Keywords

Introduction

The term “endophytes” refers to microorganisms inhabiting plant tissues for at least a part of their life cycle without causing any visible damage to their host plant. Endophytic microorganisms can be fungi or bacteria, including actinobacteria, although the most frequently encountered endophytes are fungi [1]. Endophytic fungi are a highly diverse polyphyletic group of microorganisms [2, 3].

Most plants that have been previously studied harbor endophytic microorganisms [4, 5], and there is evidence, found in fossilized tissues of plants, that endophyte–host relationships have evolved from the time high plants first appeared on the earth [6, 7].

The asymptomatic colonization of plants by endophytic microorganisms is the result of a balance of antagonisms between endophytic virulence and plant defense [8]. If destabilization in this balance occurs, the fungal strain may perish or the plant may succumb. Recently, it was suggested that the plant–endophyte interaction might be much more complex than the balanced antagonism hypothesis, because in the same way that plants have to defend themselves against the fungal virulence factors , the endophytes also should have resistance mechanisms to counter the toxic secondary metabolites produced by the host plant [2].

While in a symbiotic relationship, both endophyte and host plant are benefited. The plant provides to endophytic microorganism protection, nutrients, and dissemination to the next generation of hosts [9–11]. On the other hand, the endophytes are believed to be responsible for the adaptation of plants to abiotic stresses such as drought, high temperature and salinity, harmful effects of light, and metal toxicity, as well as to biotic factors such as herbivores, nematodes, insects, and pathogens [12–21]. The host protection is achieved mainly by natural products produced by endophytic microorganisms [3, 22]. Besides that, endophytes are also suggested to be capable of inducing host defense mechanisms [23].

The discovery of some endophytes producing biologically important secondary plant metabolites and their analogues—such as the anticancer drugs paclitaxel (1, Fig. 8.1) [24], camptothecin (2, Fig. 8.1), 9-methoxycamptothecin (3, Fig. 8.1), and 10-hydroxycamptothecin (4, Fig. 8.1) [25–28]; the anticancer drug lead compounds podophyllotoxin(5, Fig. 8.1) and deoxypodophyllotoxin (6, Fig. 8.1) [29–31]; the antidepressant hypericin (7, Fig. 8.1) along with emodin (8, Fig. 8.1) [32, 33]; and the natural insecticides azadirachtin A (9, Fig. 8.1) and B (10, Fig. 8.1) [34]—have fueled the investigation on these groups of microorganisms. Although endophytes capable of synthesizing plant compounds are continually being discovered, it has not yet been possible to utilize them for the sustained production of the desired plant compounds [35].

Abb. 8.1
figure 1

Secondary metabolites common to endophytes and host plants

Full size image

There are many hypothesized mechanisms proposed for the production of plant secondary metabolites by associated microorganisms. In some cases, it was suggested that the interactions between endophytes and their plant host contribute to the production of these bioactive molecules [36]. In others, it has been shown that biosynthetic pathways of the same compounds evolve independently in fungi and plants [36–39], evidencing the unprecedented metabolic arsenal owned by fungal endophytic strains.

Indeed, endophytes are a remarkable reservoir of genetic diversity and their secondary metabolism may be activated by the metabolic interactions with the host, making the endophytic microorganisms a rich source of new biologically active natural products [6]. There are many specialized reviews covering the biological activities of the new compounds that have been isolated from endophytic and associated marine derived fungi [2, 3, 6, 15, 22, 23, 40–50].

Here, we reviewed the novel compounds isolated from these groups of microorganisms from 2012 to April 2014, mentioning their biological activities (Table 8.1). The novel metabolites were classified into four major groups based on their biosynthetic pathways , such as polyketide and fatty acid , phenylpropanoid and terpenoid derivatives as well as N-containing compounds. In the “N-containing compounds” group, compounds from nonribosomal peptide pathway and alkaloid derivatives were included, including those from polyketide pathway whose oxygen atom was substituted by nitrogen. Regarding other classification groups, compounds from mixed biosynthetic origin were not particularized, thus being included in one of the matching biosynthetic groups. In the text, only secondary metabolites containing any interesting structural novelty or relevant biological activity were highlighted.

Table 8.1 Novel secondary metabolites from endophytic fungi
Full size table

Novel metabolites with biological or chemical relevance

Five new decalactone derivatives, coryoctalactones A–E (29–33, Fig. 8.2), were obtained from Corynespora cassiicola JCM 23.3, an endophyte of the mangrove plant Laguncularia racemosa (Combretacaeae). These new polyketides share a carbon skeleton containing an aromatic ring attached to an octalactone ring system, which have not been reported and are unusual. None of them were active in a panel of bioassays to evaluate cytotoxic activity against murine lymphoma cells, L5178Y cells, antimicrobial activity against several pathogenic microorganisms, and antitrypanosomal activity [51].

Abb. 8.2
figure 2figure 2figure 2figure 2figure 2figure 2figure 2

a–g New polyketide and fatty acid derivatives from endophytic fungi

Full size image

Several metabolites were obtained from Microsphaeropsis arundinis PSU-G18, endophytic fungus from the leaves of Garcinia hombroniana, including one new modiolin, microsphaerodiolin (71, Fig. 8.2), and seven novel phthalides, microsphaerophthalides A–G (72–78, Fig. 8.2). Interestingly, compounds 74–78, which are 3-oxygenated phthalides, are rare natural products. Only compounds obtained in sufficient amount were submitted to biological tests. Thus, compounds 72, 75, and 76 were tested for antifungal activity. Compound 72 was moderately active against Microsporum gypseum SH-MU-4 with a minimum inhibitory concentration (MIC) value of 64 µg/mL, whereas compound 76 exhibited moderate antifungal activity against Cryptococcus neoformans with a MIC value of 64 µg/mL. Additionally, compound 76 showed mild activity against M. gypseum with a MIC value of 200 µg/mL [52].

Five new metabolites, epicocconigrones A and B (82–83, Fig. 8.2), 3-methoxyepicoccone B (84, Fig. 8.2), 3-methoxyepicoccone (85, Fig. 8.2), and 2,3,4-trihydroxy-6-(methoxymethyl)-5-methylbenzaldehyde (86, Fig. 8.2) were obtained from Epicoccum nigrum, an endophytic fungus isolated from the leaves of Mentha suaveolens. These compounds were evaluated for their inhibitory activity against a panel of 16 protein kinases. Compound 82 was active against all tested enzymes, except MEK1 wt. Compounds 84 and 86 inhibited only some kinases. A preliminary structure–activity relationship proposal suggested that a β-hydroxyl-α,β-unsaturated carbonyl moiety, present in 82, 84, and 86 but absent in inactive compounds (83 and 85), is necessary for the inhibition of protein kinases. Compound 82 showed also strong inhibition against histone deacetylase (HDAC), becoming a promising compound for the development of anticancer drugs [53].

Chaetosidone A (88, Fig. 8.2), a new depsidone, was isolated from Chaetomium sp., an endophytic fungus from the leaves of Zanthoxylum leprieurii. Interestingly, compound 88 is indeed the parent compound in the series of more than 60 orsellinic acid-derived depsidones. This compound exhibited moderate inhibitory activity against Bacillus subtilis and Staphylococcus aureus at a concentration of 40 μg per paper disk, and also moderate cytotoxicity towards brine shrimp larvae (Artemia salina) [54]. Compound 96, named Corynesidone D, corresponds to the same natural product 88, however, it was isolated from C. cassiicola, an endophytic fungus from Gongronema latifolium, and reported by the same time [55].

A new depsidone, excelsional (102, Fig. 8.2), and a new decaline derivative, 9-hydroxyphomopsidin (103, Fig. 8.2), were obtained from Phomopsis sp. CAFT69, an endophytic fungus from Endodesmia calophylloides. These two novel compounds exhibited strong motility inhibition and lysis of zoospores of grapevine pathogen Plasmopara viticola. Although further studies are necessary for understanding the biological mechanisms of motility inhibitory and lytic effects, it is suggested that these metabolites might play a role in the protection of the host plant [56].

The novel glycolipid fusaroside (107, Fig. 8.2) was obtained from Fusarium sp. LN-11, an endophytic fungus isolated from the fresh leaves of the tree Melia azedarach L. Based on 2D NMR experiments, the presence of two hexoses in their pyronose forms was revealed. Both of them were glucose residues having α anomeric configurations. Compound 107 is made of an unusual branched fatty acid and an α,α-trehalose, which is unique in nature since this family of glycolipids has not been reported previously in literature. This glycolipid exhibited moderate toxicity against brine shrimp larvae (A. salina), with the mortality rate of 47.6 % [57] .

Two new dihydroanthracenone derivatives, diaporthemins A and B (109–110, Fig. 8.2), were obtained from Diaporthe melonis, an endophytic fungus isolated from Annona squamosa. Compound 109 was identified as a heterodimer and the first compound possessing a C7–C5ʹ linkage between their monomeric subunits. Besides that, the planar structure of these two compounds was established suggesting they are stereoisomers. The mirror image of their circular dichroism (CD) spectra suggested that they were atropodiastereomers differing in the axial chirality. None of them showed antibacterial activities against the multi-resistant clinical isolate of S. aureus 25697, a susceptible strain of S. aureus ATCC 29213, and Streptococcus pneumoniae ATCC 49619. Curiously, it was proposed that a C7–C5ʹ linkage present in these compounds seems to abolish antibacterial activity [58].

Chlorinated diphenyl ethers have seldom been found as fungal metabolites and three of them were isolated from Pestalotiopsis sp. PSU-MA69, an endophytic fungus from a branch of the mangrove plant Rhizophora apiculata. Pestalotethers A–D (124–127, Fig. 8.2) were isolated and tested against Candida albicans NCPF3153 and C. neoformans ATCC90112 for antifungal activity. Compound 126 was not obtained in sufficient amount for biological tests. Compounds 124 and 125, containing a chlorine atom, exhibited antifungal activity against C. neoformans (MIC value of 200 µ]g/mL) while 127 was inactive. These compounds were also inactive against C. albicans [59].

Annulosquamulin (131, Fig. 8.2), a dihydrobenzofuran-2,4-dione backbone possessing one alkyl side chain and a γ-lactone ring is rarely found when compared to other metabolites from the genus Annulohypoxylon. This compound was isolated from Annulohypoxylon squamulosum BCRC 34022, an endophytic fungus from the stem bark of medicinal plant Cinnamomum sp. Compound 131 exhibited significant cytotoxic activity against human breast adenocarcinoma (MCF-7), non-small cell lung cancer (NCI-H460), and glioblastoma (SF-268) cell lines with IC50 values < 4 µg/mL [60].

Two new members of the naphthoquinone spiroketal family, palmarumycin EG1 (146, Fig. 8.2) and preussomerin EG4 (147, Fig. 8.2), were obtained from Edenia gomezpompae, an endophytic fungus isolated from the leaves of Callicarpa acuminata. Compound 147 exhibited phytotoxic effect when evaluated for its ability to inhibit the seed germination, root elongation, and seedling respiration of Amaranthus hypochondriacus, Solanum lycopersicum, and Echinochloa crus-galli [61].

A novel compound, named as 2,3-didehydro-19α-hydroxy-14-epicochlioquinone B (149, Fig. 8.2), together with two new griseofulvin derivatives, 6-O-desmethyldechlorogriseofulvin and 6ʹ-hydroxygriseofulvin (150–151, Fig. 8.2), were isolated from Nigrospora sp. MA75, an endophytic fungus from the stem of semi-mangrove plant Pongamia pinnata. Compound 149 showed antibacterial activity against methicillin-resistant S. aureus (MRSA, MIC 8 µg/mL), Escherichia coli (4 µg/mL), Pseudomonas aeruginosa (4 µg/mL), Pseudomonas fluorescens (0.5 µg/mL), and Staphylococcus epidermidis (0.5 µg/mL). Interestingly, the activity against E. coli, P. fluorescens, and S. epidermidis was stronger than ampicillin, used as positive control [62].

A new compound containing an inedited skeleton was isolated from an antimicrobial fraction of the ethyl acetate extract of E. nigrum, endophytic from Saccharum officinarum [63]. This unique natural product named epicolactone (157, Fig. 8.2) is a quasisymetrical molecule containing an unprecedented pentacyclic ring system exclude this final part. Analyses of X-ray crystallographic data showed that epicolactone may crystallize as a racemic mixture [63, 64].

Two new azaphilone derivatives, biscogniazaphilone A and B (158–159, Fig. 8.2), were isolated from Biscogniauxia formosana BCRC 33718, an endophytic fungus from the bark of medicinal plant Cinnamomum sp. Although both compounds exhibited antimycobacterial activity against Mycobacterium tuberculosis strain H37Rv, it was observed that 159, possessing one γ-lactone group, was twofold (MIC ≤ 2.52 µg/mL) stronger than 158 (MIC ≤ 5.12 µg/mL), suggesting that the presence of that group plays a possible role in the antimycobacterial activity [65].

Six novel unique spiroketals, chloropestolides B–G (183–188, Fig. 8.2), were obtained from Pestalotiopsis fici, an endophytic fungus from the branches of Camellia sinensis (Theaceae). These new compounds are biosynthesized by naturally occurring Diels-Alder reactions, including reverse electron demand Diels-Alder (REDDA) for compounds 183–185 and normal electron demand Diels-Alder (NEDDA) for 186–188. Despite their novelty, only compound 183 exhibited cytotoxic activity against the stable oncoprotein LMP1 integrated nasopharyngeal carcinoma cells (CNE1-LMP1), malignant melanoma cells (A375), and MCF-7 with IC50 values of 16.4, 9.9, and 23.6 µM, respectively [66].

Dothideomycetide A (193, Fig. 8.2), which is the first polyketide possessing a tricyclic 6,6,6 ring system, similar to that of a terpenoid, was isolated from Dothideomycete sp., an endophytic fungus from the roots of Tiliacora triandra. This compound was isolated together with two new compounds, dothideomycetone A and B (194–195, Fig. 8.2), which are diastereomers and probably derive from an azaphilone [67]. According to the biosynthetic proposal, compound 193 is likely derived from 194. Compound 194 exhibited weak cytotoxicity against acute lymphoblastic leukemia cancer cell line (MOLT-3). Compound 193 exhibited weak cytotoxicity against human cholangiocarcinoma (HuCCA-1), human lung carcinoma (A549), human hepatocellular liver carcinoma (HepG2), and MOLT-3 cell lines. Besides that, compound 193 also exhibited moderate to weak antibacterial activity against S. aureus ATCC 25923 (MIC value of 128 µg/mL) and MRSA ATCC 33591 (MIC value of 256 µg/mL) [67].

Three new C25 steroids, named as norcyclocitrinol A (214, Fig. 8.3), erythro-11a-hydroxyneocyclocitrinol (215, Fig. 8.3), and pesudocyclocitrinol A (216, Fig. 8.3) were isolated from Penicillium chrysogenum P1X, an endophyte from Huperzia serrate. These compounds belong to a class of rare steroids featuring an unusual A/B bicyclic ring system, possibly originated from ergosterol by a carbon-skeleton rearrangement. Compound 214 possesses a tetracyclic C23-steroid skeleton, featuring a previously unreported bisnor C-atom side chain. Compounds were evaluated for their cytotoxic activities against HeLa (adenocarcinoma) and HepG2 cell lines. However, none exhibited a significant cytotoxicity at 20 μM [68].

Abb. 8.3
figure 3figure 3

a, b New terpenoid derivatives from endophytic fungi

Full size image

A new sesquiterpenoid, diaporol A (218, Fig. 8.3), and eight new drimane sesquiterpenoids, diaporols B-I (219–226, Fig. 8.3), were isolated from Diaporthe sp., an endophytic fungus from the mangrove plant Rhizophora stylosa. Compound 218 possesses a new tricyclic framework, with an unusual six-membered lactone. All the compounds had their cytotoxicity against four cell lines assessed, but none of them was active at the concentration of 20 μM [69].

Three novel eremophilane sesquiterpenoids derivatives, MBJ-0011, MBJ-0012, and MBJ-0013 (251–253, Fig. 8.3) were produced by the endophytic fungus Apiognomonia sp., isolated from an unidentified Japanese plant. Compound 251 is an eremophilane derivative possessing an uncommon tetrahydro-α-methyl-5-oxo-2-furanacetic acid moiety. The cytotoxic activities of all compounds against human ovarian adenocarcinoma SKOV-3 cells were assessed. After 72 h of treatment, compound 251 exhibited moderate cytotoxic activity with the IC50 value of 3.4 µM [70].

Pestalotiopens A and B (255–256, Fig. 8.3), produced by Pestalotiopsis sp., an endophyte from Rhizophora mucronata, are novel types of natural products with unprecedented hybrid carbon skeletons derived from a drimane-type sesquiterpene and a polyketide. Thus, compound 255 is a sesquiterpene and cyclopaldic acid-derived hybrid, whereas compound 256 also contains an additional triketide subunit linked through a cyclic acetal. In antimicrobial assays against a panel of six bacterial strains, compound 255 exhibited moderate antimicrobial activity against Enterococcus faecalis (MIC value between 125 and 250 μg/mL), whereas compound 256 was inactive [71].

Asperterpenoid A (258, Fig. 8.3) was isolated from a salt rice solid culture of the mangrove fungus Aspergillus sp., endophyte from Sonneratia apetala, and was identified as a novel sesterterpenoid with a new carbon skeleton, containing a planar 5/7/(3)6/5 pentacyclic structure. Its chemical structure was confirmed by single-crystal X-ray diffraction experiments. The inhibitory activity of compound 258 against M. tuberculosis protein tyrosine phosphatase B (mPTPB) was evaluated, showing it as a strong inhibitor, with an IC50 value of 2.2 μM [72].

New cyclohexenone–sordaricin derivatives xylarinonericins A–C (259–261, Fig. 8.3) were isolated from Xylaria plebeja PSU-G30, an endophytic fungus from G. hombroniana. In addition to peculiar tetracyclic moiety, which forms the known sodaricin structure [73], these compounds possess an unusual ester moiety at C6 of the sordaricin skeleton instead of a carboxylic acid. Besides that, compound 261 has a unique feature with an ester unit instead of an ether group at C19. All these compounds were tested against C. albicans ATCC 90028 and C. neoformans ATCC 90113, but none was active against both fungal strains at a concentration of 200 µg/mL [74].

Emericellenes A−E (263–267, Fig. 8.3) were obtained based on a bioactivity-guided fractionation of extracts of Emericella sp., an endophytic fungus from Astragalus lentiginosus. These new sesterterpenoids possess a scaffold similar to that of verticillane-type diterpenoids, with a 12-membered cyclic fused to another cyclic moiety. In addition, these compounds hold an isoprene unit, and compounds 264–267 hold a carboxylic acid, while 263 possesses a carbonyl moiety. Thus, the structures of these compounds represent a unique class of sesterterpenoid metabolites bearing a novel emericellane-type bicarbocyclic ring system. Their cytotoxic activities were evaluated against six tumor cell lines. All compounds were not cytotoxic up to a concentration of 5.0 μM [75].

The new butyrolactone derivative 283 (Fig. 8.4), isolated from Aspergillus terreus var. boedijnii (Blochwitz), an endophyte from red marine alga Laurencia ceylanica J. Agardh, displayed a considerable inhibitory activity against the enzyme β-glucuronidase (IC50 6.2 μΜ), this activity being stronger than that provided by the positive control glucosaccharo-(1,4)-lactone (IC50 value of 48.4 μΜ) [76].

Abb. 8.4
figure 4

New phenylpropanoid derivatives from endophytic fungi

Full size image

The novel cyclic depsipeptide pullularin E (286, Fig. 8.5) and the new linear peptide pullularin F (287, Fig. 8.5) were produced by Bionectria ochroleuca, an endophytic fungus isolated from the mangrove plant Sonneratia caseolaris. The cytotoxicity assays in L5178Y cell line showed that antiproliferative properties were prevalent among some pullularin cyclic analogues, with IC50 values ranging between 0.1 and 6.7 μg/mL, whereas the linear compound 287 did not exhibit any cytotoxic activity at the tested dose of 10 μg/mL [77].

Abb. 8.5
figure 5figure 5figure 5

a–c New N-containing compounds from endophytic fungi

Full size image

The new spirotryprostatin A derivative 295 (Fig. 8.5), produced by Aspergillus fumigatus , an endophyte from Erythrophloeum fordii Oliv., is related to diketopiperazines derived from amino acids proline and tryptophan. However, unlike those generally reported diketopiperazines, compound 295 possesses a spirocyclic moiety, typical in the spirotryprostatin class. Neither significant anti-inflammatory activity, nor detectable cytotoxicity (IC50 > 10 μM) toward five human tumor cell lines were displayed by this compound during the bioassay [78].

A novel diketopiperazine derivative containing an isopentenyl moiety attached (299, Fig. 8.5) was isolated from P. chrysogenum MTCC 5108, an endophytic fungus from the mangrove plant Porteresia coarctata. The crude extract of P. chrysogenum had been active against Vibrio cholerae MCM B-322, a pathogen causing cholera in humans. Then, the antibacterial activity of the pure compound against this pathogen was also tested, indicating that it is comparable to that of the standard antibiotic streptomycin [79].

The new thiodiketopiperazines phomazines A–C (300–302, Fig. 8.5) were produced by Phoma sp., endophyte from the mangrove plant Kandelia candel. Compounds 300 and 301 are biosynthesized from two phenylalanine residues with the uncommon oxidation of only one phenyl nucleus. In biological assays, compound 301 showed moderate to weak cytotoxicity against HL-60 (acute promyelocytic leukemia), HCT-116 (human colon carcinoma), K562 (chronic myelogenous leukemia), MGC-803 (human gastric cancer), and A549 tumor cell lines, while 300 and 302 were inactive [80].

The bioactivity-guided fractionation of extracts of Emericella sp., an endophytic fungus from A. lentiginosus, led to the isolation of secoemestrin D (303, Fig. 8.5). Compound 303 is a new epipolythiodioxopiperazine analogue harboring a tetrasulfide moiety. Compounds possessing disulphide bridge are recognized to their potentially toxic effects due to the reaction of thiol groups with proteins, and to the generation of reactive oxygen species [81]. Therefore, the potential anticancer activity of compound 303 was assessed using a panel of six human tumor cell lines: NCI-H460, SF-268, MCF-7, PC-3 M (metastatic prostate adenocarcinoma), MDA-MB-231 (breast adenocarcinoma), CHP-100 (neuroblastoma), and normal human fibroblast cells (WI-38). Compound 303 exhibited strong cytotoxicity against all the cancer cell lines with IC50 values of 0.15, 0.06, 0.14, 0.17, 0.06, and 0.10 μM, respectively. It was also toxic to normal cells (IC50 0.24 μM), but showed a moderate selectivity for SF-268 and MDA-MB-231 cell lines [75].

The new farinomaleins C–E (306–308, Fig. 8.5), isolated from the fungus AMO 3-2 (unidentified), an endophyte from the mangrove plant Avicennia marina, are structurally related to the farinomalein class, of which only two compounds have been reported previously [82, 83]. Compounds 306–308 were not cytotoxic against L5178Y cells at the concentration of 10 μg/mL. Besides, none of them exhibited significant antimicrobial activities against S. aureus ATCC 29213, S. pneumoniae ATCC 49619, and E. coli ATCC 25922, at the concentration of 64 μg/mL [84].

Three new aspochalasins, named trichalasin E, F, and H (314–316, Fig. 8.5), were isolated from the fungus Trichoderma gamsii, an endophyte from Panax notoginseng (BurK.) F.H. Chen. Compound 314 contains a unique hydroperoxyl group, which has not been reported before in the aspolachalasin class. Besides, compound 316 is stereoisomer of a known compound (aspergillin PZ) [85], possessing a rare 6/5/6/6/5 pentacyclic skeleton. Compounds 314–316 displayed weak cytotoxicity against three tumor cell lines (A549, MDA-MB-231, and PANC-1- human pancreatic carcinoma) [86].

Chaetoglobosin Vb (317, Fig. 8.5), isolated from a culture of Chaetomium globosum, an endophytic fungus from the medicinal plant Ginkgo biloba, possesses a very rare fusion into the macrocycle moiety forming a cyclopentenone ring. The absolute stereochemistry of this compound was determined based on CD spectrometry showing that it is a stereoisomer of a known compound (chaetoglobosin V, [87]). Compound 317 did not show antimicrobial activity against a panel of bacteria and fungi at the concentration of 100 μg/mL. Interestingly, its stereoisomer exhibited moderate to weak toxicity against Alternaria solani, Bacillus cereus, and P. aeruginosa [87].

New quinazolinone alkaloids were isolated from Aspergillus nidulans MA-143, an endophytic fungus from the marine mangrove plant R. stylosa. Those compounds, named aniquinazolines A–D (318–321, Fig. 8.5), own a remarkable structural diversity. The structure of 318 was confirmed by single-crystal X-ray diffraction analysis. All compounds were more toxic to brine shrimp larvae (LD50 values of 1.27, 2.11, 4.95, and 3.42 μΜ, respectively) than the positive control colchicine (LD50 values of 88.4 μΜ). However, none of those compounds displayed cytotoxic activity against four cell lines tested. Compounds were also inactive against E. coli and S. aureus [88].

The new polyketide-containing-nitrogen cryptosporioptide (322, Fig. 8.5) was produced by Cryptosporiopsis sp., an endophytic fungus from Viburnum tinus. Compound 322 possesses an unprecedented tetracyclic structure, holding a chromone nucleus merged with a five-membered cycle, in which an eight-membered cycle is attached. Its biological activities were assessed and compound 322 showed significant lipoxygenase inhibitory activity, while it was inactive against acetylcholinesterase and butyrylcholinesterase. In the antimicrobial assays, compound 322 was active against Bacillus megaterium, but inactive against the bacteria E. coli, the fungi Microbotyrum violaceum and Botrytis cinerea, and the alga Chlorella fusca [89].

The new indole-terpenes mycoleptodiscins A and B (323–324, Fig. 8.5) were isolated from Mycoleptodiscus sp., an endophytic fungus from Desmotes incomparabilis. These compounds have new skeletons that are uncommon in nature. The terpenoid moiety is an indole ring forming a fused pentacyclic alkaloid. Compound 324 was tested against the four cancer cell lines H460, A2058 (human melanoma), H522-T1 (non-small cell lung cancer), and PC-3 (prostate cancer), showing strong cytotoxicity, with IC50 values ranging from 0.60 to 0.78 μM. However, this compound was also strongly cytotoxic against nonproliferating normal cells (IMR-90, IC50 0.41 μM), indicating an indiscriminant cytotoxicity [90].

Citriquinochroman (325, Fig. 8.5), a novel compound with an unknown quinolactacide–isochroman skeleton, was isolated from rice cultures of Penicillium citrinum, an endophytic fungus from Ceratonia siliqua. Compound 325 showed cytotoxicity against L5178Y cells (IC50 6.1 μM) comparable with the positive control kahalalide F (IC50 4.3 μM). In the antibacterial assays, it was not active against S. aureus ATCC 29213, S. pneumoniae ATCC 49619, and E. coli ATCC 25922 at the concentration of 64 μg/mL [91].

Bipolamides A and B (332–333, Fig. 8.5) are new compounds and were isolated from Bipolaris sp., an endophyte from Gynura hispida. Compound 332 possesses an acyloin and triene fatty acid secondary amide moieties, neither of which is reported in nature. The last mentioned moiety is also present in compound 333, making them rare natural products. Both compounds were inactive against four bacterial strains at the concentration of 512 μg/mL, and compound 333 showed only mild toxicity against some fungal strains tested [92].

Two new alkaloids named as embellicines A and B (337–338, Fig. 8.5) were obtained from Embellisia eureka CATS2, isolated from healthy stem tissues of Cladanthus arabicus (Asteraceae). It was observed that compound 338 was completely converted to 337 during storage, suggesting a direct intramolecular dehydration process from 338 to 337. Besides, chemical correlation and biogenetic considerations suggest homochirality of these two compounds. Both compounds exhibited cytotoxicity against K562 cells, being able to induce cell death with an IC50 lower than 10 µM. It was also observed that compound 338 was highly active due to its cytotoxic/cytostatic potential. Interestingly, compound 338 was 5–10 times more active than compound 337 against K562 cells, indicating that the hydroxylation pattern in the pyrrolidinone ring is more important for its cytotoxicity than the presence of a C17-C18 double bond [93].

Triggering Biosynthesis of Novel Secondary Metabolites

Taking into account that endophytic microbes interact with their host plant and other associated microbes in the environment, all those interactions should trigger the production of secondary metabolites [2]. The lack of external stimulus under unnatural conditions may lead to a minimal or absent production of many interesting microbial natural products, which could be produced by the endophytes while interacting in their natural habitat.

Many strategies of cultivation have been used to stimulate the production of microbial secondary metabolites under laboratory conditions. These strategies include variations in media composition, pH, temperature, aeration, or shape of culturing flask; biotic elicitation by coculture of different strains; abiotic elicitation by physical or chemical stresses; and epigenetic modulation by chemical epigenetic modifiers [94–98].

Those approaches have been also applied to the endophytes cultures [99–106] and the following examples confirm that the secondary metabolism remodeling by cultivation-dependent approaches may yield new metabolites [103–105].

Six novel benzophenone derivatives, cephalanones A–F (167–172, Fig. 8.2), were obtained from Graphiopsis chlorocephala, an endophytic fungus from the leaves of Paeonia lactiflora. It was found that cultivation of this endophytic fungus in the presence of nicotinamide, HDAC inhibitor, yielded benzophenone production, including two uncommon chlorinated derivatives, 167 and 168. This result is the first evidence that NAD+-dependent HDAC inhibitors are an effective epigenetic strategy to access new natural products from endophytic fungi [107].

The new sesquiterpenes 270 and 271 (Fig. 8.3) were produced in response to abiotic stress elicitation when the endophytic fungus Pestalotiopsis sp. Z233, isolated from the alga Sargassum horneri, was grown in culture medium supplemented with CuCl2. The tyrosinase inhibitory activities of those compounds were evaluated, showing that they are potent inhibitors, with IC50 values of 14.8 μM and 22.3 μM, respectively. The IC50 value of kojic acid, used as a control, was 21.2 μM [105].

The production of two new linear depsipeptides, subenniatins A and B (288–289, Fig. 8.5), was induced during the mixed culture of Fusarium tricinctum and Fusarium begonia , both endophytes from the plant Aristolochia paucinervis. Interestingly, these compounds are suggested to be biogenetic building blocks of cytotoxic enniatins produced by F. tricinctum in pure culture. The compounds were inactive against E. coli, S. aureus, and P. aeruginosa at 64 μg/mL, and did not display cytotoxicity against L5178Y (IC50 > 10 μg/mL) [104].

The cocultivation of two marine-derived mangrove endophytic fungi led to the isolation of the new compounds marinamide and methyl marinamide (310–311, Fig. 8.5), whose structures were revised, indicating that they are pyrrolyl 4-quinolone analogues [108]. Compounds 310–311 were active against Pseudomonas pyocyanea and S. aureus, and exhibited potent cytotoxicity against HepG2, 95-D (lung), MGC832 (gastric), and HeLa tumor cell lines [103, 108].

Conclusion

Undoubtedly, the endophytic fungi are a remarkable source of novel and biologically active compounds. Nevertheless, the huge potential of endophytes to produce new natural products is not fully exploited. The understanding of chemical ecology of these microorganisms with their natural environment will assist the human beings during this unraveling process.