secondary metabolites in the host plant (Tawfike et al. 2019). Because plant-associated endophytic fungi are rich sources of novel bioactive compounds (Andrés et al. 2017), they provide an important alternative to exploiting the plant itself (Obermeier and Christina 2019). This is of particular interest when (a) the bioactive secondary metabolites are not commercially available, (b) the bioactive secondary metabolites are derived from slow-growing or rare plants, or (c) total synthesis of the bioactive secondary metabolite is difficult or expensive due to its high molecular weight or complex structure.

Further research is needed to elucidate which partner (host-plant or endophytic strain) contributes to the biosynthesis of secondary metabolites, and to what extent. For example, it remains unclear which secondary metabolites are exclusively produced from plants, from the endophytic strain, or from both partners (Ludwig-Müller 2019). We do know that particular secondary metabolites can be exchanged with the partner and then further metabolized, either by the plant or by the endophytic fungus (Ludwig-Müller 2015). It has also been confirmed that endophytic strains can transform host-synthesized metabolites into multi-functional products (Schouten 2019c), and therefore particular fungi could be used as catalysts during biotransformation processes producing oxygenated compounds (Wang and Dai 2011).

The structurally unique natural compounds produced by plant endophytic fungi include alkaloids (Panaccione et al. 2014; Rustamova et al. 2019), terpenoids (Souza et al. 2011), various polyketides (El-Neketi et al. 2013), isocoumarins (Saeed 2016), flavonoids (Wang et al. 2011), lactones (Shen et al. 2015), and other such compounds (Martelli and Giacomini 2018). Endophytic secondary metabolites and their biological properties have been studied in terrestrial plants as well as marine organisms (Hou et al. 2019). Biochemistry and molecular biology techniques (e.g., biotransformation, exploration of biosynthetic pathways, and heterologous expression of enzymes in microorganisms) have been developed for the large-scale production of natural products. For example, between 2014 and 2016 there was a continual increase in the number of new secondary metabolites isolated from mangrove-associated fungi (103 in 2014, 133 in 2015 and 149 in 2016), mostly from endophytic species (Carroll et al. 2019). These data illustrate the potential of secondary metabolites isolated from endophytic strains.

Over the past decade the chemistry and biology of plant-associated endophytic fungi have emerged as topics of increasingly vital importance to our understanding of plant and microbial ecosystems, as well as to their potential applications in the pharmaceutical and other industries. Understanding of the chemistry of these fungi leads directly to better knowledge of their phytochemistry vis-a-vis isolation, analysis, biosynthesis, biotransformation and diversity of fungal components, while biological studies allow improvement in methods of strain isolation, microbiological characterization, and understanding of mechanisms of biological diversity and plant-fungal symbiosis. A literature search of Scopus (www.scopus.com) and Web of Science (www.webofknowledge.com) databases covering the period of January 2018 to June 2019 and using the search terms “endophyte” “endophytic fungi,” “endophytic fungus,” and “secondary metabolites from endophytic fungus” as keywords revealed nearly 1000 peer-reviewed scientific reports, including up to 150 research studies dedicated to the phytochemistry and medicinal chemistry of plant-associated natural compounds produced by endophytic fungi. This review significantly expands this body of work by summarizing 221 new secondary metabolites isolated from plant-associated endophytic fungi, along with their taxonomy (Fig. 1) and discussion of their biological properties.

Fig. 1

Locations and ratios of the endophytic fungi that have been collected (a), percentages of endophytic fungi belonging to various phyla (b) and the number of endophytic fungi studied in each genus (c)

Terpenes and terpenoids

Structurally unique and biologically active terpenes and terpenoids provided in this section include sesquiterpenes/sesquiterpenoids, diterpenes/diterpenoids, triterpenoids, and meroterpenoids, as well as steroids exhibiting various biological activities. All of the natural compounds we’ve isolated and characterized fall within these categories, are novel, and were isolated from endophytic strains belonging to defined fungal genera.

Sesquiterpenoids

Novel phenolic bisabolane sesquiterpenoids, (7R,10S)-7,10-epoxysydonic acid (1), (7S,10S)-7,10-epoxysydonic acid (2), (7R,11S)-7,12-epoxysydonic acid (3), (7S,11S)-7,12-epoxysydonic acid (4), 7-deoxy-7,14-didehydro-12-hydroxysydonic acid (5), (Z)-7-deoxy-7,8-didehydro-12-hydroxysydonic acid (6), and (E)-7-deoxy-7,8-didehydro-12-hydroxysydonic acid (7), were obtained from a culture of the endophytic fungus Aspergillus sp. xy02 isolated from the leaves of a Thai mangrove Xylocarpus moluccensis (Fig. 2) (Wang et al. 2018a). Four derivatives (2, 3, 5 and 7) showed moderate antibacterial activities against Staphylococcus aureus ATCC 25,923 with IC₅₀ values ranging from 31.5 to 41.9 μM, with none of the novel sesquiterpenoids demonstrating DPPH scavenging antioxidant potential.

Fig. 2

The structures of novel sesquiterpenoids (1–25) isolated from endophytic fungi

Another fourteen novel secondary metabolites [one 2,3-seco-protoilludane-type sesquiterpenoid (8), eight protoilludane-type sesquiterpenoids (9–16), four illudalane-type sesquiterpenoids (17–20), and one botryane-type sesquiterpenoid (21)] were obtained from the strain Phomopsis sp. TJ507A, an endophytic fungus isolated from the leaves of the medicinal plant Phyllanthus glaucus (Fig. 2) (Xie et al. 2018b). All sesquiterpenoids were evaluated for their BACE1 inhibitory activities. Among them, eight isolates 8–14 and 16 showed BACE1 inhibitory activities ranging from 19 to 43% at a concentration of 40 μM, while the positive control LY2811376 revealed an inhibitory activity of 38.6%.

Sesquiterpenes, including three cyclonerane sesquiterpenes, 11-methoxy-9-cycloneren-3,7-diol (22), 10-cycloneren-3,5,7-triol (23), and methyl 3,7-dihydroxy-15-cycloneranate (24), as well as one acorane sesquiterpene, 8-acoren-3,11-diol (25), were isolated from a culture of Trichoderma harzianum X-5, an endophytic fungus obtained from the marine brown alga Laminaria japonica (Fig. 2) (Song et al. 2018b). The isolates have been evaluated for their antimicroalgal activities against Chattonella marina, Heterosigma akashiwo, Karlodinium veneficum, and Prorocentrum donghaiense phytoplankton species, as well as their antibacterial activity against Vibrio anguillarum, Vibrio harveyi, Vibrio parahemolyticus, and Vibrio splendidus marine-derived pathogenic bacteria. Compound 22 was the most active against C. marina (IC₅₀ = 0.66) and K. veneficum (IC₅₀ = 2.2) compared to the other obtained products. All four metabolites failed to demonstrate significant activity against bacterial strains of the Vibrio genus, with the exception of V. anguillarum which displayed only weak potency toward compounds 22–25.

The endophytic fungus Cytospora sp., isolated from the hypocotyls of the Chinese mangrove Ceriops tagal yielded seiricardine D, a new bicyclic sesquiterpene (26) (Fig. 3) (Deng et al. 2018). In vitro antimicrobial activity of metabolite 26 against the human pathogens Escherichia coli, methicillin-resistant S. aureus (MRSA), Pseudomonas aeruginosa, Candida albicans, and the plant pathogens Bacillus subtilis, Colletotrichum gloeosporioides, and Magnaporthe oryzae revealed that this compound exhibited weak inhibitory activity only against M. oryzae with an MIC of 839 µM.

Fig. 3

The structures of new sesquiterpenes (26–46) isolated from endophytic fungi

The fungal strain Aspergillus fumigatus, an endophyte of Ligusticum wallichii, produced two new sesquiterpenes, fumagillene A (27) and fumagillene B (28) (Fig. 3) (Li et al. 2018a). The cytotoxicity evaluation of the novel metabolites against MDA-ME-231 and MV4-11 cancer cell lines revealed that both secondary metabolites showed modest growth inhibition, with derivative 27 displaying IC₅₀ values of 8.4 and 14.3 µM and compound 28 having IC₅₀ values of 11.2 and 17.3 µM, respectively.

Two novel sativene-type sesquiterpenoids, bipolenins G and H (29, 30, Fig. 3), were obtained from Bipolaris eleusines, an endophytic fungus isolated from fresh potatoes (Li et al. 2018c). These metabolites did not demonstrate activity (IC₅₀ > 40 μM) against three human cancer cell lines (HL-60, A-549 and MCF-7). However, isolate 29 exhibited anti-NO production activity with an IC₅₀ value of 23.8 μM. Another new terpenenoid, the first example of 14-nordrimane-type sesquiterpene, phomanolide (31, Fig. 3), was obtained from the culture broth of an endophytic fungus of the Phoma sp. isolated from the roots of Aconitum vilmorinianum (Liu et al. 2019c). Antiviral screening revealed that sesquiterpene 31 showed anti-influenza activity on influenza A virus (A/Puerto Rico/8/34, H1N1) with an IC₅₀ value of 2.96 μg/mL.

The endophytic fungus Xylaria sp. GDG-102, isolated from the leaves of Sophora tonkinensis, yielded one new eremophilane sesquiterpene, xylareremophil (32, Fig. 3) (Liang et al. 2018b). In order to evaluate the antibacterial potential of compound 32, several strains were selected for the antibacterial assay, including S. aureus, Micrococcus lysodeikticus, Micrococcus luteus, B. subtilis, Enterobacter aerogenes, Salmonella paratyphi B, and Proteus vulgaris. Metabolite 32 exhibited moderate inhibition of M. luteus and P. vulgaris with an MIC value of 25 μg/mL.

Three novel sesquiterpene lactones, purpurolides A–C (33–35, Fig. 3), with significant inhibitory activity against pancreatic lipase, were produced from the strain Penicillium purpurogenum IMM003, an endophytic fungus obtained from fresh and healthy leaves of Edgeworthia chrysantha (Wang et al. 2018c). The new metabolites 33–35 potently inhibited the pancreatic lipase demonstrating IC₅₀ values of 2.83, 5.45, and 6.63 μM, respectively, compared to the standard kaempferol (1.50 μM).

Preussia isomera XL-1326, an endophyte isolated from the stems of Panax notoginseng, produced a pair of norsesquiterpenoidal enantiomers, (+-(36) and (−-(37) preuisolactone A (Fig. 3) (Xu et al. 2019a). These metabolites failed to demonstrate cytotoxic activity against the human A549, Huh7, MGC803, HCT116, and LN229 cancer cell lines (IC₅₀ > 100 μM). However, they showed moderate to weak antibacterial activities against M. luteus and Bacillus megaterium with MIC values of 10.2 and 163.4 μM, respectively. Notably, the antimicrobial screening led to the selection of a total of 16 microbial strains, including eight bacterial and eight fungal species.

The endophytic fungus Bipolaris eleusines, isolated from potato, also yielded two novel anti-phytopathogenic (Alternaria solani) sesquiterpenoid-xanthone hybrids, namely bipolins I and J (38 and 39, Fig. 3) (He et al. 2019). These hybrids were evaluated for their anti-phytopathogenic activities against four plant pathogens including Phytophthora infestane, A. solani, Rhizoctonia solani, and Fusarium oxysporum. Both, metabolites 38 and 39 displayed potent inhibitory activity towards A. solani, with MIC values of 8 and 16 µg/mL, respectively. Compound 38 also weakly inhibited F. oxysporum with an MIC value of 64 µg/mL.

Two new triquinane-type sesquiterpenoids, named cerrenins D (40) and E (41) (Fig. 3), were isolated from endophytic Cerrena sp. A593, which was obtained from the plant Pogostemon cablin (Liu et al. 2019a). The new metabolite 40 was evaluated for its cytotoxic activity in four cancer cell lines (SF-268, MCF-7, NCI-H460 and HepG-2) and compared with cisplatin as positive control drug. The results revealed that cerrenins D displayed moderate activity against the breast cancer MCF-7 cell line with an IC₅₀ value of 14.43 µM. Only weak cytotoxic activities were observed toward the other cell lines.

Novel secondary metabolites, including two trichothecene sesquiterpenoids, trichothecrotocins A (42) and B (43), and merosesquiterpenoid racemate, (±)-trichothecrotocin C (44 and 45), were detected in the potato (Solanum tuberosum) endophytic fungus Trichothecium crotocinigenum (Fig. 3) (Yang et al. 2018a). All isolated sesquiterpenoids were evaluated for their anti-phytopathogenic properties against four potato pathogens, including Phytophthora infestans (late blight), A. solani (early blight), R. solani (black scurf), and F. oxysporum (blast). It should be noted that several potato diseases are caused by the plant pathogens described above. Additionally, they are known to cause substantial problems in the production of potatoes. Notably, (±)-trichothecrotocin C weakly inhibited P. infestans and R. solani, while all metabolites of the new isolates demonstrated potent inhibitory activities against A. solani (MIC = 12, 16, 8 μg/mL) and F. oxysporum (MIC = 32, 16, 16 μg/mL).

An endophytic fungus, Pestalotiopsis adusta, collected from the stem bark of the wild and rare medicinal plant Sinopodophyllum hexandrum (Royle), produced one novel sesquiterpene pestalustaine A (46, Fig. 3), with an unusual 5/6/7-fused tricyclic system (Xiao et al. 2018). The cytotoxic activities of the new isolate against HeLa, HCT116, and A549 cell lines were weak to moderate, with the IC₅₀ values ranging from 29.58 to 58.75 µM.

Diterpenoids

Drechmerin H, a novel 1(2), 2(18)-disecoindole diterpenoid (47), and new indole diterpenoid, 2′- epiterpendole A (48), was reportedly produced by the endophytic fungus of Drechmeria sp. SYPF 8335, isolated from the root of Panax notoginseng (Fig. 4) (Zhao et al. 2018a). The isolates were assayed for agonistic effects on the pregnane X receptor (PXR) and metabolite 47. A significant agonistic effect on PXR was measured with an EC₅₀ value of 134.91 nM. Further, seven new indole diterpenoids, drechmerins A–G (49–55, Fig. 4) were derived from the fungal strain Drechmeria sp., which is an endophyte isolated from the root of Panax notoginseng (Zhao et al. 2018b). The antimicrobial screening revealed that drechmerin B (50) displayed significant inhibition of C. albicans, with an MIC value of 12.5 µg/mL, while other metabolites demonstrated only weak antimicrobial effects.

Fig. 4

The structures of novel diterpenoids (47–60) produced by endophytic fungi

Three new diterpenes, koninginols A–C (56–58, Fig. 4), produced by the endophytic fungus Trichoderma koningiopsis A729, were derived from the fresh branches of the medicinal plant Morinda officinalis (Chen et al. 2019b). Regarding their biological screening, the novel isolates 56 and 57 exhibited significant antibacterial activity against B. subtilis with MIC values of 10 and 2 μg/mL, respectively. Furthermore, in their antitumor evaluation using HepG-2, MCF-7, SF-268, and A549 cell lines, only diterpene 57 displayed an antiproliferative activity in A549 with an IC₅₀ value of 46.6 μM.

One harziane diterpene, 3R-hydroxy-9R,10R dihydroharzianone (59), and one proharziane diterpene, 11R-methoxy-5,9,13-proharzitrien-3-ol (60), were obtained from endophytic Trichoderma harzianum X-5 of the marine brown alga Laminaria japonica (Fig. 4) (Song et al. 2018b). In anti-microalgal and antibacterial assays, compound 60 exhibited modest activity against C. marina. This compound also showed weak activity against V. harveyi, with an IC₅₀ value of 6.8 µM at 20 μg/disk.

Triterpenoids

Nine novel triterpenoids, named as kadhenrischinins A–H (61–68) and 7β-schinalactone C (69) were isolated from Kadsura angustifolia fermented by a symbiont endophytic fungus, Penicillium sp. SWUKD4.1850 (Fig. 5) (Qin et al. 2019). This was the first report on the fermentation of Kadsura angustifolia and the first discovery of highly oxygenated schitriterpenoids using microbial technology. All metabolites were evaluated for their cytotoxicity in human hepatocellular liver carcinoma cells (HepG2). The triterpenoids 61–68 demonstrated moderate cytotoxic activity with IC₅₀ values ranging from 14.3 to 21.3 μM, while derivative 69 displayed weak activity (IC₅₀ > 40 μM) in the HepG2 cells.

Fig. 5

The structures of novel triterpenoids (61–73) isolated from endophytic fungi

Two new tetracyclic triterpenoids, integracide E (70) and isointegracide E (71), were obtained from the mycelium of the endophytic fungus Hypoxylon sp. 6269, which was isolated from medicinal plant Artemisia annua (Fig. 5) (Liang et al. 2018a). These novel secondary metabolites were tested for their anti-HIV-1 integrase potential in coupled and strand transfer assays. Herein, derivative 70 displayed weak activity in both assays with IC₅₀ values of 31.63 and 29.06 μM, respectively, while metabolite 71 was inactive in all performed tests.

One new pentanortriterpenoid, 23,24,25,26,27-pentanorlanost-7,9(11)-dien-3b,22-diol (72), and one new triterpenoid, lanost-8-en-3b,22S,23S-triol (73), were derived from the endophytic fungus Diplodia cupressi, isolated from the healthy tissues of Polytrichum commune (Fig. 5) (Liu et al. 2019d). The cytotoxicity of these compounds in human cancer cell lines (A549, Hep G2, Hepa 1c1c7, and HeLa) was evaluated, and derivative 73 displayed weak inhibitory activity with an IC₅₀ value of 35.0 µM against the proliferation of Hepa 1c1c7 cells.

Meroterpenoids

The endophytic fungus Fusarium sp. YD-2, obtained from the twigs of Santalum album, produced two new spiromeroterpenoids, namely fusariumin A (74) and B (75) (Fig. 6) (Yan et al. 2018). These terpenoids were evaluated for their potential anti-inflammatory and antibacterial properties. Fusariumin A exhibited significant activity against S. aureus and P. aeruginosa with an MIC value of 6.3 µg/mL, while fusariumin B showed modest anti-inflammatory activity in vitro by inhibiting the nitric oxide (NO) production in lipopolysaccharide (LPS)-activated RAW264.7 cells with an IC₅₀ value of 50 µM. For antibacterial screening, eight bacterial species (S. aureus, Staphylococcus albus, B. subtilis, Bacillus cereus, M. luteus, E. coli, P. aeruginosa, Salmonella enteritidis) were selected for the evaluation of 74 and 75.

Fig. 6

The structures of new natural meroterpenoids (74–88) isolated from endophytic fungi

The chemical investigation of the fungal strain Talaromyces amestolkiae YX1, an endophyte isolated from healthy leaves of the marine mangrove Kandelia obovata, produced four new meroterpenoids, amestolkolides A-D (76–79, Fig. 6) (Chen et al. 2018). Meroterpenoids were tested for their inhibitory activities against LPS-activated NO production in RAW264.7 cells using the Griess assay, with indomethacin as the positive control (IC₅₀ = 26.3 µM). Amestolkolide B (77) displayed strong inhibitory effects on the NO production with IC₅₀ values of 1.6 µM, while amestolkolide A (76) demonstrated weaker activity (30 µM).

The fungal strain Emericella sp. TJ29, an endophyte derived from the root of the plant Hypericum perforatum, produced emeridones A–F (80–85, Fig. 6), six new 3,5-demethylorsellinic acid-based meroterpenoids (Li et al. 2019). The anticancer screening of the novel secondary metabolites in five human cancer cell lines (HL-60, SMMC-7721, A549, MCF-7, and SW-480) revealed that meroterpenoids 81, 83, and 85 displayed modest cytotoxic activities against SMMC-7721 cells with IC₅₀ values of 18.80, 8.19, and 17.49 μM, respectively. These three isolates also demonstrated cytotoxicity towards SW-480 cells (IC₅₀ = 18.35, 14.67, and 16.84 μM, respectively), while emeridone D (83) was active against A-549 cells with an IC₅₀ of 11.33 μM.

The endophytic fungus Penicillium sp. sh18 was isolated from the fresh stems of Isodon eriocalyx var. laxiflora. Three novel meroterpenoids, isopenicins A–C, were derived from this endophyte (86–88, Fig. 6), which contained terpenoid-polyketide hybrid skeletons (Tang et al. 2019a). These secondary metabolites were evaluated for their inhibitory activity towards the Wnt/β-catenin signaling pathway using HEK293 cells stably transfected with Wnt3a, Renilla, and SuperTopflash luciferase (ST-Luc). Metabolite 86 was identified as a potent inhibitor of the Wnt signaling pathway. The Wnt signaling inhibitory activity of metabolite 86 was also investigated in Wnt-dependent colorectal cancer cells, SW620 and HCT11610. The elevated ST-Luc activity was significantly decreased by derivative 86 in both SW620 and HCT116 cells. Further experiments evaluating the effect of compound 86 on the expression of endogenous Wnt target genes revealed that the expression of Axin 2, c-myc, and survivin was considerably suppressed in SW620 and HCT116 cells exposed to isopenicin A.

Steroids

The Fusarium sp. is a rich source of ergosterol derivatives and other metabolites of diverse classes. Three new ergosterol derivatives [Fusaristerol B ((22E,24R)-3-palmitoyl-19(10 → 6)-abeo-ergosta-5,7,9,22-tetraen-3β-ol) (89), Fusaristerol C [(22E,24R)-ergosta-7,22-diene-3β,6β,9R-triol] (90) and Fusaristerol D [(22E,24R)-ergosta-7,22-diene-3β,5α,6β,9α-tetraol 6-acetate] (91)] were isolated from the endophytic fungus Fusarium sp., separated from the interior tissues of the Mentha longifolia L. (Labiatae) roots (Fig. 7) (Khayat et al. 2019). The 5-lipoxygenase (5-LOX) inhibitory activity of the secondary metabolites from Fusarium sp. was assessed, with isolate 89 displaying strong 5-LOX inhibitory activity with the IC₅₀ value of 3.61 μM compared to that of indomethacin (IC₅₀ 1.17 μM), while isolates 90 and 91 demonstrated moderate activity with IC₅₀ values of 7.01 and 4.79 μM, respectively. Therefore, ergosterol derivatives could be lead compounds in the development of 5-LOX inhibitory agents to treat various inflammatory disorders.

Fig. 7

The structures of new steroids (89–91) endophytic fungi

Polyketides

Many of the secondary metabolites produced from endophytic fungi are polyketides. These are widely distributed in other microorganisms and plants, and many of them exhibit various biological activities. This class of compounds includes coumarins, isocoumarins, α-pyrones, chromens, quinones, anthrones, and other related polyketides. This section describes recent developments in the chemistry and biology of polyketides in phytochemistry along with their medicinal uses.

Coumarins

One rare coumarin derivative, pestalustaine B (92, Fig. 8), with an unprecedented 6/6/5/5-fused tetracyclic system was isolated from the endophyte Pestalotiopsis adusta of the plant Sinopodophyllum hexandrum (Xiao et al. 2018). The anticancer activity of this coumarin has been evaluated in HeLa, HCT116, and A549 cancer cell lines, and moderate cytotoxic activities against all selected cell lines was observed with IC₅₀ values of 21.18, 21.04, and 37.33 µM, respectively.

Fig. 8

The structures of coumarins 92, isocoumarins 93–110 produced by endophytic fungi

Isocoumarins

Pestalotiopsis sp., an endophytic fungus isolated from the fresh, healthy branches of the Chinese mangrove plant Rhizophora stylosa, yielded one new isocoumarin derivative, pestalotiopisorin B (93, Fig. 8) (Xu et al. 2019b). For antimicrobial screening, five types of microorganisms were selected (P. aeruginosa, Enterococcus faecalis, MRSA, E. coli and C. albicans). This secondary metabolite displayed moderate antibacterial activity towards E. coli and P. aeruginosa with MIC values of 12.5 and 50 µg/mL, respectively.

Seven new dihydroisocoumarins, including five brominated (94–98) and two chlorinated (99 and 100) metabolites were produced by the endophytic fungus Lachnum palmae, isolated from the fresh tissue of the surface-sterilized Przewalskia tangutica (Fig. 8) (Zhao et al. 2018c). All novel isolates were evaluated for antimicrobial activity against Cryptococcus neoformans, Penicillium sp., C. albicans, B. subtilis, and S. aureus strains. Coumarin 98 possessed antimicrobial activities against all the tested strains with MIC values in the range of 10–55 mg/mL. However, HepG2 cells revealed only weak cytotoxicity with an IC₅₀ value of 42.8 µM. Regarding the anti-inflammatory properties, palmaerones A and E (94 and 98) exhibited moderate inhibitory effects on NO production in LPS-induced RAW 264.7 cells, with IC₅₀ values of 26.3 and 38.7 µM, respectively, with no obvious toxicities observed at 50 µM.

Ten new isocoumarins, named peniisocoumarins A–J (101–110, Fig. 8), were obtained through the fermentation of an endophytic fungus Penicillium commune QQF-3, isolated from fresh fruits of the mangrove plant Kandelia candel (Cai et al. 2018). These new coumarins were tested for their inhibitory activities against α-glucosidase in vitro, and metabolites 101, 105, 107, and 108 demonstrated potential inhibitory activity comparable to the positive control acarbose. The IC₅₀ values ranged from 38.1 to 78.1 μM. In addition, all coumarins were tested for their MptpB inhibitory activity, and cytotoxicity using human tumor cell lines including A549, HepG2, HeLa, MCF-7, and HEK293T. Herein, isolate 105 showed potent MptpB inhibitory activity with an IC₅₀ value of 20.7 μM compared to the positive control oleanolic acid. However, none of the metabolites displayed cytotoxicity against the tested human cell lines at 100 μM.

α-Pyrones and chromens

In continuing pursuit of new bioactive metabolites from endophytic fungi of endemic plants around the Three Gorges area, four new α-pyrone type derivatives (111–114, Fig. 9) were obtained from an ethyl acetate extract of Xylariales sp. (HM-1), an endophytic fungi isolated from the healthy leaves of Distylium chinense (Guo et al. 2018). The preliminary cytotoxicity of the novel isolates in four cancer cell lines (A549, HepG2, Caski and MDA-MB-231) failed to reveal significant activity. However, xylariaopyrones A–D (111–114) displayed antimicrobial inhibitory activity with MIC values ranging between 20.5 and 50.6 μg/mL. In addition, the results of the brine shrimp inhibiting activity from four new metabolites displayed inhibition percentages ranging from 42 to 82%.

Fig. 9

The structures of novel α-pyrones (111–122), and chromens (123–130) isolated from endophytic fungi

Three new 3,4,6-trisubstituted α-pyrone derivatives, namely 6-(2′rhydroxy-3′E,5′E-diene-1′-heptyl)-4-hydroxy-3-methyl-2H-pyran-2-one (115), 6-(2′S-hydroxy-5′E-ene-1′-heptyl)-4-hydroxy-3-methyl-2H-pyran-2-one (116), and 6-(2′S-hydroxy-1′-heptyl)-4 -hydroxy-3-methyl-2Hpyran-2-one (117) were derived from the endophytic fungi Penicillium ochrochloronthe associated with the roots of Taxus media (Fig. 9) (Zhao et al. 2018d). Although these secondary metabolites exhibited minimal cytotoxic activity in seven human cancer cells (A549, LN229, MGC, LOVO, Huh7, MHCC97H and MDA231), all the novel α-pyrones demonstrated significant antifungal activity when tested in fungal strains including Cercospora arachidicola Hori, A. solani, Bipolaris carbonum Wilson, Fusarium graminearum, Cylindrocladium parasiticum, Alternaria alternata f. sp. mali, Cercospora personata, and Botrytis cinerea Pers. with MIC values ranging between 12.5–50 μg/mL, and weak to modest antibacterial activity against 20 fungal and 10 bacterial strains.

Three new α-pyrone derivatives, namely (S)-6-(sec-butyl)-5-(hydroxymethyl)-4-methoxy-2H-pyran-2-one (118), (5S,7R)-7-ethyl-4,5-dimethoxy-7-methyl-5,7-dihydro-2H-furo[3,4-b]pyran-2-one (119) and (5R,7R)-7-ethyl-4,5-dimethoxy-7-methyl-5,7-dihydro-2H-furo[3,4-b]pyran-2-one (120), were induced by chemical epigenetic manipulation of the endophytic fungus Penicillium herquei, isolated from the fruiting body of Cordyceps sinensis (Fig. 9) (Guo et al. 2019). The cytotoxicity of these metabolites in MDA-ME-231 and MV4-11 cancer cell lines revealed that α-pyrones 118–120 exhibited weak growth inhibition in the MV4-11 cell line with IC₅₀ values of 90.09, 74.16 and 70.00 µM, respectively.

Two new pyranones, Wortmannine F (121) and G (122) (Fig. 9), were isolated from Talaromyces wortmannii LGT-4, which is an endophytic fungus derived from Tripterygium wilfordii (Zhao et al. 2019). These new isolates were tested for monoamine oxidase (MAO), AChE, and phosphoinositide 3-kinase α (PI3Kα) inhibitory activities, and metabolites 121 and 122 demonstrated potent PI3Kα inhibition with IC₅₀ values of 25 and 5 μM, respectively.

Novel isochromanes, named (S)-3,6-dihydroxy-8-methoxy-3-methylisochroman-4-one(123) and (R)-3,6-dihydroxy-8-methoxy-3-methylisochroman-4-one(124), as well as 6-methoxy-3-methylisochromane-3,8-diol (125), 3,6-dimethoxy-3-methylisochroman-8-ol (126) and 3,6,7,8-tetramethoxy-3-methylisochromane (127), were derived from the endophytic fungus Aspergillus fumigatus, isolated from the fruiting body of Cordyceps sinensis (Fig. 9) (Li et al. 2018b). The antitumor activity of these novel metabolites was evaluated in MV4-11 and MDA-ME-231 cancer cell lines. Compounds 123 and 125 demonstrated intermediate activity in the MV4-11 cell line with the IC₅₀ values of 38.39 μM and 30.00 μM, respectively.

The potato endophytic fungus, Bipolaris eleusines, yielded a novel chromone (S)-5-hydroxyl-2-(1-hydroxyethyl)-7-methylchromone (128, Fig. 9) when the fungal strain was cultured on potato dextrose agar (PDA) medium (He et al. 2018). This secondary metabolite displayed weak inhibitory activity towards S. aureus subsp. with an inhibition rate of 56.3%, at a concentration of 128 μg/mL (penicillin G: 99.9% at 5 μg/mL).

A pair of enantiomers (+)- and (−)-alternarilactone A (129 and 130, Fig. 9) were derived from the fungal strain Alternaria sp. hh930. This endophyte was isolated from fresh stems of Isodon sculponeatus (Tang et al. 2019b). These enantiomers were evaluated for their inhibitory potential in corticosterone-induced apoptosis in PC12 cells, together with their antimicrobial activities. Both derivatives failed to demonstrate significant antibacterial or antifungal effects against the Gram-positive pathogen S. aureus, the Gram-negative pathogen Salmonella typhi, and the plant pathogenic fungi Gaeumannomyces graminis and Verticillium cinnabarium. However, these metabolites possessed weak cytoprotective activity (cell survival (%) of 129 and 130 revealed as 64.86 and 61.86%, respectively) against corticosterone-induced apoptosis in PC12 cells. These secondary metabolites are the first examples of dibenzo-α-pyrones possessing a diepoxy-cage-like moiety.

Quinones, anthrones and related secondary metabolites

A new hydroanthraquinone, nigrosporone A (131), and a new naturally isolated nigrosporone B (132) (Fig. 10) were produced by the strain Nigrospora sp. BCC 47789, an endophytic fungi isolated from the leaf of Choerospondias axillaris (Roxb.) (Kornsakulkarn et al. 2018). The two novel isolates were subjected to assays to assess their antiplasmodial (P. falciparum, K1), antimycobacterial (M. tuberculosis H37Ra), antibacterial (B. cereus and Enterococcus faecium) activities and cytotoxic potential in cancer cell lines (KB, MCF-7, and NCI-H187) and non-cancerous Vero cells. Metabolite 132 showed significant biological activities against P. falciparum (IC₅₀ 10.81 μM), M. tuberculosis (MIC 172.25 μM), B. cereus (MIC 21.53 μM), and E. faecium (MIC 10.78 μM). It also demonstrated selective cytotoxicity in NCI-H187 and Vero cells with IC₅₀ values of 0.25 and 0.72 μM, respectively. Additionally, isolate 131 demonstrated only moderate to weak cytotoxicity in MCF-7 and NCI-H187 cancer cells (IC₅₀ 110.36 and 13.69 μM).

Fig. 10

The structures of novel quinones (131–135, 136–138) isolated from endophytic fungi

The fungus Neofusicoccum austral SYSU-SKS024, an endophyte isolated from the branches of the mangrove plant Kandelia candel, produced three new ethylnaphthoquinone derivatives, neofusnaphthoquinone A (133), 6-(1-methoxylethy1)-2,7-dimethoxyjuglone (134), and (3R,4R)-3-methoxyl-botryosphaerone D (135) (Fig. 10) (Cui et al. 2018). These metabolites were evaluated for their indoleamine 2,3-dioxygenase (IDO) inhibitory activity. All isolates displayed promising effects against IDO. In particular, compound 134 revealed superior cytotoxicity with an IC₅₀ value of 0.11 μM.

A new anthraquinone (136, Fig. 10), isolated and identified from the cultures of Colletotrichum sp. JS-0367, an endophytic fungus isolated from the leaves of the mulberry tree Morus alba L. (Song et al. 2018a), and an analogue of this metabolite, evariquinone (Song et al. 2018a), which was also derived from Colletotrichum sp., demonstrated potent neuroprotection against excessive glutamate-induced apoptosis in the immortalized murine HT22 hippocampal neuronal cell line. With regards to the difference between metabolite 136 and evariquinone, compound 136 contains a methoxy substituent at position C-2, while evariquinone has a hydroxyl group at the same position.

Two new cryptic 3,4-dihydronaphthalen-(2H)-1-one (1-tetralone) derivatives, aspvanicin A (137) and its epimer aspvanicin B (138) (Fig. 10) were obtained from the ethyl acetate extract of a co-culture of the endophytic fungus Aspergillus versicolor KU258497 (an endophyte isolated from fresh, healthy leaves of Eichhornia crassipes, family Pontederiaceae, from the shores of the River Nile in Mansoura, Egypt) and the bacterium B. subtilis 168 trpC2 on a solid rice medium (Abdelwahab et al. 2018). The isolates were assessed for their antiproliferative activity in the mouse lymphoma cell line L5178Y. Aspvanicin B (138) demonstrated cytotoxic potential with an IC₅₀ value of 22.8 µM when compared to kahalalide F as a standard antiproliferative agent (IC₅₀ = 4.3 µM).

Other polyketides

A new xanthoquinodin B9 (139, Fig. 11) was isolated from the endophytic fungus Chaetomium globosum 7s-1, obtained from the leaves of Rhapis cochinchinensis (Tantapakul et al. 2018). The antibacterial (against Gram-positive bacteria B. cereus, S. aureus, and MRSA, as well as Gram-negative bacteria E. coli, P. aeruginosa, and Salmonella typhimurium), antimalarial (against Plasmodium falciparum), anti-mycobacterial (against Mycobacterium tuberculosis H37Ra) and antitumor (in KB, MCF7, and NCI-H187) activities of this novel xanthoquinodin were evaluated. Compound 139 showed significant activity against B. cereus with an MIC value of 0.87 µM, as well as against S. aureus and MRSA with MIC values ranging from 0.87 to 1.75 µM. Moreover, the antimalarial activity against Plasmodium falciparum (IC₅₀ = 2.57 µM) demonstrated satisfactory inhibition, with only weakly activity in the anti-TB assay. Isolate 139 showed promising cytotoxicity in the selected cancer cell lines (KB, MCF-7, and NCI-H187) with IC₅₀ values of 7.04, 18.40, and 0.98, respectively.

Fig. 11

The structures of novel polyketides (139–149) isolated from endophytic fungi

Aspergillus sp. TJ23, isolated from fresh leaves of Hypericum perforatum L., yielded a new polyketide, asperetide (140), and a new prenylxanthone derivative, asperanthone (141) (Fig. 11) (Qiao et al. 2018). These two novel metabolites have been evaluated for their anticancer activities in B16, MDA-MB-231, 4T1, HepG2, and LLC cells. Compound 141 displayed a weak inhibitory activity in HepG2 cells with an IC₅₀ value 35.5 μM.

Four novel polyketide heterodimers, cytorhizins A–D (142–145) (Fig. 11), were obtained from the endophytic fungus Cytospora rhizophorae A761, isolated from the plant Morinda oficinalis (Liu et al. 2019b). These polyketides were evaluated for cytotoxicity in human cancer cell lines (HepG-2, MCF-7, SF-268, and NCIH460). Cytorhizin A and cytorhizin D displayed weak cytotoxic activity with IC₅₀ values ranging from 29.4 to 68.6 μM. In the E. coli and S. aureus antibacterial screening, these isolates failed to demonstrate any significant antimicrobial activity at a concentration of 100 μM.

The fungal isolate Emericella sp. XL029 was obtained from the leaves of Panax otoginseng and yielded four novel polyketides, emericelactones A–D (146–149) (Fig. 11) (Pang et al. 2018). The one strain-many compounds (OSMAC) approach was applied to Emericella sp. XL029, indicating that more diverse secondary metabolites were produced, including compounds 146–149, when cultivated in the solid medium of rice plus malt extract broth. All isolated secondary metabolites demonstrated moderate antimicrobial activities against three agricultural pathogenic fungi (Verticillium dahliae Kleb, R. solani, and Gibberella saubinetii) and two human pathogenic bacteria (M. lysodeikticus and S. typhi) with MIC values ranging between 25 and 50 µg/mL.

Four new polyketides, namely bellidisins A–D (150–153, Fig. 12), were isolated from the rice fermentation extract of the fungus Phoma bellidis, an endophyte isolated from the healthy leaf tissue of Tricyrtis maculate (Wang et al. 2019b). All novel secondary metabolites evaluated in human cancer cell lines (HL-60, A549, SMMC-7721, MCF-7, and SW480). Derivative 153 demonstrated considerable cytotoxicity with IC₅₀ values ranging from 3.40 to 15.25 μM, stronger than that of cisplatin (4.86–27.70 μM).

Fig. 12

The structures of novel polyketides (150–166) isolated from endophytic fungi

Paraconiothyrium sp. SW-B-1, an endophytic fungus isolated from the seaweed Chondrus ocellatus Holmes, produced five new polyketides, designated as paralactonic acids A–E (154–158) (Fig. 12) (Suzuki et al. 2019). All metabolites were evaluated for potential antimicrobial activity against Gram-positive and Gram-negative bacteria, yeast, and other fungal strains by the agar diffusion assay. Only paralactonic acid E (158) showed moderate antibacterial activity against S. aureus NBRC 13276 (24 mm) at a concentration of 100 μg/disk (MIC value: 3.2 μg/mL). In addition, all isolates were tested for their inhibitory effect against Ca²⁺ signal transduction using the hypersensitive drug screening procedure based on the YNS17 [Saccharomyces cerevisiae (zds1Δ erg3Δ pdr1Δ pdr3Δ)] strain. Notably, only derivative 158 exhibited a dose-dependent growth-restoring activity around the inhibition zone in the mutant yeast strain YNS17.

Two new secondary metabolites, the polyketide paecilin D (159) and the maleic anhydride cordyanhydride A methyl ester (160), were obtained from fungal strains Talaromyces stipitatus DgCr2 2.1b and Talaromyces sp. VrTrb2 1.1, respectively (Fig. 12) (Silva et al. 2018). The endophyte of Talaromyces stipitatus DgCr2 2.1b was isolated from the medicinal plant roots of Duguetia stelechantha, while the endophyte of Talaromyces sp. VrTrb2 1.1 strain was isolated from Victoria amazonica, collected in the Amazonian rainforest. Novel metabolites have been isolated from the antimicrobial extracts of the examined strains. Paecilin D (159) was reportedly active against C. albicans and Candida tropicalis (MIC: 15.6 and 31.3 μg/mL).

The endophytic fungus Byssochlamys spectabilis, isolated from leaf tissue of the traditional Chinese medicinal plant Edgeworthia chrysantha, yielded a polyketide-derived octaketide dimer with a novel carbon skeleton, designated bysspectin A (161), and two new precursor derivatives, bysspectins B and C (162 and 163) (Fig. 12) (Wu et al. 2018). Only derivative 163 displayed weak inhibition on strains including E. coli and S. aureus with MIC values of 32 and 64 µg/mL, respectively. The novel octaketide dimer (161) possessed highly hydrophobic properties in terms of its two long and bulky aliphatic ketone carbon chains coupled with a 2-phenylbenzo[b]furan core. Therefore, compound 161 was assayed for its inhibitory effects towards human carboxylesterases hCE1 and hCE2. Notably, bysspectin A (161) was a novel and highly selective inhibitor against hCE2 with an IC₅₀ value of 2.01 µM.

Two new prenylated polyketides, ascomfurans C (164) and ascomarugosin A (165), were isolated from the endophytic fungus Ascomycota sp., obtained from the mangrove plant Kanelia candel (Fig. 12) (Liu et al. 2018a). Ascomarugosin A exhibited weak anti-inflammatory activity with an IC₅₀ value of 72.3 µM. Isoshamixanthone (166, Fig. 12), a new stereoisomeric pyrano xanthone was produced from the endophytic fungal strain Aspergillus sp. ASCLA obtained from leaf tissues of the medicinal plant Callistemon subulatus (Kamel et al. 2019). This metabolite demonstrated moderate to high activities (11–16 mm) against microbial strains including S. aureus, P. aeruginosa, C. albicans, S. cerevisiae, B. cereus, and B. subtilis ATCC 6633. However, in the antitumor screening, compound 166 displayed no cytotoxic effects in KB-3-1 and its multidrug resistant subclone, KB-V1.

Eleven new polyketones named diaporthsins A–K (167–177) (Fig. 13) were obtained from the fermentation of Diaporthe sp. JC-J7, isolated from the stems of Dendrobium nobile (Hu et al. 2018). Amongst new isolated metabolites, compounds 167–169, 171, 174 and 176 were evaluated for their antihyperlipidemic activities. Diaporthsin E (171) exhibited lipid lowering effects on triglycerides in steatotic L-02 cells, with an inhibition ratio of 26% at a concentration of 5 µg/mL Lovastatin (31.80%) used as positive control drug. All other screened isolates displayed inhibition ratios of < 10%. In addition, most of the new polyketones were esterified products of diaporthsin B (168), which occurs in fungal biotransformation.

Fig. 13

The structures of new polyketides (167–186) produced by endophytic fungi

The chemical investigation on the medicinal mangrove derived endophytic fungus Dothiorella sp., isolated from the stem of the mangrove Xylocarpus granatum Koenig, resulted in three new cytosporone derivatives, dothiorelones K–M (178–180) (Fig. 13) (Zheng et al. 2019). Their inhibitory activities against α-glucosidase, antibacterial activity and cytotoxic activity have been evaluated. In an α-glucosidase inhibitory assay, the new metabolites 178 and 180 exhibited inhibitory activities with IC₅₀ values of 22.0 and 77.9 μg/mL, respectively. Moreover, these two compounds demonstrated moderate antibacterial activities against S. aureus (MIC = 50 μg/mL). However, none of the new dothiorelones compounds demonstrated cytotoxic activity in the three human cell lines (A549, HeLa, and HepG2) at a concentration of 10 μg/mL.

The symbiotic strain Fusarium oxysporum ZZP-R1 is an endophytic fungus derived from the coastal plant Rumex madaio Makino, one of the traditional Chinese medicinal herbs used to treat inflammation and intoxication. This fungus produced two novel secondary metabolites, fusariumins C (181) and D (182) (Fig. 13) (Chen et al. 2019a). Compound 182 displayed a moderate inhibitory effect against S. aureus with an MIC value of 25.0 μM, while metabolite 181 showed potent activity against S. aureus with an MIC value of 6.25 μM.

Three novel compounds, identified as xylarolide A (183), diportharine A (184), and xylarolide B (185) (Fig. 13), were isolated from the endophytic fungus Diaporthe sp., which was obtained from Datura inoxia (Sharma et al. 2018). All the derived novel secondary metabolites were screened for their antioxidant, antibacterial and cytotoxicity activities. Xylarolide A (183) showed significant growth inhibition in MIAPaCa-2 and PC-3 cells with IC₅₀ values of 20 and 14 µM, respectively. In addition, metabolite 183 displayed significant DPPH scavenging activity (EC₅₀: 10.3 µM) using ascorbic acid as the positive control drug. However, these metabolites failed to demonstrate antibacterial activity against S. aureus, P. aeruginosa, and E. coli at a concentration of 62.5 µM. A new furan derivative named 3-(5-oxo-2,5-dihydrofuran-3-yl)propanoic acid (186) (Fig. 13) was isolated from the endophytic Aspergillus tubingensis of Decaisnea insignis (Griff.) Hook.f. & Thomson (Yang et al. 2018b). This metabolite exhibited potent antifungal activity against F. graminearum (MIC = 16 µg/mL) and moderate antibacterial activity against Streptococcus lactis (MIC = 32 µg/mL).

Nine new natural compounds, dothiorelones (187–192), 5-hydroxy-7-methoxy-4,6-dimethyl-2-phenylisoindoline-1,3-dione (193), (13R)-diaporphthalide A (194) and (9S, 17R, 19S, 6Z, 10E, 14E)-diaporlactone A (195) (Fig. 14), were isolated from Diaporthe pseudomangiferaea, an endophytic fungus obtained from the leaves of the toxic Chinese plant Tylophora ouata (Liu et al. 2018b). All the secondary metabolites were investigated for anti-fibrosis, cytotoxic, antidiabetic, anti-oxidative, antibacterial, and anti-inflammatory activities. Only two metabolites, acetoxydothiorelone B (187) and dothiorelone L (190), inhibited the transforming growth factor beta (TFG-β) induced activation of the human lung MRC-5 fibroblasts by 17.4% and 59.2% at 10 μM, respectively. The isolates showed moderate to weak activities in other performed assays.

Fig. 14

The structures of new polyketides (187–201) produced by endophytic fungi

The strain, Aspergillus sp. which was isolated from the root of Tripterygium wilfordii produced four novel butenolides, terrusnolides A–D (196–199) (Fig. 14) (Qi et al. 2018). The in vitro anti-inflammatory effects of these isolates were evaluated using LPS-stimulated RAW264.7 macrophages. The butenolides exhibited promising inhibitory effects on the production of IL-1β (16.21–35.23 μM), TNF-α (19.83–42.57 μM), and NO (16.78–38.15 μM) in LPS-induced RAW264.7 cells, comparable with the positive control indomethacin. In addition, compounds 198 and 199 showed a weak inhibition against the BACE1 and AChE inhibitory activity.

Two new secondary metabolites, β-lactone polonicin A (200) and enoic acid polonicin B (201) (Fig. 14), were isolated from Penicillum polonicum obtained from the fruits of Camptotheca acuminate Decne (Wen et al. 2019). The antidiabetic properties of the isolated novel compounds were examined in L6 cells at a concentration of 30 µg/mL. Both isolates increased the rate of glucose uptake by 1.8 and 1.5 times, respectively. In addition, a L6 cell line stably expressing IRAP-mOrange was used to evaluate the effects of polonicin A on the glucose transporter type 4 (GLUT4) translocation. Incubation with derivative 200 increased the fluorescence intensity in the L6 cell membranes by 2.1-fold.

The fungus Exserohilum rostratum LPY-001 has been isolated from leaf tissue of Gymnadenia conopsea, a Tibetan medicinal plant known as wangle (Chinese), which has been traditionally used for the treatment of cough, asthma, and other syndromes. This fungus produced a new oxacyclododecindione-type macrolactone designated (13R,14S,15R)-13-hydroxy-14- deoxyoxacyclododecindione (202, Fig. 15) (Lin et al. 2018). Cytotoxicity assays for compound 202 were performed in the HCT-8, A2780, BGC-823, Bel-7402, and A549 cell lines. This secondary metabolite displayed weak selective cytotoxicity in A549 lung cancer cells with an IC₅₀ value of 9.2 µM.

Fig. 15

The structures of novel polyketides (202–210) produced by endophytic fungi

Curtachalasins A (203) and B (204) (Fig. 15) are a new family of cytochalasans with a 5/6/6/6-fused tetracyclic skeleton, obtained from the endophytic fungus Xylaria curta E10, isolated from the healthy stem tissues of potato (Solanum tuberosum) (Wang et al. 2018b). These new secondary metabolites were screened against four bacterial strains (E. coli, S. aureus, Salmonella enterica, and P. aeruginosa), as well as against four fungal species (C. albicans, Epidermophyton floccosum, Trichophyton rubrum, and Microsporum gypseum). Both compounds displayed weak antifungal activity (70.3% and 68.4% inhibitory percentage, respectively) against M. gypseum at a concentration of 200 μM. Furthermore, three novel secondary metabolites, curtachalasins C–E (205–207) (Fig. 15) were also identified from the endophytic fungus Xylaria cf. curta, derived from the healthy stem tissues of potato (Solanum tuberosum) (Wang et al. 2019a). Among these curtachalasins, curtachalasin C and curtachalasin E were screened for their antifungal activities against C. albicans with the drug resistant genes Cdr1, Cdr2 and Mdr1; however, in this case the MIC₅₀ value of fluconazole was higher than 500 μg/mL. Derivative 205 demonstrated dose-dependent resistant reversal activity when combined with 10 μg/mL fluconazole. The inhibitory ratio against this strain was close to 50%, which was a significant improvement compared to fluconazole alone. Regarding the other screened new isolates, curtachalasin E showed no or weak resistance reversal activity at a concentration of 128 μg/mL.

New chlorinated bianthrones, allianthrone A (208) and its two diastereomers (allianthrones B (209) and C (210) (Fig. 15), were isolated from the co-cultures of two different developmental stages of a marine alga-derived Aspergillus alliaceus (teleomorph: Petromyces alliaceus) strain (Mandelare et al. 2018). Metabolite 208 showed weak cytotoxic activity in HCT-116 colon cancer and SK-Mel-5 melanoma cell lines with IC₅₀ values of 9.0 and 11.0 μM, respectively.

Alkaloids and other nitrogen related compounds

Fungal endophytes are capable of forming biologically active secondary metabolites including alkaloids, various amides and other nitrogen containing compounds. In this section we describe alkaloids and their biological properties from fungal genera such as Fusarium, Penicillium, Aspergillus, and Mucor.

The endophytic fungus Fusarium chlamydosporum, isolated from the root of Suaeda glauca, yielded one new indole alkaloid 211 (Fig. 16) (Wang et al. 2018d). Compound 211 showed significant phytotoxic activity against the radicle growth of Echinochloa crusgalli with an inhibition rate above 80%, even at a concentration of 1.25 µg/mL, which was superior to the positive control 2,4-dichlorophenoxyacetic acid. Another endophytic fungus, has been identified as a Mucor sp., was obtained from the medicinal plant Centaurea stoebe. It produced a new indole compound, terezine E (212, Fig. 16) (Abdou et al. 2018). The antiproliferative and cytotoxic activities of the isolate 212 were tested in HUVEC, K-562, and HeLa cancer cell lines. In both assays, terezine E exhibited significant antiproliferative activity with GI₅₀ values of 28.02 and 27.31 µM against HUVEC and K-562, respectively. Furthermore, this indole derivative revealed a CC₅₀ value of 60.43 µM in the HeLa cancer cells. Compound 212 demonstrated only modest antifungal properties against A. terreus strain with an MIC value of 39.7 µg/mL.

Fig. 16

The structures of the alkaloids and other nitrogen related secondary metabolites (211–221) isolated from endophytic fungi

Four new aromatic butenolides, asperimides A–D (213–216, Fig. 16), were derived from solid cultures of the tropical endophytic fungus Aspergillus terreus, isolated from fresh, healthy leaves of Suriana maritima L. (Liao et al. 2018). Metabolites 215 and 216 inhibited NO production with IC₅₀ values of 0.78 and 1.26 μM, respectively, in LPS-stimulated RAW264.7 cells. Another endophytic strain, Penicillium janthinellum, isolated from Panax notoginseng, produced three new secondary metabolites, including rotational isomers brasiliamide J-a (217), brasiliamide J-b (218), and peniciolidone (219) (Fig. 16) (Xie et al. 2018a). A total of 180 fungal isolates, belonging to 20 genera and 47 species, were obtained from the roots, stems, and leaves of Panax notoginseng. Among the endophytic fungus, Penicillium janthinellum SYPF 7899 displayed the strongest antibacterial activity and has been studied for its production of secondary metabolites. Isolates 217–219 were screened for antimicrobial effects against several pathogenic bacteria including S. aureus, B. subtilis, P. aeruginosa, Klebsiella pneumonia, and E. coli. Brasiliamide J-a and J-b exhibited significant inhibitory activities against B. subtilis and S. aureus with MIC values of 15 and 18 μg/mL, respectively. Moderate inhibition against B. subtilis (MIC = 35 μg/mL) and S. aureus (MIC = 39 μg/mL) was observed with derivative 219.

Fusarithioamide B (220) (Fig. 16), a new aminobenzamide derivative with an unprecedented carbon skeleton, was isolated from the strain Fusarium chlamydosporium, an endophytic fungus obtained from Anvillea garcinii (Burm.f.) DC. leaves (Asteraceae) (Ibrahim et al. 2018). Compound 220 demonstrated selective antifungal activity against C. albicans (MIC = 1.9 µg/mL; inhibition zone diameter = 14.5 mm) when compared to clotrimazole (MIC = 2.8 µg/mL; inhibition zone diameter = 17.9 mm), as well as high antibacterial potential against strains such as S. aureus (MIC = 3.1 µg/mL; inhibition zone diameter = 17.4 mm), B. cereus (MIC = 2.5 µg/mL; inhibition zone diameter = 23.0 mm), and E. coli (MIC = 3.7 µg/mL; inhibition zone diameter = 25.1 mm) compared to ciprofloxacin (MIC = 3.4, 2.9 and 3.9 µg/mL; inhibition zone diameter = 15.3, 21.2 and 25.6 mm). In addition, the strong cytotoxic effects of 220 in KB, HCT-116, BT-549, MCF-7, SKOV-3, and SK-MEL cell lines indicated that the new metabolite displayed selectivity and potential efficacy with IC₅₀ values of 0.09, 0.21, 1.23, and 0.59 µM, respectively, compared to doxorubicin (IC₅₀ values: 0.046, 0.05, 0.321, and 0.24 µM, respectively).

A new dihydroquinolone derivative, aflaquinolone H (221, Fig. 16) was isolated from the endophytic Aspergillus versicolor, isolated from the leaves of the Egyptian water hyacinth Eichhornia crassipes (Ebada et al. 2018). This secondary metabolite exhibited moderate antiproliferative activity (IC₅₀ = 10.3 μM), reporting no potential antimicrobial activity.

Conclusions

In summary, we have outlined a total of 221 novel natural products synthesized by 67 species of endophytic fungi, which belong to three phyla, 31 genera and were collected from 10 countries or sources, along with their various biological properties. Among these 67 strains, five endophyte genera, including Aspergillus, Fusarium, Penicillium, Xylaria and Talaromyces, were most frequently studied in the year 2018 than any other isolated strains. These endophytic crude extracts (in polar solvents, normally ethyl acetate (EtOAc) extracts) were yielded after appropriate fermentation and exhibited various bioactivities. Most individually purified novel natural products demonstrated promising cytotoxic, antimicrobial, or anti-inflammatory activities. Although some isolated new compounds displayed weak activity, their unique structures could lay interesting ground for further investigations. Thus, these microbial secondary metabolites are valuable as they enrich chemical diversity of bioactive molecules and may support the drug-development process in the pharmaceutical industry. However, plant endophytes remain poorly understood with regard to their pharmaceutical properties, especially those related to their use as potentially novel drug-candidates. Because this potential has been appreciated only recently in the field of medicinal chemistry, phytochemical aspects of these metabolites are still the most thoroughly characterized. New approaches for endophytically synthesized natural compounds include studying their biosynthetic pathways and biotransformation reactions to improve product yields, relationships between host plants and endophytic fungi producing the bioactive compounds, as well as the target evaluation of new secondary metabolites from a medicinal chemistry point of view. In addition, the plethora of novel compounds produced by endophytic fungi will open new avenues to investigate their medicinal utility. We believe that this is a fertile ground for research, which will continue to grow in the coming years.