Polyketides from the Mangrove-Derived Endophytic Fungus Nectria sp. HN001 and Their α-Glucosidase Inhibitory Activity

Four new polyketides: nectriacids A–C (1–3) and 12-epicitreoisocoumarinol (4), together with three known compounds: citreoisocoumarinol (5), citreoisocoumarin (6), and macrocarpon C (7) were isolated from the culture of the endophytic fungus Nectria sp. HN001, which was isolated from a fresh branch of the mangrove plant Sonneratia ovata collected from the South China Sea. Their structures were determined by the detailed analysis of NMR and mass spectroscopic data. The absolute configuration of the stereogenic carbons for compound 4 was further assigned by Mosher’s ester method. All of the isolated compounds were tested for their α-glucosidase inhibitory activity by UV absorbance at 405 nm, and new compounds 2 and 3 exhibited potent inhibitory activity with IC50 values of 23.5 and 42.3 μM, respectively, which were more potent than positive control (acarbose, IC50, 815.3 μM).


Introduction
Diabetes mellitus, one of the most common chronic metabolic diseases, occurs when the pancreas produces insufficient levels of insulin or when the body cannot use the insulin effectively [1]. In 2015, about 415 million people had diabetes worldwide, with type II diabetes accounting for about 90% of the cases [2,3]. α-Glucosidase is an important enzyme for breaking down complex carbohydrates for absorption, and α-glucosidase inhibitors such as acarbose, miglitol, and voglibose, all originating from natural products, are widely used to treat type II diabetes, indicating that natural products are an important source of anti-diabetes drugs.
All of the isolates were evaluated for in vitro α-glucosidase inhibitory activity [10]. The results showed that compounds 2 and 3 possessed stronger activity than positive control (acarbose, IC50, 815.3 μM) with IC50 values of 23.5 and 42.3 μM, respectively. However, compounds 4-6 showed moderate activity with IC50 values ranging from 300 to 600 μM (Table 3), while compound 7 did not display inhibitory activity compared to positive control. Interestingly, although compounds 1-3 possess same carbon skeleton, their α-glucosidase inhibitory activity are different. The activity against α-glucosidase for 2 (23.5 μM) and 3 (42.3 μM) was more potent than that for 1 (121.8 μM), which suggested that esterification of terminal carboxyl group (C-12) may play a key role in the inhibitory effects. Although compounds 2 and 3 possess different configuration of the C4-C5 double bonds, they exhibited the same level of activity. Meanwhile, compounds 4 and 5 exhibited relatively stronger activity when compared to 6.
All of the isolates were evaluated for in vitro α-glucosidase inhibitory activity [10]. The results showed that compounds 2 and 3 possessed stronger activity than positive control (acarbose, IC 50 , 815.3 µM) with IC 50 values of 23.5 and 42.3 µM, respectively. However, compounds 4-6 showed moderate activity with IC 50 values ranging from 300 to 600 µM (Table 3), while compound 7 did not display inhibitory activity compared to positive control. Interestingly, although compounds 1-3 possess same carbon skeleton, their α-glucosidase inhibitory activity are different. The activity against α-glucosidase for 2 (23.5 µM) and 3 (42.3 µM) was more potent than that for 1 (121.8 µM), which suggested that esterification of terminal carboxyl group (C-12) may play a key role in the inhibitory effects. Although compounds 2 and 3 possess different configuration of the C4-C5 double bonds, they exhibited the same level of activity. Meanwhile, compounds 4 and 5 exhibited relatively stronger activity when compared to 6.

Fungal Material
The fungal strain HN001 was isolated from the branches of the mangrove plant Sonneratia ovata collected from the South China Sea in Hainan province, China. The fungus was identified by our team as Nectria sp. HN001, Nectriaceae, according to a molecular biological protocol by DNA amplification and sequencing of the ITS region [10] (deposited in GenBank, accession No. KU359411). A voucher strain was deposited in School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou, China, with the access code, 2015-HN001.

Preparation of (R)-and (S)-MTPA Esters of 4
As our previous reported method [23], 12-epicitreoisocoumarinol (4) (1 mg) was dissolved in 1 mL of pyridine and stirred at room temperature for 10 min. An excess of (R)-or (S)-MTPA chloride (10 µL) was added, and the reaction was stirred overnight at room temperature. The solvent was removed in vacuo, and the crude reaction product was purified by preparative silica gel TLC using pure dichloromethane as developing solvent.

In Vitro Inhibition Studies on α-Glucosidase
An assay of α-glucosidase inhibitory activity was performed using a reported method, with slight modifications [10]. All the assays were performed using 0.01 M KH 2 PO 4 -K 2 HPO 4 buffers, pH 7.0, and a Bio-Rad iMark microplate reader (Bio-Rad Laboratories, Inc., Kyoto, Japan). Enzyme solution was prepared to give 2.0 Units/mL in 2 mL aliquots. The assay medium contained phosphate buffer, pH 7.0 (130 µL), 10 µL of enzyme solution, 20 µL of DMSO or inhibitor (dissolved in DMSO), and 40 µL of substrate (p-nitrophenyl glycoside, 3 mg/mL). The substrate was added to the assay medium containing enzyme and buffer with inhibitor added after 15 min of incubation time at 37˝C. The activity was determined by measuring the increase in absorbance at 405 nm for a 1 min interval. Calculations were performed according to the equation: η p%q " rpB´Sq{Bsˆ100% (B stands for the assay medium with DMSO; S stands for the assay medium with inhibitor). All measurements were done in triplicate from two independent experiments. The reported IC 50 was the average value of two independent experiments.

Conclusions
Four new (1-4) and three known polyketides (5-7) were isolated and identified from the culture of the endophytic fungus Nectria sp. HN001. Compounds 2 and 3 exhibited stronger inhibitory activity on α-glucosidase than positive control [10]. To the best of our knowledge, this is the first report of the α-glucosidase inhibitory activity of the C 15 conjugated pentaene diacid derivatives. This finding can allow us to explore structural diversity of linear polyene diacid derivatives and offer new guidance to discover α-glucosidase inhibitors.