New Sorbicillinoids with Tea Pathogenic Fungus Inhibitory Effect from Marine-Derived Fungus Hypocrea jecorina H8

Four new dimeric sorbicillinoids (1–3 and 5) and a new monomeric sorbicillinoid (4) as well as six known analogs (6–11) were purified from the fungal strain Hypocrea jecorina H8, which was obtained from mangrove sediment, and showed potent inhibitory activity against the tea pathogenic fungus Pestalotiopsis theae (P. theae). The planar structures of 1–5 were assigned by analyses of their UV, IR, HR-ESI-MS, and NMR spectroscopic data. All the compounds were evaluated for growth inhibition of tea pathogenic fungus P. theae. Compounds 5, 6, 8, 9, and 10 exhibited more potent inhibitory activities compared with the positive control hexaconazole with an ED50 of 24.25 ± 1.57 µg/mL. The ED50 values of compounds 5, 6, 8, 9, and 10 were 9.13 ± 1.25, 2.04 ± 1.24, 18.22 ± 1.29, 1.83 ± 1.37, and 4.68 ± 1.44 µg/mL, respectively. Additionally, the effects of these compounds on zebrafish embryo development were also evaluated. Except for compounds 5 and 8, which imparted toxic effects on zebrafish even at 0.625 μM, the other isolated compounds did not exhibit significant toxicity to zebrafish eggs, embryos, or larvae. Taken together, sorbicillinoid derivatives (6, 9, and 10) from H. jecorina H8 displayed low toxicity and high anti-tea pathogenic fungus potential.


Introduction
The influence of bioactive compounds from natural sources on human life has challenged scientists to research new environmental contexts and the associated biological diversity [1]. The ocean, as the largest frontier in biological exploration, represents one of the most favorable reservoirs of organisms producing secondary metabolites with biological activities [2]. The deep sea is an extreme environment; in this respect, its associated micro-organisms have great potential to produce natural products with novel biological properties [3].
The tea plant (Camellia sinensis L.) is an important commercial crop all over the world. However, the tea plant suffers from biotic stresses of some pathogenic fungi [4,5], which often exhibits severe damage of the blade tissue and discoloration of the leaves, common symptoms, including blight (Exobasidium vexans Massee), brown blight (Colletotrichum camelliae Massee), and red rust (Cephaleuros parasiticus Karst). These pathogenic fungi greatly reduce the quality of tea and damage human health [6][7][8].
As part of our continuing exploration for structurally novel and biologically interesting secondary metabolites from marine microorganisms, the fungal strain Hypocrea jecorina H8 (H. jecorina H8) was isolated from mangrove sediments and showed potent inhibitory activity against tea pathogenic fungus P. theae.
Chemical investigation of H. jecorina H8 from rice medium led to the isolation of 11 compounds, including five sorbicillinoids (1)(2)(3)(4)(5) and six known sorbicillinoid analogs. Some of these compounds exhibited significant inhibitory activity against tea pathogenic fungus P. theae. and low toxicity to zebrafish. Herein, we report the isolation, structural determination, as well as antifungal activity of these isolated compounds.
As part of our continuing exploration for structurally novel and biologically interesting secondary metabolites from marine microorganisms, the fungal strain Hypocrea jecorina H8 (H. jecorina H8) was isolated from mangrove sediments and showed potent inhibitory activity against tea pathogenic fungus P. theae.
Chemical investigation of H. jecorina H8 from rice medium led to the isolation of 11 compounds, including five sorbicillinoids (1)(2)(3)(4)(5) and six known sorbicillinoid analogs. Some of these compounds exhibited significant inhibitory activity against tea pathogenic fungus P. theae. and low toxicity to zebrafish. Herein, we report the isolation, structural determination, as well as antifungal activity of these isolated compounds.
The above spectroscopic data showed high similarities to those of trichodermolide B, a known compound isolated from Trichoderma reesei (HN-2016-018) [12], except for the presence of signal for a hydroxyl group, a signal for methylene group at C-22 (δ H 4.32, δ C 62.7) and the lack of signal for a methyl group (δ C 19.1). Thus, 1 was deduced to be a hydroxylated derivative of trichodermolide B at C-22, validated by the COSY correlations of δ H 6.35 (H-21) with δ H 4.32 (H2-22) (Figure 2a).
The two double bonds in sorbyl side chain for 1 were assigned both as E configuration based on their coupling constants (J H-18/H-19 = 15.2 Hz, J H-20/H-21 = 15.4 Hz) and the NOESY correlation between H-18/H-20. For the bridged bicycle lactone ring system, it was only possible if the CH 3 -9 and CH 2 -10 were oriented equatorially. In addition, the 1  .08 for 1) suggested the same relative configurations of C-3, C-15, and C-7 in 1 as that in trichodermolide B [12].
Therefore, the relative configuration of 1 was assumed as 3S*,15R*,7R*. The ECD curve of 1 showed a negative Cotton effect around 220 nm and a positive Cotton effect around 270 nm, respectively. These were the same as trichodermolide B ( Figure S41 and experimental ECD spectra of trichodermolide B). The absolute configuration of 1 was assigned as 3S,15R,7R ( Figure 3a). As a result, the structure of 1 was determined and named as trichodermolide C ( Figure 1).
Mar. Drugs 2022, 20, x FOR PEER REVIEW 4 of 12 around 270 nm, respectively. These were the same as trichodermolide B ( Figure S41: Calculated and experimental ECD spectra of trichodermolide B). The absolute configuration of 1 was assigned as 3S,15R,7R ( Figure 3a). As a result, the structure of 1 was determined and named as trichodermolide C ( Figure 1).  Compound 2 was also obtained as yellow amorphous powder. The molecular formula was deduced to be C21H30O5 by interpretation of the HR-ESI-MS peak at m/z 363.2162 [M + H] + (calcd for 363.2093 C21H31O5 + ), implying seven degrees of unsaturation. Compound 2 presented 1 H and 13 C NMR signals similar to those of compounds 1, especially those on the bridged bicyclic ring moiety. The structural differences in the side chains could be revealed by the DEPT spectra, in which two carbonyl signals vanished and two around 270 nm, respectively. These were the same as trichodermolide B ( Figure S41: C culated and experimental ECD spectra of trichodermolide B). The absolute configurat of 1 was assigned as 3S,15R,7R (Figure 3a). As a result, the structure of 1 was determin and named as trichodermolide C (Figure 1).  Compound 2 was also obtained as yellow amorphous powder. The molecular f mula was deduced to be C21H30O5 by interpretation of the HR-ESI-MS peak at m/z 363.21 [M + H] + (calcd for 363.2093 C21H31O5 + ), implying seven degrees of unsaturation. Co pound 2 presented 1 H and 13 C NMR signals similar to those of compounds 1, especia those on the bridged bicyclic ring moiety. The structural differences in the side cha could be revealed by the DEPT spectra, in which two carbonyl signals vanished and t    H-17/CH 3 -11, and CH 3 -8/CH 2 -12 established the relative configuration of 2. Therefore, the relative configuration of 2 was assumed as 3S*,15R*,7R*. The calculated ECD curve of 3S,15R,7R-2 was consistent with the experimental data (Figure 3b), and hence the absolute configuration of 2 was assigned as 3S,15R,7R.
Compound 3 was obtained as a yellow amorphous powder. The results from the HR-ESI-MS peak at m/z 537.2090 [M + Na] + (calcd for 537.2100 C 28 H 34 O 9 Na + ) suggested that the molecular formula of 3 was C 28 H 34 O 9 , thus, implying twelve degrees of unsaturation. The 1 H and 13 C NMR data of 3 showed 30 protons and 28 carbons signals, and these carbon signals were classified into twelve quaternary carbons (four ethylenic bonds), two carbonyls, six ethylenic bonds, one oxygenated methines, two methines without an oxygen link, one methylene, and six methyls.
Comparison of the NMR data of 3 with those of 10, a known metabolite isolated from a strain of the same genus [6], indicated that 3 possessed an identical trichodimerol [13] skeleton to 10. Although compound 10 afforded only 14 signals because of a symmetric structure, some 13 C signals of compound 3 split, suggesting an asymmetric structure ( Table 2). The major difference were found at the signals due to C-8 , C-10 moiety, suggesting a hydration at C-8 /C-9 double bond in compound 3. In 13 C NMR and DEPT, the double bond signals of C-8 and C-9 in 10 turn to a methylene (δ C 40.9, C-8 ) and an oxygen-linked methine (δ C 80.7, C-9 ) in 3. The conclusion was confirmed by the COSY correlation from δ H 2.38 (H-8 ) to δ H 4.31 (H-9 ), and to δ H 5.67 (H-10 ). In the HMBC spectrum of 3, the correlations from H-8 to C-7 , C-9 were also found.
Compound 5 was obtained as a white amorphous powder with positive HR-ESI-MS ion peaks at m/z 499.2312 [M + H] + indicating 12 degrees of unsaturation. According to the HR-ESI-MS data, compound 5 and 11 shared the same molecular formula ( Figure S42). The NMR data of 5 had similar features compared to those of 11, which suggests that they are stereoisomers. The planar structure of 5 was determined by the COSY and HMBC data. The main differences of NMR signals were attributable to C-1, C-4, and C-4a, with the 13  Thus, compounds 5 and 11 should be epimers around either C-4 or C-4a. In the NOESY spectrum, key cross peaks were observed between δ H 3.63 (H-8a) and δ H 1.34 (CH 3 -1a) and δ H 1.48 (CH 3 -5a) (Figure 2e), indicating that the methyls CH 3 -1a and CH 3 -5a were at the same side with H-8a. Furthermore, NOESY were observed between CH 3 -1a and δ H 1.29 (CH 3 -4). These results indicated that the relative configuration of C-4 was same as that of 11. Therefore, we concluded that 5 is a stereoisomer of 11 on C-4a. The relative configuration of 5 was defined as shown in Figure 1.

Evaluation of Toxicity
The zebrafish is a small teleost that is becoming increasingly popular in many biomedical and environmental studies [15]. This model has shown sensitivity to a broad variety of contaminants (such as endocrine disruptors and organic pollutants), indicating their suitability as a biological method for environmental monitoring in risk assessment.
We evaluated the toxicity of compounds 5, 6, 8, 9, and 10 in a zebrafish model (Figure 4). Figure 4a showed that these compounds, except compound 8, killed zebrafish embryo less than 50% when treated with a concentration of 10 µM. When the treatment time was prolonged to 72 h (Figure 4b), the mortality rate of zebrafish embryo caused by compound 8 at 0.625 µM increased to nearly 60%, whereas the effects of compounds 5, 6, 9, and 10 did not change greatly.
In addition, the impact on the malformation of zebrafish by these compounds was observed using a Leica stereomicroscope. Figure 4c showed graphically under the same treatment that compounds 5 and 8 had greater effects than other compounds on the mortality rate and malformation of zebrafish both at a concentration of 0.625 µM for 24 h and at a concentration of 10 µM for 72 h. In summary, our data demonstrated that compounds 6, 9, and 10 were of low toxicity and could be used against tea pathogenic fungi agents and deserve further optimization.
Chemically, the configurations and absolute of these compounds were described by their NOESY and CD spectra, respectively. All the isolated compounds of anti-tea pathogenic fungus Pestalotiopsis theae activities were evaluated. Compounds 5, 6, 8, 9, and 10 had stronger inhibitory effects against the fungi assays compared with hexaconazole.
However, the security of the antifungal regents is an important factor for use in agricultural applications. Although it showed potent activity, compound 5 exhibited strong anti-proliferative effects on the embryonic development of zebrafish, and compound 8 killed zebrafish embryos more than 50% at both a concentration of 0.625 µM for 24 h and a concentration of 10 µM for 72 h, thus, indicating high toxicity. Compounds 6, 9, and 10 showed much lower toxicity to zebrafish. In summary, sorbicillinoid derivatives (6, 9, and 10) from H. jecorina H8 had low toxicity and potent potency against tea pathogenic fungus.

General Experimental Procedures
An electrospray ionization source (ESI)-equipped Q-Exactive Mass spectrometer (Thermo Fisher Scientific Corporation, Waltham, MA, USA) was used to analyse the HR-ESI-MS data. A Shimadzu UV-260 spectrometer (Shimadzu Corporation, Tokyo, Japan) and a Perkin-Elmer 683 infrared spectrometer (PerkinElmer, Inc., Waltham, MA, USA) were used to obtain the UV and IR spectra, respectively. A JASCO P-200 polarimeter (JASCO Corporation, Tokyo, Japan) with a 5 cm cell was applied to measure the optical rotation value. The NMR spectra with TMS as the internal standard were taken on a Brucker Avance III 600 FT NMR spectrometer (Bruker Corporation, Billerica, MA, USA).

Fungus Carbohydrate Fermentation
H. jecorina (H8) was isolated from the mangrove sediments collected from Zhangjiangkou Mangrove National Nature Reserve, Fujian province, China. The strain was identified as Hypocrea jecorina on the basis of the internal transcribed spaces (ITS) sequence. The ITS region of the fungus was a 636 bp DNA sequence (GenBank accession number OL376355), which had 99% identity to Hypocrea jecorina. The fungal strain has been preserved at Third Institute of Oceanography, China. P. theae (ITS GenBank accession number HQ832793) was isolated from foliar lesions of the tea leaf, and its pathogenicity to tea leaves was verified both in vitro and in vivo.

Antifungal Activity Assay
Initial evaluations of the antifungal activity of the purified compounds were conducted against tea pathogenic fungus P. theae in six-well microplates as described by Xia Yan with certain modifications [17]. The final concentrations of each compound in the wells were 80, 40, 20, 10, 5, and 2.5 µg/mL (two-fold dilutions). DMSO and hexaconazole were used as a