Fungal Endophytes: Sources of Medicines

1. Introduction

Several recent reports suggest that natural products may play a substantial role in the drug discovery and development process as a source of diverse and novel templates for future drugs [1,2,3,4]. With the rapidly evolving recognition that significant numbers of natural products are either produced by microbes or a result of microbial interactions with their hosts, the area of endophyte research for natural products is positioned to take the drug discovery and development process to the next level [5,6]. In the backdrop of the past 25 years of studies, endophytes may be defined as a polyphyletic group of unique microorganisms residing in healthy living internal tissues of the plants with covert and/or overt positive effects on their hosts. They establish a variety of intricate biological intra- and inter-relationships among them and with their hosts, respectively. Endophytes are able to produce a multitude of secondary metabolites with diverse biological activities [7,8,9]. However, merely 0.75–1.50% of known plant species has been explored for their endophytes yet. So, the opportunity to find new potential bioactive metabolites from cryptic endophytic microorganisms of nearly 374,000–400,000 plant species congruently occupying millions of biological niches is considered high [5,10]. This opportunity has increased further with the innovative discovery of biosynthesis of

Several recent reports suggest that natural products may play a substantial role in the drug discovery and development process as a source of diverse and novel templates for future drugs^[1][2][3][4]. With the rapidly evolving recognition that significant numbers of natural products are either produced by microbes or a result of microbial interactions with their hosts, the area of endophyte research for natural products is positioned to take the drug discovery and development process to the next level ^[5][6]. In the backdrop of the past 25 years of studies, endophytes may be defined as a polyphyletic group of unique microorganisms residing in healthy living internal tissues of the plants with covert and/or overt positive effects on their hosts. They establish a variety of intricate biological intra- and inter-relationships among them and with their hosts, respectively. Endophytes are able to produce a multitude of secondary metabolites with diverse biological activities^[7][8][9]. However, merely 0.75–1.50% of known plant species has been explored for their endophytes yet. So, the opportunity to find new potential bioactive metabolites from cryptic endophytic microorganisms of nearly 374,000–400,000 plant species congruently occupying millions of biological niches is considered high ^[5][10]. This opportunity has increased further with the innovative discovery of biosynthesis of

Taxus

derived anticancer compound ‘taxol’ from its endophytic fungus

T. andreanae in 1993 by Stierle et al. [11]. This discovery leads to renewed attention in endophytic fungi for isolating plant-derived medicinal compounds [12,13,14]. Later, a series of works revealed that a reasonable number of plant-derived compounds are synthesized by endophytes rather than hosts [9,15]. However, there are unsettled and contradictory reports regarding the phylogenetic origin of genes related to the biosynthesis pathway of such plant-derived compounds in host plants and their microbial endophytes [16]. The above facts prompted us to use the word “plant/host-derived” rather than “plant/host-origin” for such compounds. Nevertheless, it is now an established fact that endophytes can co-/produce, induce, and/or modify a plethora of “specific plant-derived” metabolites in-/outside of host plants [12,14,17]. Such discoveries opened the new horizons for the up-scaled production of plant-derived medicinal compounds from endophytes. The recent increase in demand for natural products and difficulties in accessing them from plants make endophytes interesting targets for the assessment and isolation of typical host-derived compounds [18,19]. Since medicinal plants are an inherent source of many therapeutic compounds, it is vital to explore their endophytes to isolate such compounds. The current review aims to provide an up-to-date overview on the globally isolated specific plant-derived bioactive compounds synthesized by fungal endophytes from the period 1993 to mid-2020. It will also focus on applications and modes of actions of such compounds. This review will also provide insights about different challenges in employing endophytes as an alternative source for the synthesis of plant-derived bioactive compounds and their application in drug discovery. Its outcome would certainly lead to strategize the use of endophytes as an efficient novel source for plant-derived metabolites.

in 1993 by Stierle et al.^[11]. This discovery leads to renewed attention in endophytic fungi for isolating plant-derived medicinal compounds ^[12][13][14]. Later, a series of works revealed that a reasonable number of plant-derived compounds are synthesized by endophytes rather than hosts^[9][15]. However, there are unsettled and contradictory reports regarding the phylogenetic origin of genes related to the biosynthesis pathway of such plant-derived compounds in host plants and their microbial endophytes ^[16]. The above facts prompted us to use the word “plant/host-derived” rather than “plant/host-origin” for such compounds. Nevertheless, it is now an established fact that endophytes can co-/produce, induce, and/or modify a plethora of “specific plant-derived” metabolites in-/outside of host plants^[17][12][14]. Such discoveries opened the new horizons for the up-scaled production of plant-derived medicinal compounds from endophytes. The recent increase in demand for natural products and difficulties in accessing them from plants make endophytes interesting targets for the assessment and isolation of typical host-derived compounds^[18][19]. Since medicinal plants are an inherent source of many therapeutic compounds, it is vital to explore their endophytes to isolate such compounds. The current review aims to provide an up-to-date overview on the globally isolated specific plant-derived bioactive compounds synthesized by fungal endophytes from the period 1993 to mid-2020. It will also focus on applications and modes of actions of such compounds. This review will also provide insights about different challenges in employing endophytes as an alternative source for the synthesis of plant-derived bioactive compounds and their application in drug discovery. Its outcome would certainly lead to strategize the use of endophytes as an efficient novel source for plant-derived metabolites.

2. Plant-Derived Bioactive Natural Products from Fungal Endophytes

A wide array of secondary metabolites in fungi is biosynthesized from very few key precursor compounds by slight variations in basic biosynthetic pathways and can be classified into nonribosomal peptides, polyketides, terpenes, and alkaloids. Nonribosomal peptides are biosynthesized by multimodular nonribosomal peptide synthetases (NRPS) enzymes using both proteinogenic and nonproteinogenic amino acids. Polyketides are biosynthesized by polyketide synthase (PKS) enzymes from acetyl-CoA and malonyl-CoA units. Terpenes consisting of isoprene subunits are biosynthesized from the mevalonate pathway catalyzed by terpene cyclase enzymes. Alkaloids are nitrogen-containing organic compounds biosynthesized as complex mixtures through the shikimic acid and the mevalonate pathways, as they are usually derived from aromatic amino acids and dimethlyallyl pyrophosphate [20]. Other classes of fungal secondary metabolites are linked with the above four groups of compounds. For ease and better understanding, we have classified different fungal secondary metabolites as alkaloids, coumarins, flavonoids, lignans, saponins, terpenes, quinones, and xanthones, and miscellaneous compounds. Coumarins are a class of lactones consisting of a benzene ring fused to a α-pyrone ring and are mainly biosynthesized by the shikimic acid pathway from cinnamic acid. Flavonoids are synthesized by the phenylpropanoid pathway from phenylalanine using enzymes phenylalanine ammonia lyase (PAL), chalcone synthase, chalcone isomerase, and flavonol reductase [21]. Lignans are low molecular weight polyphenols biosynthesized by enzymes pinoresinol-lariciresinol reductase (PLR), PAL, cinnamoyl-CoA reductase (CCR), and cinnamyl-alcohol dehydrogenase (CAD) [22]. Saponins are glycosides containing a non-sugar triterpene or steroid aglycone (sapogenin) attached to the sugar moiety. Saponins are derived from intermediates of the phytosterol pathway using enzymes oxidosqualene cyclases (OSCs), cytochromes P450 (P450s), and UDP-glycosyltransferases (UGTs) [23]. Quinones are biosynthesized through several pathways; for example, isoprenoid quinones are synthesized by the shikimate pathway using chorismite-derived compounds as precursors, terrequinone by NRPS from L-tryptophan, dopaquinone by tyrosinase from tyrosine, and benzoquinone by catechol oxidase/PKS from catechol [24]. Xanthones comprise an important class of oxygenated heterocyclics biosynthesized through the polyacetate/polymalonate pathway by the internal cyclization of a single folded polyketide chain [25].

A wide array of secondary metabolites in fungi is biosynthesized from very few key precursor compounds by slight variations in basic biosynthetic pathways and can be classified into nonribosomal peptides, polyketides, terpenes, and alkaloids. Nonribosomal peptides are biosynthesized by multimodular nonribosomal peptide synthetases (NRPS) enzymes using both proteinogenic and nonproteinogenic amino acids. Polyketides are biosynthesized by polyketide synthase (PKS) enzymes from acetyl-CoA and malonyl-CoA units. Terpenes consisting of isoprene subunits are biosynthesized from the mevalonate pathway catalyzed by terpene cyclase enzymes. Alkaloids are nitrogen-containing organic compounds biosynthesized as complex mixtures through the shikimic acid and the mevalonate pathways, as they are usually derived from aromatic amino acids and dimethlyallyl pyrophosphate^[20]. Other classes of fungal secondary metabolites are linked with the above four groups of compounds. For ease and better understanding, we have classified different fungal secondary metabolites as alkaloids, coumarins, flavonoids, lignans, saponins, terpenes, quinones, and xanthones, and miscellaneous compounds. Coumarins are a class of lactones consisting of a benzene ring fused to a α-pyrone ring and are mainly biosynthesized by the shikimic acid pathway from cinnamic acid. Flavonoids are synthesized by the phenylpropanoid pathway from phenylalanine using enzymes phenylalanine ammonia lyase (PAL), chalcone synthase, chalcone isomerase, and flavonol reductase ^[21]. Lignans are low molecular weight polyphenols biosynthesized by enzymes pinoresinol-lariciresinol reductase (PLR), PAL, cinnamoyl-CoA reductase (CCR), and cinnamyl-alcohol dehydrogenase (CAD)^[22]. Saponins are glycosides containing a non-sugar triterpene or steroid aglycone (sapogenin) attached to the sugar moiety. Saponins are derived from intermediates of the phytosterol pathway using enzymes oxidosqualene cyclases (OSCs), cytochromes P450 (P450s), and UDP-glycosyltransferases (UGTs) ^[23]. Quinones are biosynthesized through several pathways; for example, isoprenoid quinones are synthesized by the shikimate pathway using chorismite-derived compounds as precursors, terrequinone by NRPS from L-tryptophan, dopaquinone by tyrosinase from tyrosine, and benzoquinone by catechol oxidase/PKS from catechol^[24]. Xanthones comprise an important class of oxygenated heterocyclics biosynthesized through the polyacetate/polymalonate pathway by the internal cyclization of a single folded polyketide chain^[25].

2.1. Plant-Derived Alkaloids from Fungal Endophytes

After a systematic literature survey, we enlisted 19 plant-derived medicinal alkaloids that have been produced by different endophytic fungi (

Table 1

), and some important alkaloids are described below.

Table 1.

Plant-derived alkaloids produced by endophytic fungi.

Plant-Derived Alkaloids	Activities/Applications	Plant Source	Endophytic Source	Host Plant	References

Plant-derived flavonoids produced by endophytic fungi.

Plant-Derived Flavonoids	Activities/Applications	Plant Source	Endophytic Source	Host Plant	References
Aconitine	Anticancer, anti-inflammatory, anti-neuralgic, cardiotoxic	Aconitum spp.	Cladosporium cladosporioides	Aconitum leucostomum
Apigenin	Antibacterial, anticancer, antioxidant, antihyperglycaemic, lipid peroxidation, sedative, thyroid dysfunction	Cajanus cajan, Cephalotaxus harringtonia, Matricaria chamomilla, vegetables	Colletotrichum sp.[26]
			Ginkgo biloba	[	132	,133,134]	Berberine	Antibiotic, antidiabetic, antihypertensive, antiproliferative hepatoprotective, hypolipidemic, vasodilator	Berberis spp., Coscinium fenestratum, Hydrastis canadensis, Phellodendron amurense	Alternaria sp.	Phellodendron amurense	[27]
							Berberine
Chaetomium globosumFusarium solani	Coscinium fenestratum	[28,29]
Cajanus cajan	[135]	Caffeine	CNS stimulant	Coffea
Paraconiothyriu mvariabile	Cephalotaxus harringtonia	[136]	spp., Theobroma cacao	Anonymous endophytes
Cajanol	Anticancer, antimicrobial, antiplasmodial	Cajanus cajanOsbeckia chinensis, Osbeckia stellata	Hypocrealixii, Potentilla fulgens	[30]
Cajanus cajan	[	137	]	Camptothecin	Antitumor	Camptotheca acuminata, Miquelia dentata, Nothapodytes nimmoniana, Ophiorrhiza spp.	Entrophospora infrequens	Nothapodytes foetida
Chalcone	Antibacterial, antifungal, antitumor, anti-inflammatory	Cleistocalyx operculatus, Members of Leguminosae, Asteraceae, Moraceae[31]
Ceriporia lacerata	Cleistocalyx operculatus	(syns. Eugenia operculata, Syzygium operculatum)	[138]				Entrophospora infrequens	Nothapodytes foetida
Chrysin	Antiaging, anticonvulsant, antidiabetic, anti-inflammatory, antimicrobial, anxiolytic, hepatoprotective[32]
Passiflora incarnata	Alternaria alternata, Colletotrichum capsici, Colletotrichum taiwanense	Passiflora incarnata	Neurospora sp.				Nothapodytes foetida	[33]
Valsa mali	Camptotheca acuminata	[34]
Nodulisporium sp.	Nothapodytes foetida	[35]
Fusarium solani	Camptotheca acuminata	[36]
Botryosphaeria parva, Diaporthe conorum, Fusarium oxysporum, Fusarium sacchari, Fusarium solani, Fusarium subglutinans, Fusarium verticillioides, Galactomyces sp., Irpex lacteus, Phomopsis sp., Fusarium sp.	Nothapodytes nimmoniana	[37]
Xylaria sp.	Camptotheca acuminata	[38]
Fusarium solani	Apodytes dimidiata	[39]
Botryosphaeria dothidea	Camptotheca acuminata	[40]
Alternaria alternata, Fomitopsis sp., Phomopsis sp.	Miquelia dentata	[41]
Trichoderma atroviride	Camptotheca acuminata	[42]
Aspergillus sp.	Camptotheca acuminata	[36]
Fusarium oxysporum	Nothapodytes foetida	[43]
Capsaicin	Anti-inflammatory, gastro-stimulatory	Capsicum annuum	Alternaria alternata	Capsicum annuum	[44]
Homoharringtonine	Anticancer	Cephalotaxus spp.	Alternaria tenuissima	Cephalotaxus sp.	[45]
Huperzine A	Acetylcholinesterase inhibitor, Alzheimer’s treatment	Huperzia serrata	Acremonium sp.	Huperzia serrata (syn. Lycopodium serratum)	[46]
			Blastomyces sp., Botrytis sp.	Phlegmariurus cryptomerianus	[47]
			Penicillium chrysogenum	Huperzia serrata	[48]
			Shiraia sp.	Huperzia serrata	[49]
			Cladosporium cladosporioides	Huperzia serrata	[50]
				Oxytropis kansuensis	[73]

Dipteryx odorata

(

Coumarouna odorata

) [

102

]. A total of seven medicinally important specific plant-derived coumarins are produced by fungal endophytes (

Table 2

Table 2.

Plant-derived coumarins produced by endophytic fungi.

Coumarins	Activities/Applications	Plant Source	Endophytic Source	Host Plant	References
	psoriasis treatment	Balanites aegyptiaca, Citrus bergamia, Grapefruit peel	Penicillium sp.	Avicennia	[103]
	psoriasis treatment	Balanites aegyptiaca, Citrus bergamia, Grapefruit peel	Botryodiplodia theobromae	Dracaena draco	[104]
Fusarium
sp.
Phlegmariurus taxifolius
[
55
]
Isofraxidin	Anticancer, anti-obesity, cardioprotective, neuroprotective, hyper pigmentation	Acanthopanax senticosus, Sarcandra glabra	Annulohypoxylon bovei var. microspora	Cinnamomum sp.	[105,106]
Marmesin	Anticancer, antihelmintic, antioxidant, antisyphilitic, purgative	Ammi majus, Balanites aegyptiaca	Fusarium sp.	Mangrove	[107]	[139
Undifilum
sp.
Mellein	Antibacterial, antifungal, antihepatitis c, larvicidal, phytotoxic	Alibertia macrophylla, Litsea akoensis, Garcinia bancana, Moringa oleifera,	]
			Colletotrichum sp., Trichoderma sp.	Huperzia serrata	[51]
			Paecilomyces tenuis

			Fusarium
			sp.	Huperzia serrata	[56]

			Swainsona canescens

			[
			74
			]
]
Bergapten, Meranzin	Antioxidant,
Bergapten, Meranzin	Antioxidant,
Stevia lucida
	Septoria nodorum		Conifer	[108]
	Curcumin		Anti-inflammatory, antioxidant, antitumor	Curcuma spp.	Chaetomium globosum	Curcuma wenyujin	[140]
	Curcumin		Anti-inflammatory, antioxidant, antitumor	Curcuma spp.	Anonymous	Curcuma wenyujin	[141]
	Kaempferol		Antibacterial, antidiabetic, anti-inflammatory, antioxidant, antitumor	Fruits, vegetables, medicinal herbs	Annulohypoxylonboveri var. microspora, Annulohypoxylon squamulosum	Cinnamomum sp.	[106,113, Piper aduncum	[112]
					]
					Fusarium chlamydosporum	Tylophora indica	[142]	Annulohypoxylon squamulosum	Cinnamomum sp.
	Mucor fragilis		[113	Podophyllum hexandrum]
	[		143]	Nigrospora sp.	Moringa oleifera	[114]

	Huperzia serrata

[
52
]
Luteolin	Anti-inflammatory, antioxidant, immunomodulatory	Fruits, vegetables, medicinal herbs	Annulohypoxylon boveri var. microspora	Cinnamomum sp.	[106]	Arthrinium (Apiospora montagnei)	Anonymous	[115]
Luteolin	Anti-inflammatory, antioxidant, immunomodulatory	Fruits, vegetables, medicinal herbs	Aspergillus fumigatus	Cajanus cajan	[144]	Xylaria sp.	Garcinia sp.	[116]
Quercetin	Anticancer, anti-inflammatory antioxidant	Fruits, vegetables	Aspergillus nidulans, Aspergillus oryzae	Ginkgo biloba	[145	Aspergillus flavus, Mycoleptodiscus terrestris, Penicillium griseofulvum	Huperzia serrata	[53]
			Peimisine, Imperialine-3b-D-glucoside	Antiasthmatic, antitumor, expectorant	Fritillaria spp.	Fusarium sp.	Fritillaria unibracteata var. wabuensis	[57,58]
			Peimisine, Imperialine-3b-D-glucoside	Antiasthmatic, antitumor, expectorant	Fritillaria spp.	Fusarium redolens	Fritillaria unibracteata var. wabuensis	[59]
Piperine	Anti-inflammatory, anticancer,
		Penicillium janczewskii	Prumnopitys andina	[109]	antimicrobial, antidepressant, hepatoprotective	Piper longum, Piper nigrum	Periconia sp.	Piper longum	[60]
		Colletotrichum gloeosporioides	Piper nigrum	[61]
		Mycosphaerella sp.
Botryosphaeria mamane	Anonymous	[110]
A xylariaceous fungus	Sapindus saponaria	[111]
Annulohypoxylon bovei var. microspora	Cinnamomum sp.	[106]]	Pezicula sp.	Forsythia viridissima	[117]
Annulohypoxylon squamulosum	Cinnamomum sp.	[113	Xylaria cubensis	Litsea akoensis	[118]
]
Nigrospora oryzae	Loranthus micranthus	[146]	Scopoletin, Umbelliferone	Antifungal, antioxidant, anti-inflammatory	Artemisia scoparia, Scopolia carniolica (syn. Scopolia japonica), Viburnum prunifolium	Penicillium sp.	Avicennia	[

2.3. Plant-Derived Flavonoids from Fungal Endophytes

Flavonoids are pigments of edible plants consisting of two benzene rings at either side of a three-carbon ring. Multiple substitutions in this basic structure produce several classes of derivatives, such as flavones, isoflavones, flavonols, flavanones, catechins, and anthocyanins. We found 12 different biologically active plant-derived flavonoids (

Table 3

) recovered from fungal endophytes and important flavonoids are described below.

Table 3.

Rotenone
Insecticide, pesticide, piscicide
Derris elliptica
Penicillium
sp.
Derris elliptica
[
147
]
Rutin	Antioxidant, cardioprotective, neuroprotective	Aegle marmelos Ginkgo biloba, Nerium oleander, Pteris multifida, fruits, vegetables	Anonymous	Pteris multifida	[148]
			Chaetomium sp.	Nerium oleander	[149]
			Xylaria sp.	Ginkgo biloba	[
			Penicillium
sp.
Huperzia serrata
[
54	]
Piper nigrum
[
62
]
Phomopsis sp.	Oryza sativa	[63]
Cinchona alkaloids: Quinine, Quinidine, Cinchonidine, Cinchonine	Antimalarial, antiarrhythmic, analgesic	Cinchona spp.	Arthrinium, Fomitopsis, Diaporthe, Penicillium, Phomopsis, Schizophyllum	Cinchona ledgeriana	[64]
	Antimalarial, antiarrhythmic, analgesic	Cinchona spp.	Fusarium incarnatum, Fusarium oxysporum (only quinine and cinchonidine) Fusarium solani (only quinine)	Cinchona calisaya	[65]
Rohitukine	Anticancer, CDK inhibitor, cytotoxic	Amoora rohituka Dysoxylum binectariferum,	Fusarium proliferatum	Dysoxylum binectariferum	[66]
			Fusarium oxysporum, Fusarium solani	Dysoxylum binectariferum	[67]
			Gibberella fujikuroi	Amoora rohituka	[67]
Sanguinarine	Anticancer, antimicrobial, anti-inflammatory antioxidant, antihelmintic, neuroprotective	Macleaya cordata, Sanguinaria canadensis	Fusarium proliferatum	Macleaya cordata	[28,68]
Sipeimine	Antibechic, anti-ulcer	Fritillaria spp.	Cephalosporium corda	Fritillaria ussuriensis	[69]
Solamargine	Anticancer, cytotoxic	Solanum nigrum	Aspergillus flavus	Solanum nigrum	[70]
Swainsonine	Toxicosis in livestock	Astragalus, Oxytropis spp., Swainsona canescens	Embellisia sp.	Astragalus, Oxytropis spp.	[15,71]
			Undifilum cinereum, U. fulvum	Astragalus lentiginosus, Astragalus mollissimus	[72]
			Fusarium tricinctum	Oxytropis deflexa,	Vinblastine, Vincristine	Antitumor	Catharanthus roseus (syn. Vinca rosea)	Alternaria sp.	Catharanthus roseus	[75]
			Fusarium oxysporum	Catharanthus roseus
Penicillium	[76]
Fusarium oxysporum	Catharanthus roseus	[77]
sp., Xylaria sp.	Alibertia macrophylla	Talaromyces radicus	Catharanthus roseus	[78]
Eutypella spp.	Catharanthus roseus	[79	Geomyces sp.	Nerium indicum				[80]
Vincamine	Antihypertensive, vasodilator	Vinca minor	Anonymous	Vinca minor	[81]

2.2. Plant-Derived Coumarins (Benzopyrones) from Fungal Endophytes

Coumarins have been routinely employed as herbal remedies since the onset of herbal medicine. It was first isolated as a natural product from seeds of