Glycosylated coumarins, flavonoids, lignans and phenylpropanoids from Wikstroemia nutans and their biological activities

Wikstroemia nutans Champ. ex Benth., a traditional herbal medicine collected at the Lingnan region of China, was chemically investigated. A new biscoumarin glucoside, wikstronutin (1), along with three known bis- and tricoumarin glucosides (2–4), two flavonoid glycosides (5–6), and eleven lignan glucosides (7–17) were isolated from the stems and roots of W. nutans. The new structure including its absolute configuration was elucidated based on a combination of 1D and 2D NMR, UV, IR, HRESIMS spectroscopic data, as well as chemical transformation. Compounds 1–17 were first isolated from the plant species W. nutans, while compounds 1–3, 8, and 11 were reported from the genus Wikstroemia for the first time. All co-isolates were evaluated for their in vitro inhibitory effects on nitric oxide (NO) production induced by lipopolysaccharide (LPS) in murine RAW264.7 macrophage cells. The antibacterial activity of the selected compounds was also tested. Our work enriches the structure diversity of the secondary metabolites from the genus Wikstroemia.


Introduction
The genus Wikstroemia (Thymelaeaceae) contains approximately 62 species, which are widespread throughout the subtropical regions of Asia and Oceania [1]. Nineteen species of the genus Wikstroemia are found to be domestic in China, such as W. nutans, W. indica, and W. canescens [2]. Previous investigations have not only reported diverse secondary metabolites from the genus, but also promising pharmacological activities of the extracts and chemical constituents produced by Wikstroemia species, including cardiovascular, neuroprotective, hepatoprotective, anti-inflammatory, and antitumor activities [1,3]. The plant species W. nutans is widely distributed in the areas of the Guangdong and Guangxi provinces of China, and the whole plants of this species are used as a folk medicine for the treatment of arthritis, mastitis, and pain relief [2]. Interestingly, the traditional medical usages are highly consistent with the phytologically related medicinal plant species W. indica that has already been approved to use as a prescription drug in China [3]. Owing to the intriguing therapeutic effects associat- ed with W. indica, extensive phytochemical studies on W. indica have been pursed [3,4]. However, there is no report on the phytochemical and pharmacological investigations upon W. nutans.
Coumarins (2H-1-benzopyran-2-one) are a large quantity of phenolic substances found in plants and microorganisms [5]. These naturally occurring coumarins were well documented due to their diverse chemical structures and promising biological properties, such as anticancer, antitubercular, anti-inflammatory, anticoagulant, antibacterial, and neuroprotective effects [6]. As part of a continuing study of our group targeting at the identification of bioactive natural products from the medicinal plants and endophytes [7,8], the chemical constituents of the stems and roots of W. nutans were investigated. This work resulted into the isolation and identification of a new biscoumarin glucoside 1, together with three known bis-and tricoumarin glucosides 2-4, two flavonoid glycosides 5 and 6, and eleven lignan glucosides 7-17 ( Figure 1). Herein, we present the isolation and structural elucidation of these natural products and their in vitro biological activities.

Results and Discussion
Compound 1 was obtained as a yellowish, amorphous powder. Its molecular formula was determined as C 29     , as well as two oxygenated methylenes resonated at δ C 70.6, and 67.7, respectively. The aforementioned information suggested that compound 1 is likely a biscoumarin glycoside [9].
In addition, two sugar units in substructure C ( Figure 2 and of the glucopyranosyl unit. This deduction was further confirmed by a ROESY correlation between H-6'' and H-1'''. Subsequently, an acid hydrolysis of 1 afforded the products including daphnogitin, a ᴅ-glucose, and a ᴅ-xylose. The absolute configurations of glucopyranosyl and xylopyranosyl was further determined by HPLC analysis of the sugar derivatives (Supporting Information File 1, Figure S4). The linkage between the substructures A and C was revealed by the crucial HMBC correlation between the anomeric proton at Glc H-1'' (δ H 5.71)/ C-7 (δ C 154.0). Therefore, the gross structure of 1 was determined as shown and the trivial name wikstronutin was given.

Biological activity
Considered naturally occurring glycosides of phenolic metabolites usually exhibit anti-inflammatory activity in literature [26], compounds 1−17 were evaluated for their inhibitory activities against LPS-induced nitric oxide (NO) production in RAW 264.7 mouse macrophages. All of them showed mild inhibitory activities with inhibition rates of 10-20% at a concentration of 50 μM (Table 2). Since coumarin derivatives were reported to have antimicrobial activities [27], the antimicrobial activity of compounds 1-4 was also evaluated against the bacteria Escherichia coli, Staphylococcus aureus subsp. aureus, Salmonella enterica subsp. enterica, and Pseudomonas aeruginosa. However, all of them were found to be devoid of inhibitory activity (MIC >250 μg/mL).

Conclusion
In this paper, the new bis-coumarin glucoside wikstronutin (1) was isolated from the stems and roots of the medicinal plant species W. nutans, together with three known bis-and tricoumarin glucosides 2-4, two flavonoid glycosides 5 and 6, and eleven lignan glucosides 7-17. Their structures were established by extensive spectroscopic analyses, including 1D, 2D NMR spectroscopy, and HRESIMS. The relative and absolute structure of 1 was unambiguously determined based on ROESY experiments and chemical transformation. In the vitro bioassays, compounds 1-17 showed a mild inhibitory effect against nitric oxide (NO) production in LPS-stimulated RAW 264.7 mouse macrophages. The antibacterial activities of compounds 1-4 against Escherichia coli, Staphylococcus aureus subsp. aureus, Salmonella enterica subsp. enterica, and Pseudomonas aeruginosa were also tested, however, none of them showed antimicrobial activities. This is the first report of the isolation of coumarins, flavonoids, lignans and phenylpropanoid glycosides from W. nutans, while compounds 1-3, 8, and 11 was encountered from the genus Wikstroemia for the first time. Our work will enrich the chemistry and structure diversity of natural products generated by plant species from the genus Wikstroemia.

Experimental General experimental procedures
Optical rotations were measured with a Horiba SEPA-300 polarimeter. UV spectra were recorded using a Waters UV-2401A spectrophotometer equipped with a DAD and a 1 cm path length cell. Methanolic samples were scanned from 190 to 400 nm in 1 nm steps. IR spectra were obtained using a Tensor 27 spectrophotometer with KBr pellets. 1D and 2D NMR spectra were acquired on Bruker DRX-600 and DRX-800 spectrometers with TMS as internal standard. Chemical shifts (δ) were expressed in ppm with reference to the solvent signals. Mass spectra were recorded on a VG Auto Spec-3000 instrument or an API QSTAR Pulsar 1 spectrometer. Semipreparative HPLC was performed on an Agilent 1120 apparatus equipped with a UV detector and a Zorbax SB-C-18 (Agilent, 9.4

Extraction and isolation
The dried root of W. nutans (3.5 kg) was extracted using 95% aqueous ethanol (15 L × 4 times × 2 h at room temperature) with ultrasonic assistance. The combined extracts were filtered and evaporated under reduced pressure to yield a green residue (350.

Anti-inflammatory assay
The RAW 264.7 cells (2 × 10 5 cells/well) were incubated in 96-well culture plates with or without 1 µg/mL LPS (Sigma Chemical Co., USA) for 24 h in the presence or absence of the test compounds. Aliquots of supernatants (50 µL) was then reacted with 100 µL Griess reagent (Sigma Chemical Co., USA). The absorbance was measured at 570 nm using Synergy TMHT Microplate Reader (BioTek Instruments Inc., USA). In the study, L-NMMA (Sigma Chemical Co., USA) was used as a positive control. In the remaining medium, an MTT assay was carried out to determine whether the suppressive effect was related to cell viability. The inhibitory rate of NO production = (NO level of blank control -NO level of test samples)/NO level of blank control. The percentage of NO production was evaluated by measuring the amount of nitrite concentration in the supernatants with Griess reagent as described previously [28].

Antimicrobial assay
Compounds 1-4 were evaluated for their antimicrobial activities against Escherichia coli, Staphylococcus aureus subsp. aureus, Salmonella enterica subsp. enterica, and Pseudomonas aeruginosa. Antimicrobial assay was conducted by the previously described method [29]. Add the sample to be tested into the 96-well culture plate, the maximum concentration of the used compounds was 100 μM. Add bacteria liquid to each well, the final concentration is 5 × 10 5 CFU/mL. Incubate at 37 °C for 24 h, the OD value at 595 nm was measured by a microplate reader, and the medium blank control was set in the experiment.
Determination of absolute configurations of sugar units of 1 Compound 1 (2 mg) was hydrolyzed with 1 N HCl (2 mL) at 90 °C for 2 h. The residue was partitioned between EtOAc and H 2 O to give the aglycone and sugar, respectively. The aqueous residue was concentrated to dryness under N 2 . The aqueous residue, ᴅ-glucose (2 mg), and ᴅ-xylose standard (2 mg) were separately dissolved in 0.5 mL anhydrous pyridine, and ʟ-cysteine methyl ester hydrochloride (2.0 mg) was then added. Each reaction mixture was heated at 60 °C for 1 h, and then 2-methylphenyl isothiocyanate (10 μL) was added to each reaction mixture and heated further for 1 h. The reaction mixture (0.5 mL) was then analyzed by HPLC and detected at 250 nm. Analytical HPLC was performed on a Welch Ultimate XB-C18 column (4.6 mm × 250 mm, 5 microm) at 35 °C with isocratic elution of 25% CH 3 CN in 0.1% H 3 PO 4 for 40 min and subse-quent washing of the column with 90% CH 3 CN at a flow rate 0.8 mL/min. Peaks at 16.54 and 19.64 min have coincided with derivatives of ᴅ-glucose and ᴅ-xylose [30].

Supporting Information
Supporting Information File 1 NMR, MS, UV, IR spectra and HPLC chromatogram of derivative 1.