Natural Products from Endophytic Fungi Associated with Rubiaceae Species

This review presents the chemical diversity and pharmacological properties of secondary metabolites produced by endophytic fungi associated with various genera of Rubiaceae. Several classes of natural products are described for these endophytes, although, this study highlights the importance of some metabolites, which are involved in antifungal, antibacterial, anti-protozoal activities; neurodegenerative diseases; cytotoxic activity; anti-inflammatory and antioxidant activity; and hyperglycemic control.


Introduction
Natural products are small molecules from primary and secondary metabolites naturally synthesized by microorganisms, plants, or animals [1,2]. They are a continuing source of novel bioactive metabolites and have a significant impact on modern medicine [3,4]. Currently, more than 70% of antibacterial and anticancer compounds are natural products or their derivatives [5,6].
Fungi-derived natural products are considered one of the most relevant sources discovery and molecular diversity for new drugs. They are valuable source of biological metabolites that find wide-ranging applications as antibiotics, antifungal, immunosuppressants, antiparasitic and anticancer agents [7][8][9][10][11][12]. Among the microorganisms, endophytes have aroused interest in the last decades mainly for the discovery of important secondary metabolites identified from them.
The term endophyte refers to the microorganism that colonizes interior organs of plants, generally inhabiting their aerial parts such as stems and leaves, but that does not have pathogenic effects on its host [1,7,[13][14][15][16]. Endophytes are ubiquitously found in every plant species examined to date. It is worth mentioning that, of the nearly 300,000 species on earth, each plant hosts one or more endophytes, and approximately 1 million of different species of microorganisms can be found [1,17]. In their symbiotic association, the host plant protects and feeds the endophyte, which in return produces bioactive metabolites to enhance the growth and competitiveness of the host and to protect it from herbivores and plant pathogens [7,9,18].

Biological Activities
Endophyte fungi are capable of synthesizing bioactive compounds, including alkaloids, terpenoids, flavonoids and steroids. Hitherto, most of the secondary metabolites from endophytes are anticancer agents, antibiotics, biological control agents, and other bioactive compounds determined by their different functional roles. In this review, we highlight mainly bioactive natural products endophytically synthetized by endophytic fungi associated with various genera of Rubiaceae (Table 1).

Biological Activities
Endophyte fungi are capable of synthesizing bioactive compounds, including alkaloids, terpenoids, flavonoids and steroids. Hitherto, most of the secondary metabolites from endophytes are anticancer agents, antibiotics, biological control agents, and other bioactive compounds determined by their different functional roles. In this review, we highlight mainly bioactive natural products endophytically synthetized by endophytic fungi associated with various genera of Rubiaceae (Table 1).
Crude extracts of S. formicum from D. hirsuta showed specific activity against Staphylococcus aureus. Stelliosphaerols A (192) and B (193) were subsequently isolated by bioassay-guided isolation as causative agents of this activity. Following it, the growth inhibition assays revealed minimum inhibitory concentration (MIC) values for stelliosphaerols A and B of approximately 250 µg mL −1 [142]. On the other hand, the meroterpene guignardone I (171), guignardone B (173) and the fatty acid glucoside (174) produced by the endophytic fungal from S. hydrophyllacea showed modest inhibitory effects on S. aureus and methicillin-resistant S. aureus (MRSA) [132,133].
The compounds from D. lithocarpus, another endophyte also isolated from M. officinalis, were tested for their cytotoxic activity by the sulforhodamine B method on four human tumor cell lines (SF-268, MCF-7, HepG-2 and A549). The compounds lithocarin B (102), lithocarin C (103), and tenellone H (105) presented IC 50 values ranging from 30 to 100 µM in the four tumor cell lines selected [114].
The PI3Kα inhibitory activity of compounds isolated from C. gloeosporioides, an endophytic fungus from U. rhynchophylla, was evaluated. The phosphoinositide 3-kinases (PI3Ks), a family of lipid kinases, showed a crucial regulatory role in many cellular processes, including cell proliferation, especially PI3Kα as one of the main targets for therapeutic intervention in cancer [154]. Hence, compounds from C. gloeosporioides were tested for their phosphoinositide 3-kinase (PI3Kα) inhibitory activity. The compounds cyclo(L-leucyl-l-leucyl) (149) and brevianamide F (150) showed potent PI3Kα inhibitory activity with IC 50 values of 38.1 and 4.8 µM, respectively, while the other compounds showed weak activity at a concentration of 20 µg·mL −1 [127].

Anti-Inflammatory and Antioxidant Activity
Xylarenones C, D, F, and G (14,15,17,18)  were similar to those of quercetin and apocynin, an efficient inhibitor of the NADPH (nicotinamide adenine dinucleotide phosphate) oxidase complex. Furthermore, the compounds 14, 15, 17 and 18 were also evaluated for their radical scavenging properties in different analytical methods, such as scavengers of superoxide anions (the first ROS produced via the NADPH oxidase complex by stimulated neutrophils), HOCl (the main strong oxidant produced by myeloperoxidase (MPO)), and MPO enzymatic activity, however, the compounds were inactive and had IC 50 values of >100 µM [100].
The in vitro assay results suggest that cercosporin (177) is highly active against Plasmodium falciparum (IC 50 = 1.03 µM), L. donovani (IC 50 = 0.46 µM), and Trypanosoma cruzi (IC 50 = 1.08 µM). Nevertheless, the bioactivity profile observed for cercosporin indicated that it was not specific for any the assayed parasites [127,128]. Compound 178, identified as a seven-membered dioxepane ring-opened analogue of cercosporin, showed a significant reduction in activity in all these biological assays (IC 50 >10 µg mL −1 ), indicating the importance of the methylenedioxy functionality to the biological properties of compound 177 [136]. On the other hand, the alkaloid quinine (118) produced by Diaporthe sp. is a well-known antimalarial drug that is effective against the erythrocyte stage of the parasite P. falciparum [120,121,146].

Other Activities
Proteases are relevant enzymatic targets because these proteins control the formation of functional peptides that participate in physiological processes [155]. The protease inhibitory activity of compounds xylarenones C-E (14-16) was evaluated in vitro using the enzymes subtilisin and pepsin. A potent inhibitory activity for the pepsin and subtilisin in protease assays was observed for compound 14 with an IC 50 of 0.288 and 0.462 µM, respectively. However, compounds 15 and 16 displayed no inhibitory activity on subtilisin (<10%) at any of the four concentrations tested (1.00, 0.1, 0.01, and 0.001 µM) [153].

Conclusions
As demonstrated in this paper, an increasing number of publications revealed a significant interest in endophytes from the Rubiaceae family in recent years due to pharmacological activities. This review presents the chemical diversity and pharmacological properties of secondary metabolites produced by endophyte fungi associated with various genera of Rubiaceae. Several classes of natural products are described for this endophyte, although this study highlights the importance of some metabolites which are involved in antifungal, antibacterial, and anti-protozoal activities; neurodegenerative diseases, cytotoxic activity, anti-inflammatory and antioxidant activity; and hyperglycemic control.
Funding: This research received no external funding.