Screening of metabolites from endophytic fungi of some Nigerian medicinal plants for antimicrobial activities

Due to the relationships that endophytes seem to have with their host plants, they make a myriad of biologically active compounds. These compounds can be classified as antibiotics, antioxidants, anticancer agents, volatile antimicrobial agents, immunosuppressive compounds, plant growth promoting agents, and insecticides (1). It has been reported that endophytes possess the ability to produce the same or similar chemicals as those originating from their host plants (2, 3). This presents endophytic microorganisms as potential alternatives to plants in the search for biologically active molecules.

Newbouldia laevis (Bignoniaceae), Ocimum gratissimum (Lamiaceae), and Carica papaya (Caricaceae) are medicinal plants commonly found in Nigeria. These plants have been used ethnomedicinally in the treatment of several disease conditions including ulcers, burns, diarrhea, bleeding, haemorrhoids, dysentery, abdominal pains, cough, colds, pruritus, stress, headache, pneumonia, conjunctivitis, diabetes mellitus, epilepsy and convulsions (4, 5, 6, 7, 8, 9, 10, 11, 12).

Various scientific studies have shown that N. Laevis possesses antimicrobial, antidiabetic, antioxidant, and antimalarial activities (11, 12, 13, 14). O. gratissimum has been reported to show antidiabetic, antioxidant, anxiolytic, sedative, anti-inflammatory, hepatoprotective, antitumor, gastroprotective, hypolipidemic, insecticidal, nematicidal, and antibacterial activities (4,5,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27). Also, scientific reports have revealed that C. papaya possesses immunomodulatory, antimicrobial, anti-inflammatory, antihelmintic, anticancer, wound healing, anti-fertility, abortifacient, diuretic, antihypertensive and antimalarial properties (10, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39).

In our search for biologically active molecules from endophytic fungi associated with Nigerian plants, this present study was carried out to investigate the secondary metabolites of endophytic fungi isolated from three Nigerian medicinal plants N. laevis, O. gratissimum and C. papaya for antimicrobial activities, and also identify the constituents of the fungal extracts using high performance liquid chromatography (HPLC).

A total of five endophytic fungi was isolated, two from N. laevis (NL-L1 and NL-L2), one from O. gratissimum (SL-L1), and two from C. papaya (PPL-LAC and PPL-LE2). In Table 1, it can be observed that the extracts of NL-L2, SL-L1, and PPL-LE2 displayed antibacterial activity against both Gram negative and Gram positive test bacteria. PPL-LAC extract showed activity only against S. aureus, while no antimicrobial activity was recorded for NL-L1 extract. All the endophytic fungal extracts had no activity against the test fungi C. albicans and A. fumigatus HPLC analysis of the fungal extracts revealed the presence of ethyl 4-hydroxyphenyl acetate and ferulic acid in NL-L1 (Fig. 1); ruspolinone in NL-L2 (Fig. 2); Protocatechuic acid, scytalone, and cladosporin in SL-L1 (Fig. 3); indole-3-acetic acid and indole-3-carbaldehyde in PPL-LE2 (Fig. 4); and indole-3-acetic acid in PPL-LAC (Fig. 5).

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

The antibacterial activity displayed by the endophytic fungal extracts can be attributed to the antimicrobial compounds present in the extracts. As can be observed in Table 2, compounds with antimicrobial properties detected by HPLC analysis of the fungal extracts include: ferulic acid, cladosporin, indole-3-carbaldehyde, and protocatechuic acid. The other detected compounds (ethyl 4-hydroxyphenyl acetate, ruspolinone, scytalone, and indole-3-acetic acid) possess diverse biological activities that include cytotoxic/anticancer, antioxidant, anti-inflammatory activities, etc. (42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86).

Ferulic acid, a derivative of cinnamic acid, is a predominant natural phenolic compound first isolated from the plant Ferula foetida (45). It is present in plant seeds and leaves as a component of lignocelluloses, conferring rigidity to the cell wall by making the crosslink between polysaccharides and lignin (45, 87). Ferulic acid has been detected in and/or isolated from culture extracts of endophytic fungi like Annulohypoxylon stygium (88) and Aspergillus austroafricanus (89). Ferulic acid has been reported to show several pharmacological activities including antioxidant anti-diabetic, antihypertensive, anticancer, anti-inflammatory, hepatoprotective, and antimicrobial activities. Other reported biological activities of ferulic acid are anti-allergic, increase sperm viability, antithrombotic, antiviral and vasodilatory actions, metal chelation, modulation of enzyme activity, activation of transcriptional factors, gene expression and signal transduction activities (43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54).

Protocatechuic acid (3,4-dihydroxybenzoic acid) is a widely distributed naturally occurring phenolic acid present in most edible and medicinal plants (53, 90, 91, 92). The compound has also been reported to be produced by several species of bacteria and fungi (40, 57, 93, 94, 95). Protocatechuic acid has been reported to show antioxidant, antimicrobial, anticancer, anti-ulcer, antidiabetic, anti-ageing, antifibrotic antiviral, anti-inflammatory, analgesic activity, anti-atherosclerotic, cardiac, hepatoprotective, neurological and nephroprotective activities (57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71).

Cladosporin is an isocoumarin derivative previously isolated from Cladosporium cladospo rioides (72, 75) and Eurotium sp. (76). Cladosporin has been reported to show antiplasmodial, anti microbial, insecticidal and antitumor activities (72, 73, 74, 75, 76).

Indole-3-carbaldehyde (indole-3-carboxaldehyde), an indole alkaloid, had been isolated from an endophytic Lasiodiplodia sp. associated with Viscum coloratum (96), from endophytic actinomycetes (85, 97), from Pseudomonas sp. (98), and from a Red Sea sponge Hyrtios erectus (86). Indole-3-carbaldehyde showed moderate cytotoxic (86) and antibacterial activities (85). In plants, indole-3-carbaldehyde is associated with the innate immunity to microbial pathogen infections (84,85).

Indole-3-acetic acid is the most abundant and well known plant hormone of the auxin class which regulates various aspects of plant growth and development (99, 100, 101). Many bacterial and fungal species have been reported to be able to produce indole-3-acetic acid (40, 99, 101, 102). Indole-3-acetic acid has been reported to possess cytotoxic/anticancer, antioxidant, anti-inflammatory activities (81, 82, 83).

Ethyl-2-(4-hydroxyphenyl) acetate (ethyl p-hydroxyphenyl acetate) has been isolated from Ixeris chinensis (103), Osmanthus fragrans (104). Ethyl p-hydroxyphenyl acetate has been used as an intermediate for the preparation of anti-inflammatory agents (42).

Ruspolinone, a pyrrolidine alkaloid, was isolated from the plant Ruspolia hypercrateriformis (105, 106). Pyrrolidine alkaloids are hazardous for humans and animals, but exhibit a host of biological activities, and are used as organocatalysts and building blocks in organic synthesis (55, 56).

Scytalone, a tetralone derivative, has been previously isolated from several endophytic fungi which include Penicillium cf. glaucoalbidum (107), Cladosporium tenuissimum (108), Annulohypoxylon sp. (109), Phomopsis sp. (78), and Scytalidium sp. (110). Scytalone has been known as an intermediate in the biosynthesis of melanin, the dark pigment of many phytopathogenic fungi which aid their hyphal (appresoria) penetration into leaves and cell walls (78, 79, 80). Scytalone was also reported to promote plant growth (77).

Studies of the endophytic fungal population of Nigerian plants have revealed the potentials possessed by these plants as host to endophytes that express important biological active compounds (2, 3, 40, 41, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121). These endophytes also hold key of possibilities to the discovery of novel molecules for pharmaceutical, agricultural and applications.

Endophytic fungi isolated from with three Nigerian plants Newbouldia laevis, Ocimum gratissimum, and Carica papaya produced compounds with diverse biological activities. These plants and their associated endophytes could be an excellent source of novel biologically active compounds with pharmaceutical or industrial importance.