New steroid produced by Periconia pseudobyssoides K5 isolated from Toona sureni (Meliaceae) and its heme polymerization inhibition activity

Abstract A new ergostane-type steroid named (22E)-3α,6α,9α-ergosta-7,22-diene-3,6,9-triol (1), along with six known steroids 5α,8α-epidioxy-24-ethyl-cholest-6-en-3β-ol (2), ergosterol-5,8-peroxide (3), cerevisterol (4), isocyathisterol (5), 6β-hydroxystigmast-4-en-3-one (6), 6β-hydroxy-4-campesten-3-one (7), were isolated from the fermented unpolished rice media by Periconia pseudobyssoides K5 (Periconiaceae), an endophytic fungus from medicinal plant Toona sureni (Meliaceae). The fermentation takes at 28 ± 2 °C for 30 days. The structure of new steroid (1) was elucidated by extensive spectroscopic measurements (IR, HR-ESI-TOFMS, and 1D and 2D NMR) analyses. The isolated compounds (1–7) were evaluated for heme polymerization inhibition assay (HPIA). The IC50 HPIA value of 1 is 8.24 ± 0.03 mg/ml.


Introduction
Endophytes fungi are endosymbiont microorganisms with ecological benefits for their host [1,2].In interaction with the plant, endophytes promote better nutrient uptake and protection against pathogens and improve the plant's ability to adapt to biotic and abiotic stress [3,4].Subsequently, endophytes can produce various secondary metabolites, such as polyketides, phenols, alkaloids, terpenes, and steroids [5,6].Some of these compounds have been reported to possess various pharmacological properties, including antioxidant, antibacterial, antifungal, antidiabetic, and antimalarial activity [5][6][7][8].Endophytes have increased the interest in natural product exploration for the last decades [9,10].

Results and discussion
The crude extract from rice media fermented by P. pseudobyssoides K5 was successfully separated with n-hexane and ethyl acetate.The n-hexane fraction was separated by various chromatographic techniques including the normal and reversed phase using silica gel 60 and C18-ODS.The purification process guided by TLC silica GF 254 led to the isolation of seven steroids (1-7) (Figure 1 and Figure S1).
Compound 1 was obtained as a white amorphous powder (in CHCl 3 ).The molecular formula of C 28 H 46 O 3 was determined by HR-ESI-TOFMS, which showed a molecular ion peak at m/z 453.3355 [M þ Na] þ , encompassing six degrees of unsaturation.The IR spectrum showed major absorption bands corresponding to the presence of hydroxyl (3429 cm À1 ), aliphatic (2955 and 2870 cm À1 ), and alkene groups (1458 cm À1 ).The 1 H NMR spectrum of 1 (Table     .Moreover, the configuration of hydroxyl group at C-9 was deduced by the biogenetic approach of ergosterol derivatives as well as by a comparison of 13 C NMR between 1 and its analogs.Yaoita et al. [15] stated the ergosterol compounds with a-hydroxyl group at C-9 (75.1-77.2ppm), so C-9 (76.1 ppm) in 1 was an a-orientation.Further analysis and the literature investigation revealed that compound 1 had a similar structure to the known compound (22E,24R)-ergosta-7,22-diene-3b,5a,6a,9a-tetrol [15].However, the main differences are the absence of a hydroxyl group at C-5 and the b-configuration of hydroxyl group at C-3 in 1.Therefore, compound 1 was determined as a new steroid, namely (22E)-3a,6a,9a-ergosta-7,22-diene-3,6,9-triol.

General experimental procedures
Optical rotations were calculated with an ATAGO AP-300 automatic polarimeter (ATAGO, Saitama, Japan).Melting points were measured with a Fisher-Johns melting point apparatus (Vernon Hills, USA) and are uncorrected.IR spectra were recorded on a KBr plate with a Perkin Elmer Spectrum 100 FT-IR spectrometer (Perkin Elmer, Shelton, USA).The high-resolution time-of-flight mass analyzer (HR-TOFMS) was determined on a Water Xevo Q-TOF direct probe/MS system using ESI mode and microchannel plates MCPs detector (Milford, MA, USA).NMR spectra were recorded on a JEOL JNM-ECX500R/S1 spectrometer (JEOL, Tokyo, Japan) and a Bruker Topspin spectrometer (Karlsruhe, Germany), both 500 MHz for 1 H and 125 MHz for 13 C, using TMS as an internal standard.Column chromatography (CC) Table 2. Heme polymerization inhibition activity (HPIA) of compounds 1-7, n-hexane-extract and chloroquine diphosphate (positive control) (IC 50 ± SD, mg/ml) a .

Collection of fungal and plant material
The plant material was collected from Kuningan District, West Java, Indonesia (-69-6.941812,108.438740).The plant was determined as T. sureni (Blume) Merr by Herbarium Staff at the Plant Taxonomy Laboratory, Department of Biology, Universitas Padjadjaran, Indonesia.The fungi were isolated from the inner cortical tissue by a surface sterilization method.This endophytic fungus was identified by molecular analysis of the internal transcribed region (ITS) by staff at the Division of Biological Activity, Central Laboratory, Universitas Padjadjaran Indonesia, and is known as P. pseudobyssoides K5 strain (GenBank Accession number KC954161.1).
3, and 135.3, four quaternary carbons (including one oxygenated and one olefinic carbons) at d C 76.1 and 144.1, implying an ergosterol-type steroid.The above data explained the presence of two unsaturated degrees, indicating four additional ring systems related to the tetracyclic core structure of the steroid group.Furthermore, the 1 H-1 H COSY experiment of 1 (bold bonds in Figure 2) established the key coupling relationships of H-1/H-2/H-3/H-4/H-5/H-6/H-7 in the A and B rings, which confirmed the position of hydroxyls at C-3 and C-6 as well as D 7,8 double bond.This partial structure was also reinforced by the HMBC correlations of H-2/H-4 to C-3 (d C 67.8), H-5/H-6 to C-7 (d C 117.5), and H-6/H-11/H-14 to C-8 (d C 144.5), as demonstrated in Figure 2 (red arrows).The hydroxyl's attachment at C-9, Me-28 at C-24, and D 22,23 double bond were proved by
the HMBC correlations of H-1/H-5/Me-19 to C-9 (d C 76.1), H-21 to C-22 (d C 135.3), and H-28 to C-23 (d C 132.2), C-24 (d C 42.9), C-25 (d C 33.0), along with 1 H-1 H COSY cross-peaks of H-20/H-22/H-23/H-24/(H-28)/H-25, which generated the planar structure of 1.Additionally, the E-configuration of D 22,23 double bond moiety was deduced by the large J coupling values of these two protons at 15.2 Hz for each.A NOESY experiment was conducted to determine the relative configuration of the stereocenters in 1 (Figure3).The small J coupling values of H-3 (sep, 5.0 Hz) and H-6 (t, 6.2 Hz) indicated that they were equatorially bonded toward the b-orientation.The observed cross-peaks of Me-19 (b-oriented)/Me-18, Me-19/H-6, and H-6b/H-3 confirmed the b-orientations of H-3 and H-6 and the attachment of hydroxyls at these carbons were conversely established as an a-orientation.The strong cross-peak of Me-21/Me-28 and no observed correlations between these methyls (Me-21/Me-28) and b-protons were not enough to determine the relative configuration of Me-21/Me-28 due to the nature of free rotation of the single bond.The a-configuration of Me-21 and Me-28 was then determined by a partial comparison of Me-21 (d C 21.1) and Me-28 (d C 17.6) in the side chain of compound 1 with those of reported ergostanes [Me-21a (d C 20.3-21.1)and Me-28a (d C 17.2-17.8)][15]