2. Structural and Biological Activity Studies
2.1. Terpenoids
Seven new potent phytotoxic harziane diterpenes harzianelactones A and B (
1 and
2), harzianones A–D (
3–
6) and harziane (
9) were isolated from the soft coral-derived fungus
T. harzianum XS-20090075
[13]. Compounds
1 and
2 belonged to a unique class of terpenes with a 6-5-7-5-fused carbocyclic core and a lactone ring. Harzianones A–D (
3–
6) consisted of a fused tetracyclic 6-5-7-4-fused tetra-cyclic skeleton. Chemical epigenetic manipulation was applied to activate silent genes of
T. harzianum XS-20090075 by appending a histone deacetylase (HDAC) inhibitor. With this experimental technique, two new diterpenoids harzianone E (
7) and harzianolic acid A (
41), and one new sesquiterpenoid 3,7,11-trihydroxy-cycloneran (
16) were isolated from the same strain
T. harzianum XS-20090075. At the same time, 11 known sesquiterpenoids, methyl 3,7-dihydroxy-15-cycloneranate (
17), catenioblinc (
18), ascotrichic acid (
19), cyclonerotriol (
20), (10
E)-12-acetoxy-10-cycloneren-3,7-diol (
21), cyclonerodiol (
22), cyclonerodiol oxide (
27), epicyclonerodiol oxide (
28), ent-trichoacorenol (
29), trichoacorenol (
30), and ophioceric acid (
40) were isolated from
T. harzianum XS-20090075
[14]. It was the first time for obtaining cleistanthane diterpenoid from
T. harzianum XS-20090075. Trichodermanins C–H (
10–
15) were new diterpenes with a rare fused 6-5-6-6 ring system, and have been isolated from a fungus
T. harzianum OUPS-111D-4
[15][16]. This strain was separated from a piece of sponge
Halichondria okadai. Compounds
10–
15 were evaluated for their cytotoxicity by using murine P388 leukemia, human HL-60 leukemia, and murine L1210 leukemia cell lines. Compound
10 with a fused 6-5-6-6 ring system exhibited potent cytotoxic activity
[15], and compounds
12 and
13 exhibited modest activity
[16]. Six new terpenes, including one harziane diterpene, 3
R-hydroxy-9
R,10
R-dihydroharzianone (
8), three cyclonerane sesquiterpenes, methyl 3,7-dihydroxy-15-cycloneranate (
17), 11-methoxy-9-cycloneren-3,7-diol (
23), 10-cycloneren-3,5,7-triol (
25), and one acorane sesquiterpene, 8-acoren-3,11-diol (
36), and one cyclonerane 11
R-methoxy-5,9,13-proharzitrien-3-ol (
42), together with four known sesquiterpenes, cyclonerodio (
22), 9-cycloneren-3,7,11-triol (
24), trichoacorenol (
30) and trichoacorenol B (
37) were isolated from
T. harzianum X-5
[17]. The strain X-5 was an endophytic fungus isolated from the marine brown alga
Laminaria japonica. The above six new compounds (
8, 17, 23, 25, 36, and
42) were evaluated to inhibit four marine phytoplankton species and four marine-derived pathogenic bacteria
[17]. Compounds
23 and
42 exhibited potent inhibition activity
[17]. Harzianoic acid A (
38) is a sesquiterpene, and harzianoic acid B (
39) is a norsesquiterpene with a cyclobutane nucleus. They were isolated from a sponge-isolated fungus,
T. harzianum LZDX-32-08
[18], and were found to have new natural scaffolds to exert anti-HCV activity for their capability to inhibit multi-targets, including those for virus replication and entry
[18]. (10
E)-12-Acetoxy-10-cycloneren-3,7-diol (
21) and 12-acetoxycycloneran-3,7-diol (
26) were two new cyclonerane sesquiterpenoids, which were isolated from the marine sediment-derived fungus
T. harzianum P1-4
[9]. A new acorane-type sesquiterpene, 15-hydroxyacorenone (
31), was isolated from
T. harzianum [19], together with acorenone (
32), acorenone-B (
33), 4-epiacorenone (
34), and 4-epiacorenone-B (
35). Stigmasta-7,22-dien-3
β,5
α,6
α-triol (
43) was isolated from
T. harzianum XS-20090075, cultivated by the Czapekʹs culture
[20]. Compound
43 exhibited antifouling activity with an EC
50 value of 39.2 μg/mL and Topo I inhibitory activity with an MIC value of 50.0 μM
[20]. Two fungal strains of
T. harzianum T-4 and
T. harzianum T-5 were obtained from Palampur, Himachal Pradesh (India). Stigmasterol (
44) and
β-sitosterol (
45) were isolated from
T. harzianum T-4
[21]. Ergosterol (
46) was isolated from
T. harzianum T-5
[21]. Trichosordarin A (
47), a unique norditerpene aglycone, was isolated from
T. harzianum R5
[22]. Compound
47 was toxic to the marine zooplankton
Artemia salina with an LC
50 value of 233 µM
[22] (
Figure 1).
Figure 1. Chemical structures of terpenoids (1–47) from T. harzianum. * Means marine source compounds.
2.2. Polyketides
The fermentation of a sponge-associated fungus
T. harzianum HMS-15-3 led to the isolation of four pairs of new C
13 lipid enantiomers harzianumols A–H (
48–
55)
[23]. Four polyketides, trichoharzin B (
56), methyl-trichoharzin (
57), trichoharzin (
58), and eujavanicol A (
59), were isolated from
T. harzianum XS-20090075
[20], which was fermented in rice medium by one strain many compounds (OSMAC) strategy. New naphthalene compound
57, and known naphthalene compound
58 exhibited antifouling activity with the EC
50 values of 29.8 and 35.6 μg/mL
[20]. Six new tandyukisins, tandyukisins A–F (
60–
65), were isolated from
T. harzianum OUPS-111D-4
[11][24][25], which were initially derived from the sponge
Halichondria okadai. Among the tandyukisins A–F (
60–
65), compounds
60,
64 and
65 exhibited cytotoxicity against murine P388 leukemia, human HL-60 leukemia, and murine L1210 leukemia cell lines inferior to the control 5-fluorouracil
[24]. Compounds
61–
63 showed slightly selective growth inhibition against the central nervous system cancer SNB-75 cell line in the HCC panel
[25]. Compounds
64 and
65 exhibited significant cytotoxicity against the cancer cell lines P388, HL-60, and L1210
[24]. The structure-activity relationship may be relevant to the terminals of the side chains.
T. harzianum T-4 was obtained from Palampur, Himachal Pradesh in India, and a polyketide palmitic acid (
66) was isolated from the T-4
[21]. Harzianum A (
67), was a new trichothecene isolated from the soil-borne fungus
T. harzianum in 1994
[26]. Harziphilone (
68) was a new polyketide isolated from
T. harzianum WC 47695
[27], which was isolated from sandy soil with plant debris collected in Fort Lauderdale. The REV/RRE binding assay and HIV assay revealed that compound
68 showed inhibitory activity against REV-protein binding to RRE RNA with IC
50 values of 2.0 μM. In contrast, this compound did not show protection against HIV infection at concentration levels up to 200 μg/mL. The cytotoxicity assay on the murine tumor cell line M-109 showed that
68 exhibited cytotoxicity at 38 μM
[27]. Seven polyketides, keto triol 3 (
69), keto diol 7 (
70), keto diol 6 (
71), keto diol 8 (
72), triacetate 9 (
73), triol 10 (
74) and acetal diol 2 (
75) were isolated from
T. harzianum [28]. One new trichoharzin (
58), and two known compounds, tribenzoate (
76) and triacetate (
77), were isolated from
T. harzianum Rifai in 1993
[29]. A new polyketide, T22azaphilone (
78), was isolated from
T. harzianum T22
[30]. A new compound, trichoharzianol (
79), isolated from
T. harzianum F031, exhibited antifungal activity against
Colletotrichum gloeosporioides with a MIC of 128 μg/mL
[31]. Three novel polyketides trichodenones A–C (
80–
82) were isolated from
T. harzianum OUPS-N115
[32]. This strain was separated from the sponge
Halichondria okadai. Trichodenones A–C (
80–
82) showed cytotoxicities against P388 cell line with the ED
50 values of 0.21, 1.21, and 1.45 μg/mL, respectively. Homodimericin A (
83) was isolated from
T. harzianum WC13
[33][34]. In their model, compound
83 was the biologically inert aftermath of a fungal counter to a bacterial attack. The discovery of cryptenol (
84) from
T. harzianum WC13
[34] indicated that the interactions among microbes in a termite nest were not bipartite but a multipartite system.
The structure and activity relationships of anthraquinones (AQs) in
T. harzianum have been studied. AQs represent an important class of SMs occurring in
T. harzianum strains, which exhibited a variety of biological functions
[12]. The alkylating functionalities in the AQs maximize the anticancer activity by binding tightly with DNA to disrupt the DNA function
[35]. Moreover, anthraquinone derivatives were proposed to have an anticancer function by inhibiting protein kinase CK2
[36]. Pachybasin (
85) and chrysophanol (
86) were isolated from
T. harzianum ETS 323
[37]. 1,7-Dihydroxy-3-hydroxymethyl-9,10-anthraquinone (
87), 1,5-dihydroxy-3-hydroxymethyl-9,10-anthraquinone (
88), emodin (
89), and
ω-hydroxypachybasin (
90) were isolated from
T. harzianum strain Th-R16
[38]. These compounds exhibited effective antifungal activity against
Botrytis cinerea (Ascomycete) and
Rhizoctonia solani (Basidiomycete). At a 500 μg/mL concentration, compound
88 showed comparatively higher activity against
R. solani and
B. cinerea than
89 [38]. Phomarin (
91), (+)-2′
S-isorhodoptilometrin (
92), 1,6-dihydroxy-3-(hydroxymethyl)anthracene-9,10-dione (
93), harzianumnone A (
94) and harzianumnone B (
95) were isolated from the soft coral-derived fungus
T. harzianum XS-20090075
[12]. Compounds
94 and
95 were identified as a pair of epimers, the first example of hydroanthraquinones from
T. harzianum XS-20090075. Compound
92 with Topo I inhibition activity, was further assessed for cytotoxic activity against human tumor cell lines. It exhibited cytotoxic activity against HepG2 cell line with an IC
50 value of 2.10 µM, and showed cytotoxicity against Hela cell with an IC
50 value of 8.59 µM
[12] (
Figure 2 and
Figure 3).
Figure 2. Chemical structures of polyketides (48–68 and 76) from T. harzianum. * Means marine source compounds.
Figure 3. Chemical structures of polyketides (69–75 and 77–95) from T. harzianum. * Means marine source compounds.
2.3. Peptides
Peptaibols are linear antibiotic peptides consisting of 5 to 20 amino acids
[39]. It could be biosynthesized by
T. harzianum. Peptaibols were characterized by the structures of alpha-aminoisobutyric acid (Aib), and C-terminal hydroxylated amino acid. Two new series peptaibols, trichokindins (TKs) and trichorozins (TZs), were isolated from
T. harzianum collected at Nara in Japan. TKs and TZs comprised 18 and 11 amino acid residues, respectively, while TKs were rich in isovaline (Iva). TK-VII (
106) is the most hydrophobic of TKs with 18-residue peptides. Compound
106 induced Ca
2+-dependent catecholamine secretion from bovine adrenal medullary chromaffin cells
[40]. TKs (
96–
106), with a single peak on HPLC and typical IR absorptions at 3300, 1600, and 1530 cm
−1, were confirmed as peptaibols by polarization transfer spectra
[40]. With incubating 10 μM of TK-VII (
106), 27% of the total catecholamines in bovine adrenal chromaffin cells were secreted in the presence of the Ca
2+. In contrast, only 5% of the total catecholamines were secreted without Ca
2+ [40]. Hydrophobicity is vital to the interaction between membranes and peptaibols
[41]. HB I (
107) was isolated from
T. harzianum M-903603
[42]. Trichorzins HA (
108–
113) and MA (
114–
116) were isolated from
T. harzianum M-903602 and
T. harzianum M-922835, respectively. Compounds
108–
116 are a series of 18-residue peptides
[43]. Bioassays on the antifungal activity of trichorzins and harzianins on the phytopathogenic fungus
Sclerotium cepivorum revealed that trichorzins were more potent (75% inhibition at 100 μg/mL) than harzianins (40% inhibition at 100 μg/mL)
[44]. Research on the structured-activity relationships (SARs) revealed that the peptide chain length and superhydrophobicity played an essential part in the peptide/membrane interaction and the subsequent permeability by perturbing the ironic balance of the cell
[44]. As new membrane-modifying peptides isolated from
T. harzianum, trichorozins I–IV (
117–
120), belonged to peptaibols with 11 residues. It was reported that compounds
117–
120 exhibited voltage-dependent ion channel-like activity in lipid bilayers
[45]. Eleven peptides were isolated from
T. harzianum M-903603, and named harzianins HC (
121–
131)
[46]. The detailed study of such proline-rich 14-residue peptaibols revealed that harzianins HC increased the permeability of liposomes and improved voltage-dependent conductance
[46]. An exogenous amino acid supply simplified the microheterogeneous peptide mixtures when Aib, Glu, or Arg was added to the fermentation media of
T. harzianum M-902608. Harzianin PC
U4 (
132), trichorzin PA
U4 (
133), trichorzin PA II (
134), trichorzin PA IV–VIII (
135–
139) and trichorzin PA IX (
140) were isolated from this
T. harzianum M-902608
[47]. When cultured in the Aib-enriched media, compounds
132 and
133 were isolated, while trichorzins PA was obtained from the standard culture media
[47]. Trichorzianines A (TA) and B (TB) are peptaibols isolated from
T. harzianum. TA IIIc (
141) induced the growth inhibition and lysis of the amoeba
Dictyostelium [48]. With the aid of positive ion FAB mass spectrometry, COSY and NOESY experiments, seven peptides of trichorzianines B isolated from
T. harzianum were identified, and these peptides included trichorzianine TB IIa (
142), trichorzianine TB IIIc (
143), trichorzianine TB IVb (
144), trichorzianine TB Vb (
145), trichorzianine TB VIa (
146), trichorzianine TB VIb (
147) and trichorzianine TB VII (
148)
[49]. From a mangrove-derived fungus,
T. harzianum D13, a novel heterocyclic dipeptide trichodermamide G (
149), two known biogenetically related compounds, trichodermamide A (
150) and aspergillazin A (
151) were isolated. A unique sulfur bridge was observed in the structures of compounds
149 and
151 [50] (
Figure 4).
Figure 4. Chemical structures of peptides (149–151) from T. harzianum. * Means marine source compounds.