Norisoprenoids from the Brown Alga Sargassum naozhouense Tseng et Lu

A new C11-norisoprenoid derivative, sargassumone (1), has been isolated from Sargassum naozhouense together with six known norisoprenoids and a highly oxygenated cyclopentene: (2R,6S,8S,9S)-hexahydro-2,9-dihydroxy-4,4,8-trimethyl-6-acetyloxy-3(2H)-benzofuranone (2), (6S,8S,9R)-hexahydro-6,9-dihydroxy-4,4,8-trimethyl-2(2H)-benzofuranone (3), (6S,8S,9R)-hexahydro-6,9-dihydroxy-4,4,8-trimethyl-2(2H)-benzofuranone (4), loliolide (5), (+)-epiloliolide (6), spheciospongones A (7), and (+)-kjellmanianone (8). Compound 1 was identified on the basis of nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis, and the absolute stereochemistry was defined by NOESY spectroscopy, minimizing energy calculation, and circular dichroism (CD) spectra. The known compounds 2–8, isolated from S. naozhouense for the first time, were identified by comparison of their physical and spectroscopic data with those reported in the literature. Compound 6 was tested for its inhibitory activity against protein tyrosine phosphatase 1B (PTP1B), antioxidant activity with 1,1-diphyl-2-picrylhydrazyl (DPPH) free radicals, and antimicrobial activity against resistant clinical isolates of Candida albicans, methicillin-resistant Staphylococcus aureus (MRSA), and Escherichia coli.


Introduction
Sargassum naozhouense Tseng et Lu-one species of the Sargassum genus (phylum Phaeophyta, class Cyclosporeae, order Fucales, family Sargassaceae), called "black vegetable"-is distributed widely in the coastal region of the Leizhou Peninsula of China, and can be cultivated commercially under the northern South China Sea's high-temperature condition [1][2][3][4]. It has a series of pharmaceutical functions in Chinese folk medicine, such as treating infections, laryngitis, dieresis, and other ailments [3]. However, there have been few studies of the chemical constituents that reported primarily carbohydrates, proteins, minerals, dietary fiber, sulfated polysaccharide, and phlorotanns from the brown seaweed S. naozhouense [3,5,6]. In order to fully utilize this species and obtain new bioactive compounds from seaweeds being able to produce a great variety of secondary metabolites characterized by a broad spectrum of biological activities, we investigated the brown alga S. naozhouense. Previous chemical research on this species has resulted in the isolation of 1-O-hexadecanoyl glycerol, hexadecanoyl glycerol, pheophytin a, β-sitosterol, mannitol, uracil, thymine, p-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 4-hydroxyphthalide, and 2′-deoxythmidine [2]. Now, in our further chemical investigation on the EtOH extract of S. naozhouense, a new C11-norisoprenoid derivative (namely sargassumone (1)), and six known norisoprenoids (2)(3)(4)(5)(6)(7), together with a known highly oxygenated cyclopentenone (8) (Figure 1) were obtained. Compounds 2-8 were also isolated from S. naozhouense for the first time.

General
Optical rotations were measured with a Perkin-Elmer model 341 polarimeter (Perkin Elmer Corporation, Boston, MA, USA). NMR spectra were recorded on a Bruker Avance 500 NMR spectrometer (Bruker Corporation, Faellanden, Switzerland). HR-ESI-MS spectra were recorded on a Bruker maXis impact mass spectrometer (Bruker Corporation, Bremen, Germany). EI-MS data were measured on a Thermo Scientific DSQ mass spectrometer (Thermo Fisher Scientific Corporation, New York, NY, USA). CD spectra were recorded on a Chirascan CD spectropolarimeter (Applied Photophysics Ltd., Surrey, UK). Column chromatography was performed on silica gel (100-200 and 200-300 mesh, Yantai Jiangyou Silica Gel Development Co., Ltd., Yantai, China) and Sephadex LH-20 (Pharmacia, Uppsala, Sweden). Thin-layer chromatography (TLC) was performed on pre-coated silica gel GF 254 plates (Yantai Jiangyou Silica Gel Development Co., Ltd., Yantai, China), visualization under UV light, or by heating after spraying with 5% H 2 SO 4 -EtOH (v/v). Organic solvents for column chromatography were either spectral grade or analytical reagents and obtained from Guangzhou Dongju Test Instrument Co., Ltd. (Guangzhou, China).

Plant Materials
The brown seaweed S. naozhouense was collected in July 2012 from Leizhou Peninsula of Guangdong Province, China. The specimens were identified by Professor Enyi Xie of the College

Bioactivity Assay
3.4.1. PTP1B Inhibition Assay PTP1B (protein tyrosine phosphatase 1B) was expressed and purified in E. coli, and the enzymatic activities were measured at 30 • C by monitoring the hydrolysis of pNPP [15]. The enzymatic activities of PTP1B catalytic domain were also determined at 30 • C by monitoring the hydrolysis of pNPP. Dephosphorylation of pNPP generates p-nitrophenol, which can be monitored spectrophotometrically at 405 nm. In a 100-µL assay mixture containing 50 mM MOPS, pH 6.5, 2 mM pNPP, and recombinant enzymes, PTP1B activities were continuously monitored and the initial rate of the hydrolysis was determined from the early linear region of the enzymatic reaction kinetic curve as described by Zhang et al. [15].
The inhibitory activity of compound 6 was tested against PTP1B by the colorimetric assay described by Zhang et al. Briefly, the compound was solubilized in DMSO at 20 µg/mL, and then added to a 96-well clear polystyrene plate (Corning, Action, MA, USA) with oleanolic acid (12.5 mM) as the positive inhibition. After adding an assay mixture (88 µL), 10 µL 300 nM GST-PTP1B was added to initiate the reaction. Then, the catalysis of pNPP was continuously monitored on SpectraMax 340 microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 405 nm for 2 min at 30 • C in a final 100 µL volume containing 50 mM MOPS, pH 6.5, 2 mM pNPP, 30 nM PTP1B, and 2% DMSO. The IC 50 value was calculated from the non-linear curve fitting percent inhibition (% inhibition) to inhibitor (1)

DPPH Free Radical Scavenging Assay
The free radical scavenging potential of compound 6 was evaluated using the DPPH free radical scavenging assay described by Sharma and Bhat [16]. A total of 200 µL of the reaction mixture in a 96-well plate was composed of 100 µL of 0.1 mM DPPH (Sigma Aldrich, St. Louis, MO, USA) in methanol and 100 µL of different concentrations of test compound. The reaction mixture was then shaken well and incubated for 30 min in darkness at 37 • C. Meanwhile, a sample composed of 100 µL of methanol with 100 µL of 0.1 mM DPPH was taken as a control, while vitamin C (Shanxi Yishengtang Pharmaceutical Co., Ltd., Tongchuan, China) was taken as a reference material. The absorbance of reaction solution was measured at 517 nm with a plate reader (Thermo Multiskan MK3 spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA), and the percentage of scavenging capacity (%) was calculated by the following equation, where A Control stands for the absorbance of the control and A Treatment stands the absorbance of the treatment: Scavenging capacity (%) = (1 − A Treatment /A Control ) × 100. ( The DPPH IC 50 value is the concentration required to scavenge DPPH radical by 50%.

Antimicrobial Activity
Antimicrobial test for compound 6 was carried out against resistant microbes such as Candida albicans, MRSA, and Escherichia coli by disc diffusion method [17]. Briefly, a Petri dish was prepared with a base layer of Muller Hinton (MH) agar (10 mL), and then 0.1 mL culture with the