Abstract

Endophytes are plant inhabiting microorganisms that possess a big and untapped source of natural products with unique chemical structures and biological activities for pharmaceutical and agriculture industry. About, several hundred of endophytes are associated with plants. Metabolites isolated from endophytes with diverse structures belong to alkaloids, steroids, terpenoids, flavonoids, phenols, flavonoids and others. The discovery of an array of therapeutic products has diverted the scientist’s interest from plants to these microorganisms. Hundreds of natural compounds have been obtained and structurally classified while other new active metabolites are under investigation. This present review is an approach to summarize some chemical classes of bioactive compounds obtained from these microorganisms and paving the way for future work.

1. Introduction

Endophytes are microorganisms (fungi, bacteria and actinomycetes) inhabiting healthy plant intercellularly and/or intracellularly without causing any visible symptoms of disease ^{1, 2}. De Bary was first to introduce the term “Endophyte” in 1866 ³, which was initially referred to all those organisms that found within a plant causing nonvisible infections entirely within plant tissues showing no apparent symptoms of disease ¹. Endophytes dwell all plant species reported to date throughout the globe in a symbiotic relation ⁴.

During the last decades, endophytes have been searched out for important metabolites. It is evident from research that endophytes are new source and gold-mine of novel natural products to treat different ailments in human and plants. They are synthesizers of chemical entities that are extensively used as agrochemicals, antibiotics, immunosuppressant, antiparasitics, and anticancer agents ⁵. As a routine work, medical herbs are directly used for therapeutic components to isolate and characterize. However, the discovery of plant endophytes with potential of medicinal compounds tremendously attracted the interest of chemists to search for new drugs. Among these microbes, endophytic fungi have been found bountiful of unexploited biologically active small molecules ⁶.

Based on the studies about microorganisms, actinomycetes and fungi have been reported to be the most productive source of therapeutic agents. Fungi are vital for a balanced ecosystem and biodiversity conservation ^{7, 8}. The endophyte was first discovered in 1904 ⁹. Endophytes attracted dramatic attention when their ability of synthesizing biologically active components having structures were realized. Hence, for the last 20 years, over 100 endophytes of various hosts have been investigated for chemical and biological assessment of a huge number of metabolites. Among them, many have been found to have unique structures and interesting biological functions. Gusman and Vanhaelen first isolated natural compounds from 38 endophytic fungi and studied their pharmacology ¹⁰.

About 138 natural products of endophytes were communicated by Tan and Zou ¹¹. These secondary metabolites may be mainly classified as; alkaloids, flavonoids, phenolic acids, quinones and many others ¹². Schulz et al. ¹³. Strobel ^{14, 15} and Strobel et al. ¹⁶ have explained their own research on endophytes. This review aims to present some previous and new interventions related to isolation of metabolites from endophytes.

Endophytes have been described as; Bacterial endophytes are found best producers of different therapeutic compounds. About 76% of them obtained from Streptomyces ¹⁷. They are either gram-positive or gram-negative, such as, Acinetobacter, Agrobacterium, Bacillus, Microbacterium and Pseudomonas etc. ¹⁸. More than 200 genera serve as endophytes ¹⁹.

Actinomycetes possessing mycelium like fungus and forms spores. These microorganisms were thought out transitional forms between the fungi and bacteria ^{20, 21}. They are bacteria like but actinomycetes have thin cells and an organized chromosome in a prokaryotic nucleoid and a peptidoglycan cell wall. This class has demonstrated synthesis of numerous chemical entities of unique structures that have significant medicinal value ^{22, 23}.

Fungi are heterotrophic organisms with various life cycles that inhabit symbiotically a wide variety of autotrophic organisms ²⁴. Endophytic fungi have been branched into clavicipitaceous and non-clavicipitaceous endophytes. The first are inhabitants of some grasses while the other of non-vascular plants and higher plants restricted to Ascomycota or Basidiomycota group. Most of known antibiotic and anticancer drugs were derived from these organisms. For example, Penicillenols and Taxol were potent drugs isolated from Penicillium sp. and Taxomyces andreanae, respectively. More than 20,000 bioactive products having unprecedented structures have been reported in the literature ²⁵.

Endophytes are ubiquitous and almost all vascular plant species studied to date were found to anchorage endophytic bacteria and/or fungi ^{26, 27}. Endophytes play a vital role in biodiversity of microbes ²⁸. They are host specific and a plant may harbor many species. The population of endophytes also depends on environmental conditions of the host plants ²⁹, and the tropical areas may have variegated endophyte profile. Tropical endophytes may be hyper diverse and unevenly scattered in a specific area ²⁷. Genotypic diversity is also found in some endophytes of conifers and grasses. Hence, endophytes are omnipresent depending on host and location for their population ^{30, 31, 32, 33}.

2. Isolation and Identification

Different methods of isolation like Agar Plate Method and Standard Moist Blotter method are used to isolate mycoflora from medicinal plants ^{34, 35, 36}. Cultivation-independent assays and in situ hybridization-confocal laser scanning microscopy are used for their detection ^{37, 38}. Mostly isolation from sterilized tissue of inhabitant’s plant is used to detect endophytes. Different methods of isolation have also been described by Rehman et al. ³⁹ and Karunai & Balagengatharathilagam ⁴⁰.

Morphological characteristics and biochemical tests are also usually used to identify bacteria, fungi, and actinomycetes ^{41, 42}. Due to recent advancement in molecular studies, ribosomal DNA Internal Transcribed Spacer (ITS) sequence technique is a frequent practice to identify microorganisms ⁴³. Primarily, Pleurostoma and Chaetomium along with others were identified as endophytes by ITS technique ⁴⁴.

3. Review: Bioactive Compounds

During the last few years a number of secondary metabolites originated from endophytes have been documented. They are indispensable due to their chemical and diverse biological properties. Some of the bioactive compounds are reviewed under the following groups.

Alkaloids are naturally occurring nitrogenous chemical compounds of plant origin. They have pronounced physiological effects on humans and other animals.

Camptothecin (1, CPT) is topoisomerase inhibitor and most potent cytotoxic compound ^{45, 46} having anticancer activities. CPT and its derivatives such as 10-hydroxycamptothecin (2) and 9-methoxycamptothecin (3) were derived from Fusarium solani; inhabitant of Apodytes dimidiata ⁴⁷. Eupenicillum sp. was isolated from Murraya paniculata and produced alanditrypinone (4), alantryphenone (5), alantrypinene B (6) and alantryleunone (7) ⁴⁸. Asperfumoid (8) is a new alkaloid and was isolated from Aspergilus fumigatus CY018, an endophyte of Cynodon dactylon. This metabolite caused inhibition of Candida albicans ⁴⁹. Aspernigerin (9) was produced by Aspergillus niger IFB-E003 (having same host), demonstrated growth inhibition of tumor cells ⁵⁰.

Phomoenamide (10) was isolated from fungus Phomopis sp. PUS-D15; an endophyte of Garcinia dulcis Kuiz leaves. The compound has antibacterial activity against Mycobacterium tuberculosis H37Ra ⁵¹. An endophyte Xylaria sp. was cultured from Ginkgo biloba L., which produced a compound 7-amino-4-methylcoumarin (11) having strong antibacterial and antifungal activities ⁵². A novel product; 7-O-methylvariecolortide A (12) was produced by an endophyte found inside the stems of Hibiscus tiliaceus ⁵³. Phomopsichalasin (13), a novel alkaloid produced by fungus Phomopsis living on Salix gracilostyla. The compound proved to have antibacterial and antifungal properties ⁵⁴.

Chaetoglobosins A (14) and C (15), were obtained from C. globosum; an endophyte of Ginkgo biloba. These two compounds proved to have antibacterial activity against Mucor miehei. ⁵⁵. Fungus Cladosporium (CLJ-2) derived from young C. ledgeriana stems resulted a metabolite, named cinchonine (16). The compound has cytotoxic potency for 95-D and HepG2 cell lines ⁵⁶. Herquline B (17) having antimicrobial activities was derived from endophytic fungus Talaromyces pinophilus of Arbutus unedo ⁵⁷. Vincristine (18) and vinblastine (19) were detected in the extracts of endophyte Penicillium sp.-VE89L and three strains identified as Alternaria sp.-VM84L, Cladosporium sp.-VE92L and A. amstelodami-VR177L, respectively. These endophytes were isolated from leaves of V. erecta. Both compounds have shown pronounced growth inhibition with cultured cells of HBL-100 ⁵⁸.

A steroid is a biologically active organic compound with four rings of carbon atoms arranged in a specific molecular configuration. Examples include lipid cholesterol and anti-inflammatory drug dexamethasone etc. ⁵⁹. Isolation of novel compounds, (14β,22E)-9,14-dihydroxyergosta-4,7,22-triene-3,6-dione (20) and (5α,6β,15β,22E)-6-ethoxy-5,15-dihydroxyergosta-7,22-dien-3-one (21) was achieved from Phomopsis sp. of Aconitum carmichaeli ⁶⁰. Both compounds showed moderate antifungal properties. A novel (22E, 24R)-3-acetoxy-19(10→6)-abeo-ergosta-5,7,9,22-tetraen-3beta-ol (22) was resulted by Colletotrichum sp. obtained from Ilex canariensis ⁶¹. Two new steroids namely 3β, 5α-dihydroxy-6β-acetoxyergosta-7, 22-diene (23) and 3β, 5α-dihydroxy-6β-phenyl- acetyloxyergosta-7, 22-diene (24) were characterized from the liquid culture of fungal endophyte Colletotrichum sp. of A. annua, having antifungal activities ⁶².

A novel polyhydroxylated C₂₉-sterol, named globosterol (25), together with one known tetrahydroxylated ergosterol (22E,24R)-ergosta-7,22-diene-3β,5α,6β,9α-tetraol (26) has been isolated from the cultures of an endophytic fungus, Chaetomium globosum ZY-22 originated from the plant Ginkgo biloba ⁶³. A steroid named 5α, 8α-epidioxyergosterol (27) was derived from Nodulisporium sp. residing on Juniperus cedre ⁶⁴. Solanioic acid (28) was produced by Rhizoctonia solani inhabiting tubers of Cyperus rotundus. It caused strong inhibition of Gram-positive bacterial strains ⁶⁵. Novel metabolites, named fusaristerols B (29), C (30) and D (31) were discovered from Fusarium sp. inhabiting Mentha longifolia L. (Labiatae) roots. The compounds possessed 5-LOX inhibitory activity to treat different inflammatory disorders ⁶⁶.

Terpenoids are a large and diverse class of naturally occurring organic chemicals derived from five-carbon isoprene units. Terpenoids comprise the largest group of natural products. A number of terpenoids have been isolated from plant endophytes. The xylarenones A (32), B (33) and xylarenic acid (34) were obtained from the endophytic fungus Xylaria sp. NCY2, obtained from Torreya jackii. The compounds exhibited moderate antitumor activities against HeLa cells ⁶⁷. Endophyte Phomopsis cassiae associated with Cassia spectabilis resulted two diastereoisomeric compounds named (7S, 9S, 10S and 7S, 9R, 10S)-3,9,12-trihydroxycalamenenes (35, 36), 3,12 dihydroxycalamenene (37), 3,12-dihydroxycadalene (38) and 3,11,12-trihydroxycadalene (39) ⁶⁸. Botryosphaerins A-E (40-44) along with acrostalidic acid (45), acrostalic acid (46), agathic acid (47) and isocupressic acid (48) were identified from Botryosphaeria sp. MHF derived from Maytenus hookeri ⁶⁹. Endophyte Xylaria sp. PSU-D14 resulted xylaroside A (49) and B (50). While Eutypella scoparia PSU-D44 produced scopararanes A (51), B (52) and diaportheins A (53), B (54). Both species are associated with Garcinia dulcis ^{70, 71}.

Endophytic fungus Phomopsis sp. XZ-26 collected from Camptotheca acuminate produced a new monoterpene termed as dihydroxysabinane (55) ⁷². The preaustinoid B2 (56), preaustinoid A3 (57), austinolide (58) and isoaustinone (59) were identified from fungi Penicillium sp. found in the root bark of Melia azedarach ⁷³. Fungi P. brasilianum (inhabiting same host) resulted novel compounds, called preaustinoid A1 (60), A2 (61) and B1 (62) ⁷⁴. Guanacastepene A (63) and guanacastepene (64) were characterized from endophytes of Daphnopsis americana. Whereas, periconicin A (65) and perieoniein B (66) were discovered from Periconia sp. These four novel diterpenoids proved as antibiotics ⁷⁵. Three new metabolites, namely lithocarin B (67), C (68) and D (69) were obtained from the endophytic fungus Diaporthe lithocarpus A740 derived from Morinda officinalis. Compounds B and C showed weak inhibitory activities against four tumor cell lines ⁷⁶.

Quinones are a class of cyclic organic compounds containing two carbonyl groups, > C = O, either adjacent or separated by a vinylene group, −CH = CH−, in a six-membered unsaturated ring.

Herbarin (70) and its analogue herbaridine A (71) have been isolated from an endophytic fungus D. nanum derived from F. religiosa ⁷⁷. Three new compounds named preussomerin J (72), K (73) and L (74) having antibacterial and antifungal activities were synthesized by an endophytic fungus Mycelia sterile isolated from the roots of Atropa belladonna. ⁷⁸. Ampelomyces sp., an endophyte of Urospermum picroides, produced 3-O-methylalaternin (75) and tetrahydroanthraquinone altersolanol A (76). Both compounds were active against the bacteria S. aureus, S. epidermis and E. faecalis at different MIC levels ⁷⁹.

A bioactive compound, termed as 4-dehydroxyaltersolanol A (77) was produced by an endophytic fungus, Nigrospora oryzae, isolated from leaves of Combretum dolichopetalum. The compound proved to be cytotoxic against mouse lymphoma cells ⁸⁰. Bipolaris sorokiniana A606 derived from Pogostemon cablin resulted novel metabolites termed as isocochlioquinones D and E (78, 79) and cochlioquinones G and H (80, 81). These metabolites showed cytotoxicity towards tumor cell lines ⁸¹. Endophyte Fusarium solani was originated from the roots of Aponogeton undulatus Roxb, resulted two cytotoxic compounds, named 7-desmethylscorpinone (82) and 7- desmethyl-6-methylbostrycoidin (83). Eurotium rubrum; an endophyte living on Suaeda salsa L., produced rubrumol (84). The compound inhibited Top I relaxation activity ⁸².

Flavonoids are phenolic secondary metabolites containing 15 carbon atoms and a heterocyclic ring. They are beneficial nutrients for health and treatment of different disorders.

Kaempferol (85) has been obtained from endophytic fungi Mucor fragilis collected from rhizomes of Sinopodophyllum hexandrum ⁸³. Luteolin (86) from endophyte Aspergillus fumigatus of Cajanus cajan displayed remarkable antioxidant activity ⁸⁴. Two new flavonoids, 7- methoxy-3,3',4',6-tetrahydroxyflavone (87) and 2',7-dihydroxy-4',5'-dimethoxyisoflavone (88) with one known compound fisetin (89) were produced by Streptomyces sp. BT01 isolated from root tissues of Boesenbergia rotunda ⁸⁵. Apigenin (4′, 5, 7-trihydroxyflavone) (90) has also been reported to be produced by Chaetomium globosum from the roots of C. cajan ⁸⁶.

Three secondary metabolites, silybin A (91), silybin B (92), and isosilybin A (93) were reported for the first time from Aspergillus iizukae cultured from S. marianum leaves ⁸⁷. Naringenin (94) having antioxidant and neuroprotective effect was detected in the extract of the endophytic fungus Verticilium sp. isolated from Lippia sidoides Cham ⁸⁸. Quercetin (95) was predicted in four endophytic bacterial strains of Kenikir leaves. It has shown anticancer, antibacterial and antifungal activities due to its toxicity ⁸⁹.

Peptides consist of two or more α-amino acids linked in a chain; the carboxyl group of each acid being joined to the amino group of the next by a bond of the type -OC-NH. Bioactive peptides have demonstrated potential for application as health promoting agents. Endophytic fungus of Castaniopsis fissa produced cyclo-(L-Val-L-Leu-L-Val-L-Leu) (96) and cyclo-(L-Leu-L-Ala-L-Leu-L-Ala) (97) ^{90, 91}. The compounds showed anticancer properties. Further, curvularides A-E (98-102) were isolated from Curvularia geniculata originated from Catunaregam tomentosa ⁹². For the first time, cycloaspetide A (103) was obtained from Penicillium janczewskii K. M. Zalessky collected from Prumnopitys andina. The compound was less toxic against human lungs fibroblasts ⁹³.

Trichomides A (104) and B (105) are two new cyclodepsipeptides isolated from the fungus Trichothecium roseum IFB-E066, which dwells in the roots of Imperata cylindrical. Trichomide A proved to have immunosuppressive effect ⁹⁴. Endophyte Penicillium sp. of Acrostichum aureurm origin resulted two new antibacterial compounds named cyclo (Pro-Thr) (106) and cyclo (Pro-Tyr) (107) ⁹⁵.

Phenol, also known as carbolic acid, is an aromatic organic compound with the molecular formula C₆H₅OH. They are sometime called phenolics and contribute many benefits to human health.

A novel phenolic compound, 4-(2,4,7-trioxa-bicyclo [4.1.0] heptan-3-yl) (108) with antifungal and antibacterial activity, was isolated from Pestalotiopsis mangiferae, an endophytic fungus of Mangifera indica Linn. ⁹⁶. Isolation of an antioxidant compound; graphislactone A (109) was carried out from Cephalosporium sp. IFB-E001 hosted by Trachelospermum jasminoides, ⁹⁷. A compound, 2-methoxy-4-hydroxy-6-methoxymethyl-benzaldehyde (110) was isolated from Pezicula sp. strain 553 residing on coniferous trees ⁹⁸. Three phenolic compounds, p-hydroxyphenylacetic acid (111), tyrosol (112) and protocatechuic (113) acid were isolated from endophytic fungi Pseudofusicoccum sp. derived from Annona muricata. All these compounds exhibited antioxidant activities ⁹⁹. Pestalachlorides A-C (114-116) as new chlorinated benzophenone derivatives isolated from a plant endophyte Pestalotiopsis adusta. Compounds A and B demonstrated significant antifungal activities ¹⁰⁰. A new antimicrobial tridepside colletotric acid (117) was produced by Colletotrichum gloeosporioides cultured from Artemisia mongolica ¹¹. A new cytotoxic benzophenone derivative, tenllone I (118) was isolated from the endophytic fungus Diaporthe li-thocarpus A740 of Morinda officinalis ⁷⁶.

Those organic compounds whose carbon atoms are linked in open chains, either straight or branched, rather than containing a benzene ring, are known as aliphatic compounds.

An antimicrobial compound, brefeldin A (119) was obtained from an endophytic fungi Cladosporium sp. residing in Quercus variabilis ¹⁰¹. New cyclohexanone derivatives, pestalofones A-E (120-124) were obtained from Pestalotiopsis fici, an endophyte fungus inhabitant of the branches of an unidentified tree in Hangzhou, China ¹⁰². Gamahonolides A (125) and B (126) were isolated from E. typhina living on Phleum pretense ¹⁰³. A new 6-hydroxyphomodiol (127) metabolite was characterized from an endophytic Phomopsis sp. xz-01 isolated from Camptotheca acuminate. The compound showed selective activity against HepG2 cancer cell lines ¹⁰⁴. A novel azaphilone derivative, chaetomugilin D (128) was derived from C. globosum that resides on Ginkgo biloba. The compound proved to cause significant growth inhibition of brine shrimp and Mucor miehei ⁵⁵.

4. Conclusion and Future Perspectives

It is evident from the literature and data available about the active metabolites produced by plant endophytes that the field is of great interest for Chemists and Biologists. Biology of endophytic microorganisms has developed as a new arena to hunt for novel compounds. A number of bioactive compounds have been isolated and reported so far. Endophytes are continuously under investigation to report many novel compounds of high biological importance.

The existence behavior of endophytes needs better understanding to select and study relevant plants for microfloral compounds. The progress to find new endophytes like Muscodor albus, and Pestalotiopsis microspora, Piriformospora indica, and Taxomyces andreanae has made scientists attracted towards new drugs and technological development ¹⁰⁵. Searching for bioactive compounds, based on endophytes would be a possible way to avoid drug resistance of bacterial strains ¹⁰⁶. Hence, new acquisitions in this field will be fundamental in order to exploit microbial strains for highly effective and large-scale production of plant derived drugs ¹⁰⁷.

Acknowledgments

This work was supported by the Key Research and Development Project of Hainan Province (ZDYF2019165), the Innovative Research Team Project of Ministry (IRT-16R19), and the National Natural Science Foundation of China (Nos. 21662012 and 41866005).

References