**3. Discussion**

During the analyses of natural products from the ethyl acetate (EtOAc) extract of antibacteria from special growing environment, we discovered five new sorbicillinoids (**1**–**5**) together with six known analogs (**6**–**11**). These compounds were obtained from the marinederived *H. jecorina* (H8) from the mangrove sediments collected from Zhangjiangkou Mangrove National Nature Reserve, China.

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.

#### **4. Materials and Methods**

#### *4.1. 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).

Column chromatography was performed with silica gel (Yantai Chemical Industry Research Institute, Shandong, China), Cosmosil 75 C18-MS-II (75 μm, Nacalai Tesqye corporation, Kyoto, Japan), and Spehadex LH-20 (GE Healthcare, Danderyd, Sweden). Semi-preparative HPLC was conducted on an Aglient HPLC (Agilent Technologies Inc., Santa Clara, CA, USA) system equipped with a diode array detector via a preparative Cosmosil ODS column. The HR-ESI-MS spectra were measured using a thermo Q-Exactive Mass spectrometer (Thermo Fisher Scientific Corporation).

#### *4.2. Eletronic Circular Dichroism (ECD) Calculations*

The circular dichroism (CD) spectra were recorded on a MOS-500 dichroism spectrometer. The conformational analyses were conducted with MOE 2018 using MMFF94. All calculations were conducted with Gaussian 09 using various functionals (b3lyp/6-31+g(d) and cam-b3lyp/6-31+g(d)). The overall theoretical ECD data were weighted by Boltzmann distribution, and the ECD spectra were produced by SpecDis 1.70 software [6,16].

#### *4.3. 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.

#### *4.4. Extraction and Isolation*

The fungus H8 was cultivated on rice-artificial sea water medium, incubated at 28 ◦C for 20 days in a standing position. After 20 days of fermentation, the solid cultures were dispersed in water (H2O) and extracted with EtOAc (1:1, *v/v*) three times. The EtOAc extract was concentrated under reduced pressure at 40 ◦C to afford 22.0 g residue. The residue (21 g) was subjected to silica gel CC with petroleum ether (PE)-EtOAc (*v/v*) (20:1; 10:1; 5:1; 2:1; 1:1; 0:1) and chloroform (CDCl3)-methanol (MeOH) (*v/v*) (100:1; 50:1; 20:1; 10:1; 5:1; 100% MeOH) to yield nine fractions (Fr. 1–9).

Fr. 2 (400 mg) was subjected to octadecylsilyl (ODS) chromatography and eluted with MeOH-H2O (80–100%) to give 10 subfractions (Fr. 2A–2J). Then, Fr. 2G was purified using preparative reversed-phase HPLC C18 column (pre. Rp-C18) and isocratic eluted with MeOH-H2O (85%) to obtain compound **6** (50 mg). Fr.3 (500 mg) was subjected to CC over ODS gel eluting with to MeOH-H2O (70–100%) to obtain four subfractions (Fr. 3A-Fr.3D). Subfraction Fr.3C was purified by Prep. Rp-C18 using MeOH-H2O (74%) elution to obtain **7** (11.9 mg). Fraction Fr.4 (496 mg) was subjected to CC over Sephadex LH-20 (MeOH) to yield six subfractions (Fr.4A–Fr.4F).

Compounds **1** (1.3 mg), **3** (1.1 mg), **10** (4.4 mg), and **8** (10 mg) were separated from subfraction Fr.4C by Prep. Rp-C18 (MeOH-H2O, 80%). Fraction Fr.5 was purified by CC on Sephadex LH-20 (MeOH) to yield four subfractions (Fr.5A–Fr.5D), and then fraction Fr.5B was purified by Prep. Rp-C18 isocratic eluted with MeOH-H2O (80:20) to provide **2** (5.5 mg). Fr. 5C was also purified by Prep. Rp-C18 isocratic eluted with MeOH-H2O (52:48) to obtain compound **4** (1.0 mg). Compounds **5** (9.3 mg), **9** (20 mg), and **11** (25 mg) were separated from subfraction Fr.5D by Prep. Rp-C18 (MeOH-H2O, 80%).

#### *4.5. Structrural Elucidation of the New Compounds* **1***–***5**

Trichodermolide C (**1**): yellow amorphous powder; HR-ESI-MS *m/z* 375.1798 [M + H]+ (calcd. for 375.1729 C21H27O6+) and 397.1609 [M + Na]+ (calcd. for 397.1627 C21H26O6Na+) in the positive mode; [α]30D = +54◦ (*c* = 0.5, MeOH). IR (KBr) (*ν*max): 3385, 1646, and 1436 cm<sup>−</sup>1. 1H NMR and 13C NMR data are listed in Table 1.

Trichodermolide D (**2**): yellow amorphous powder; HR-ESI-MS *m/z* 363.2162 [M + H]+ (calcd. for 363.2093 C21H31O5+) and 389.1972 [M + Na]+ (calcd. for 385.1991 C21H30O5Na+) in the positive mode; [α]30D = +105◦ (*c* = 0.5, MeOH). IR (KBr) (*ν*max): 3406, 2927, 1560, and 1430 cm<sup>−</sup>1. 1H NMR and 13C NMR data are listed in Table 1.

7, 7-, 9--Hydroxy-trichodimerol (**3**) isolated as a yellow amorphous powder; HR-ESI-MS *m/z* 51.2265 (calcd. for 515.2281 C28H35O9+) and 537.2090 [M + Na]+ (calcd. for 537.2100 C28H34O9Na+) in the positive mode; [α]30D = −35◦ (*c* = 0.5, MeOH), IR (KBr) *ν*max): 3406, 1570, and 1430 cm<sup>−</sup>1. UV (MeOH) *λ*max (log *ε*): 286 (4.39) and 359 (4.46) nm. 1H NMR and 13C NMR data are listed in Table 2.

1-(2,4-Dihydroxy-3,5-dimethylphenyl)-3,4,5-trihydroxyhexan-1-one (**4**): colorless amorphous powder; HR-ESI-MS *m/z* 285.1342 [M + H]+ (calcd. for 285.1338 C14H21O6+) in the positive mode; [α]30D = +6◦ (*c* = 0.05, MeOH), IR (KBr) (*ν*max): 3416, 2927, 1601, 1367, 1188, and 1075 cm<sup>−</sup>1. UV (MeOH) *λ*max (log *ε*): 216 (3.66), 285 (3.72), and 329 (3.41) nm. 1H NMR and 13C NMR data are listed in Table 1.

Isobisvertinol A (**5**): white amorphous powder; HR-ESI-MS *m/z* 499.2312 [M + H]+ (calcd. for 499.2326 C28H35O8+) in the positive mode, and 497.2194 [M − H]- (calcd. for 497.2181 C28H33O8−) in the negative mode; [α]30D = −46.2◦ (*c* = 0.5, MeOH), IR (KBr) (*ν*max): 3416, 1570, and 1430 cm<sup>−</sup>1. UV (MeOH) *λ*max (log *ε*): 278 (3.44) and 374 (3.52) nm. 1H NMR and 13C NMR data are listed in Table 2.

#### *4.6. 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

blank control and positive control, respectively. The ED50 (μg/mL) values were calculated statistically using Probit analyses.

## *4.7. Toxicity Evaluation*

Wild-type (AB strain) zebra fish (Danio rerio) were used in this test. Compounds **5**, **6**, **8**, **9**, and **10** were dissolved in DMSO (10 mM) and stored at −20 ◦C. Toxicity evaluations were analyzed regarding the anti-proliferative effects on embryos according to the literature [18]. In brief, 15 zebrafish embryos per condition were exposed to compounds at the concentrations of 10, 5, 2.5, 1.25, and 0.625 μM, and 0.1% DMSO was used as the vehicle control. A Leica stereomicroscope was used to observe the embryos every 24 h.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/md20030213/s1, Figures S1–S8: Spectra of **1**, Figures S9–S16: Sepctra of **2**, Figures S17–S25: Spectra of **3**, Figures S26–S32: Spectra of **4**, Spectra of **3**, Figures S33–S40: Spectra of **5**, Figure S41: ECD spectra of trichodermolide B, Figure S42: HR-ESI-MS spectra of 11. ECD calculation of **1**–**3**.

**Author Contributions:** X.-X.T. and G.-X.X. identified the fungus and performed the antifungal activity assay. W.-B.Z. performed the toxicity evaluation. S.-Z.L. and F.-M.H. isolated the compounds. Z.W. was responsible for the structural elucidation. Y.-K.Q. and M.-Y.L. supervised the project. All authors have read and agreed to the published version of the manuscript.

**Funding:** The project was supported by Key Laboratory of Marine Biotechnology of Fujian Province (2021MB02 to ML) and Asian Countries Maritime Cooperation Fund (99950410). The project was also supported by National Survey of Traditional Chinese Medicine Resources (Grant No. (2019)39) and Deep Sea Habitats Discovery Project (DY-XZ-04).

**Conflicts of Interest:** The authors have no conflict of interest to declare. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.
