Two New Alkaloids from Fusarium tricinctum SYPF 7082, an Endophyte from the Root of Panax notoginseng

Abstract Panax notoginseng (Araliaceae) is a famous traditional Chinese medicine mainly cultivated in Yunnan and Guangxi provinces of China. Two new alkaloids, rigidiusculamide E (1) and [-(α-oxyisohexanoyl-N-methyl-leucyl)2-] (2), together with two known ones, (−)-oxysporidinone (3) and (−)-4,6′-anhydrooxysporidinone (4) were isolated from the mycelia culture of Fusarium tricinctum SYPF 7082, an endophytic fungus obtained from the healthy root of P. notoginseng. Their structures were determined on the basis of extensive spectroscopic analyses. Compounds 1–4 were tested for their inhibitory effects against NO production on Murine macrophage cell line, and the new compound 2 showed significant inhibitory activity on NO production with the IC50 value of 18.10 ± 0.16 μM. Graphical Abstract Electronic supplementary material The online version of this article (10.1007/s13659-018-0171-0) contains supplementary material, which is available to authorized users.


Introduction
Panax notoginseng (Burk.) F. H. Chen (Araliaceae), known as Sanqi or Tianqi in China, is a famous traditional Chinese medicine [1], with a broad spectrum of pharmacological effects, e.g., anti-atherosclerotic [2], hemostatic and wound healing [3], antioxidant [4], anti-inflammatory [5], hypoglycemic and anti-hyperlipidemia [6], neuroprotective [7], and anti-tumor [8] activities. The plant has been cultivated and domesticated for approximately 400 years, mainly in Yunnan and Guangxi provinces, China. Continuous cultivation of P. notoginseng in the same field will led it to be attacked vulnerably by various soil-borne pathogens, like fungi, bacteria and nematodes [9]. The rhizospheric and endophytic fungal communities are considered not only of vital importance for plant health and soil fertility, but also to have positive effects on plant resistance to diseases and insects. These factors might be useful for the biological control of continuous cropping of P. notoginseng [10].
Fusarium species, a group of filamentous fungi with a number of plant pathogens in it [11], are widely distributed in soil, plants and plant-products. The secondary metabolites of which could be great resources for finding new compounds with a variety of biological activities [12]. For example, previous studies on F. tricinctum led to the identification of neosolaniol monoacetate and visoltricin from the strains of field-loss peanuts [13] and wheat kernels [14], and tricinonoic acid and tricindiol, enniatins and fusarielins, and fusartricin from the endophytic fungi from Rumex hymenosepalus [15], Aristolochia paucinervis [16,17], and Salicornia bigelovii [18], respectively.
During the research on the formation mechanism of continuous cropping obstacles of P. notoginseng, two new alkaloids, rigidiusculamide E (1) and [-(a-oxyisohexanoyl-N-methyl-leucyl) 2 -] (2), together with two known ones (3 and 4) were identified from the mycelia culture of F. tricinctum SYPF 7082, an endophytic fungus isolated from the healthy root of P. notoginseng. Their structures were determined by extensive spectroscopic analyses. Moreover, the inhibitory activities of compounds 1-4 against NO production in Murine macrophage cell line were evaluated. This paper describes the isolation, structure elucidation and results of bioassay.

Results and discussion
The EtOAc extract of the mycelia culture of F. tricinctum SYPF 7082, isolated from the root of P. notoginseng was applied to repeated column chromatography (CC) over MCI-gel CHP20P and silica gel, followed with semipreparative HPLC, to afford four alkaloids (1-4) (Fig. 1). Two of them, 1 and 2 were new compounds.
Rigidiusculamide E (1), a colorless oil, had a molecular formula of C 18 (Table 1). These NMR features are closely related to those of rigidiusculamide D, an alkaloid reported previously from Albonectria rigidiuscula [19]. However, instead of the oxygenated quaternary C-3 (d C 75.1, qC) in rigidiusculamide D, an aliphatic methine (d C 42.7, CH) was present in 1, suggesting that compound 1 was an analog of rigidiusculamide D without oxygen-substitution at C-3 position.
The structure of 1 was further confirmed by 2D NMR experiments. In the 1 H-1 H COSY spectrum, three partial structures of -C (14)  In the ROESY spectrum of 1, correlations of H-3 with H-5 (d H 3.52, m), and of H-4 with H-6a (d H 2.95, dd, J = 13.2, 4.2 Hz) and H-6b indicated that H 3 -14 and H 2 -6 were at the same side, while H-3 and H-5 were on the opposite orientation of the 4-hydroxy-3-methylpyrrolidin-2-one ring (Fig. 2), thereby established the relative configurations of 1. On the basis of the above evidence, the structure of 1 was deduced as shown.
The inhibitory activities of compounds 1-4 against NO production on Murine macrophage cell line were evaluated by Griess assay [22]. Compound 2 showed inhibition of NO production with the IC 50 value of 18.10 ± 0.16 lM, while compounds 1, 3 and 4 were inactive at the concentration of 25 lM.

General Experimental Procedures
Optical rotations were measured on a HORIBA SEPA-300 high-sensitive polarimeter, and UV spectra were recorded on a Shimadzu UV2401A ultraviolet-visible spectrophotometer. Infrared spectroscopy (IR) spectra were obtained on a Bio-Rad FTS-135 series spectrometer. HRESIMS date were obtained using API QSTAR Pular-1 spectrometer. 1 H Two New Alkaloids from Fusarium tricinctum 393 and 13 C NMR spectra were acquired with Bruker DRX-600 spectrometer, using CDCl 3 as solvent and TMS as an internal standard. Chemical shifts were reported in units of d (ppm) and coupling constants (J) were expressed in Hz. Column chromatography (CC) were carried out over silica gel (200-300 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) and MCI-gel CHP20P (75-100 lm, Mitsubishi Chemical Co. Ltd., Tokyo, Japan). An Agilent series 1260 (Agilent Technologies) were used for semipreparative HPLC with an Agilent ZORBAX SB-C18 column (5 lm, 250 9 9.4 mm), with flowing rate of 3 mL/ min.

Fungal material
The fungal strain used in this work was isolated from the healthy root of P. notoginseng, which was collected from Wen-Shan district, Yunnan province of China (104 o 19 0 17.2 00 /23 o 31 0 48.9 00 ). The RNA sequence data derived from this strain has been submitted and deposited in GenBank with the accession number MG930027. BLAST search results revealed that the isolate belongs to the genus Fusarium and had a close relationship (99% identity) with Fusarium tricinctum (KR071697). A voucher specimen (SYPF 7082) has been deposited at the School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University.

Fermentation and Isolation
The strain of Fusarium sp. SYPF7082 was cultivated on potato dextrose agar (PDA) at 25°C for seven days. Fermentation was carried out in 300 Erlenmeyer flasks (250 mL) each containing 90 g rice. Sterile water (100 mL) was added to each flask, and the contents were autoclaved at 121°C for 30 min. After cooling down to room temperature, each flask was inoculated with 20.0 mL of the spore and incubated at 25°C for 40 days.

Compliance with ethical standards
Conflicts of interest The authors declare that there are no conflicts of interest.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creative commons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.