Identification and Bioactivities of Secondary Metabolites Derived from Endophytic Fungi Isolated from Ethnomedicinal Plants of Tujia in Hubei Province: A Review

Graphic Abstract Tujia is a national minority, inhabiting in the mountainous Wuling area in China. Since 1978, Tujia medicine has been studied, summarized and developed, leading to numerous achievements by Chinese researchers, such as the publishing of approximately 30 monographs of Tujia medicine. These publications are focused on summarizing and improving the theory of Tujia medicine and developing clinical therapies from this system of medicine. The shortage of natural medicinal plants used in Tujia medicine has created the need to discover new resources to replace them and protect endangered natural plant species. Endophytic fungi are one of the conservation options, are considered a source of new bioactive natural products, and are a renewable and inexhaustible source of new drugs and agrochemicals. This review summarizes 260 compounds from endophytic fungi that have been previously isolated from the medicinal plants of Tujia. These compounds include steroids, terpenoids, meroterpenoids, polyketides, alkaloids, peptides, aliphatic compounds, aromatic compounds, and heterocyclic compounds.


Introduction
Endophytic fungi are microorganisms that inhabit in the inner healthy tissues of host plants. They typically do not induce any apparent symptoms of disease in the host [1]. Since anticancer agent paclitaxel (Taxol) was discovered in Taxomyces andreanae, an endophytic fungal strain isolated from Taxus brevifolia [2], interest in bioactive natural products derived from endophytic fungi has increased. During the past two decades, a considerable number of natural products with novel structures and interesting bioactivities have been reported, and endophytic fungi have been identified as to be a source of new bioactive natural products [3][4][5].
Interestingly, endophytic fungi can produce the same or similar bioactive metabolites as their host plants [6]. Thus, they can be used to develop a substitutable approach to producing valuable bioactive compounds to protect plant and conserve resources and the natural environment [7]. Tujia medicine is a type of Chinese medicine that has unique advantages and potential in curing different diseases, but some Tujia medicinal plants are endangered. Therefore, endophytic fungi isolated from Tujia medicinal plants of the Tujia could be a novel source of natural products, thereby protecting endangered plants.
This review summarizes metabolites, including steroids, terpenoids, meroterpenoids, polyketides, alkaloids, peptides, aliphatic compounds, aromatic compounds, heterocyclic compounds and others as well as their bioactivities of endophytic fungi isolated from the antirheumatic and anti-traumatic medicinal plant of the Tujia in Hubei province. In addition, different medicinal plant classes are described.

Cephalotaxus Fortunei Hook
Cephalotaxus fortunei is a perennial, coniferous shrub or small tree belonging to the family Cephalotaxaceae. It is mainly distributed in the subtropical regions up to the northernmost Qinling Mountains and the Huai River in central China. C. fortunei contains the anticancer alkaloid harringtonine, which has made it important for medicinal use in treating leucocythemia [8].
Trichodermanin A (1), a novel diterpenoid with skeletal carbons arranged compactly in a fused 6/5/6/6 ring system, was isolated from the subculture of endophytic fungus Trichoderma atroviride, which was obtained from the bark of C. fortunei [9].
Two sesquiterpenes, named trichoderiols A (2) and B (3), were also isolated from cultures of the same endophytic fungus T. atroviride [11]. In bioactivity studies, these three compounds showed good antifungal effects against Candida albicans, Cryptococcus neoformans, and Trichophyton rubrum as well as some antitumor activity [10]. Furthermore, compounds 2 and 3 were evaluated for their anti-inflammatory activity against nitric oxide (NO) production and showed significant NO scavenging effects, with half-maximal inhibitory concentrations (IC 50 ) values of 15.3 and 9.1 μM, respectively. The results of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay indicated that none of the concentrations used in the experiment were cytotoxic [11].
Ma et al. [14] reported a furan derivative, 5-acetoxymethylfuran-3-carboxylic acid (29), which was isolated from the endophytic fungus Aspergillus flavus in C. fortunei. This compound exhibited potent antibacterial activity against Staphylococcus Aureus, moderate antioxidant activity, and has the potential to be an antibacterial drug.

Huperzia serrata (Thunb. ex Murray) Trev
Huperzia serrata is used in the traditional Chinese medicine preparation. Qian Ceng Ta grows at an altitude of 300-2700 m in damp forests and rock crevices in China. This plant produces the alkaloid huperzine A (42), which is marketed in China as a new drug for Alzheimer's disease (AD) treatment and used in the USA as a supplement to prevent further memory degeneration [17][18][19][20]. Endophytic fungi often have the ability to produce the same or similar bioactive metabolites as their host plants. Five endophytic fungi isolated from H. serrata produced metabolites that were similar or identical to those of huperzine A. These endophytic fungi were characterized and identified as Alternaria sp., Shiraia sp., Fusarium oxysporum, and two different strains of Colletorichum gloeosporides. Moreover, Alternaria sp. produced 6-methoxy-7,4′-dihydroxyisoflavone (43) and arbutin (44) [21][22][23][24][25] (Fig. 4).
Fungal polyketides are one of the largest and most structurally diverse classes of naturally occurring compounds [29]. Four new dimeric spiro-azaplilone derivatives cochliodones E-H (66-69) were obtained from an endophytic fungus of the Chaetomium sp., which was obtained from H. serrata. Furthermore, four compounds assayed and all exhibited antibacterial activity. In particular, compound 68 inhibited E. coli growth to levels almost the same as cefotaxime did [30]. In addition, Yu et al. [31] isolated eight compounds from an endophytic fungus of the Chaetomium sp., which was collected from H. serrata, consisting of seven polyketides and one fungal toxin. They were identified as chaetoviridine F Eight diphenyl ether derivatives obtained from an endophytic fungus P. chrysogenum isolated from H. serrate were identified as penicichrysogenillide A (105), penicichrysogenillide (106), talaromyone A (107), isopencillide (108), penicillide (109), hydroxypenicillide (110), purpactin A (111) and penicichrysogenillide (112). Furthermore, in additional studies, compounds 105 and 106 showed inhibitory activity against NO production in LPS-stimulated RAW264.7 macrophage cells with IC 50 values of 76.2 and 41.2 μM, respectively [36].

Dysosma versipellis (Hance) M. Cheng ex Yang
Dysosma versipellis is an herbaceous perennial species that grows in the understory of mixed evergreen and deciduous forests in China. As an important endangered medicinal plant species, D. versipellis is restricted to eastern and southern China [42].  (162), two glycerides glycerol monooleate (163) and one nucleotide (128). In MTT assay, these compounds showed some cytotoxicity, and compound 157 showed strong cytotoxicity [44] (Fig. 14).

Celastrus anglatus Maxim
Celastrus anglatus, which is heavily distributed in the mountains of southwest China, has been exploited as a natural insecticide resource and is a popular ingredient in folk medicine because of its active ingredients [45]. The following three compounds were isolated from the endophytic fungus Oospora Wallr, which was isolated from C. anglatus: 31, cytochalasin D (164), and ducitol (165). All compounds were evaluated for their inhibitory activity against plant pathogens on spore germination at a concentration of 100 μg/mL. The inhibitory activity of compound 164 was strong, with a half-maximal effective concentration (EC 50 ) of 35.01 μg/mL against Alternaria longipes [46]. Bioassay-guided fractionation led to the isolation of five antibacterial compounds from the fermentation broth of unknown endophytic fungus isolated from C. anglatus. These compounds were identified as 3′-chlorotrypacidin (166), asterric acid (167), methylasterrate (168), methyl-4′,6′-dichloroasterrate (169), and methyl-4′-chloroasterrate (170). The inhibition rates of compounds 165 and 166 (at 500 μM) against Curvularia lunata were 100% and 67.6%, respectively, and they showed strong inhibitory activity against Bacillus subtilis [47]. An endophytic fungus isolated from the phloem of C. anglatus was identified as Fusarium proliferatum and three cyclopeptides isolated
Tan et al. [57] reported the isolation of five 10-membered macrolides and an unsaturated fatty acid and its methyl ester from the fermentation products of the endophytic  Furthermore, all compounds except 206, 208, and 210 were tested for their antimicrobial activities, but none showed a substantial effect [59] (Fig. 19).

Conclusion
It is important to point out that endophytic fungi produce highly diverse secondary metabolites and, therefore, could be used to as sources to discover novel natural products with important bioactivities. Considering the vulnerability and limitation of productivity of plants, endophytic fungi are a potential renewable and inexhaustible source of novel drugs and agrochemicals (Fig. 26).
Interactions between endophytic fungi and host plants are established through complex chemical and biological networks. Endophytic fungi inhibiting plants can colonize their internal tissues without causing disease symptoms. The plant hosts in mutualistic symbioses provide favorable conditions for endophyte development. The microorganisms can produce the same compounds found in the medicinal plants, probably because an exchange of genetic material occurs between the endophyte and plant. Studying and understanding these interactions is essential to achieving the sustainable production of natural products with significant bioactivities from endophytic fungi [72] (Fig. 27).
In Hubei province, over 2000 natural medicinal plants are used in Tujia medicine [73]. Because their overdevelopment and overuse, many medicinal plants are becoming scare, and some are facing extinction. Thus, the use of medicinal plants for the isolation of endophytic fungi is one conservation options. Moreover, endophytic fungi may significantly reduce the use of agrochemicals (fertilizers,