A new coumarin isolated from Sarcandra glabra as potential anti-inflammatory agent

Abstract One new coumarin, 3,5-dihydroxy-7-O-α-L-rhamno pyranosyl-2H-chromen-2-one (1), was isolated from the whole plant of Sarcandra glabra. The structure was elucidated by spectroscopic methods. Our results indicated that 1 significantly inhibit nitric oxide (NO) production in LPS-induced RAW264.7 macrophages. RT-PCR analysis indicated it inhibited iNOS mRNA expression. In addition, Western blot analysis showed that 1 attenuated LPS-induced synthesis of iNOS protein in the macrophages. These results suggest that 1 could be potential anti-inflammatory agent by down-regulating iNOS expression. Graphical abstract


Introduction
Coumarins are plant-derived products with various pharmacological properties including anti-inflammatory activity (Wu et al. 2009). Sarcandra glabra (Thunb) Nakai (Chloranthaceae family) is a well-known herbal medicine which mainly grows in south of China, and it is mainly used to treat inflammation and immune-related diseases such as acute respiratory infections, thrombocytopenia, pneumonia, cellulites, appendicitis, shigellosis, psoriasis and malignancies (National Commission of Chinese Pharmacopoeia 2010). As one major class of ingredients group, the coumarins found in S. glabra, such as isofraxidin, esculetin and ABSTRACT One new coumarin, 3,5-dihydroxy-7-O-α-L-rhamno pyranosyl-2H-chromen-2-one (1), was isolated from the whole plant of Sarcandra glabra. The structure was elucidated by spectroscopic methods. Our results indicated that 1 significantly inhibit nitric oxide (NO) production in LPS-induced RAW264.7 macrophages. RT-PCR analysis indicated it inhibited iNOS mRNA expression. In addition, Western blot analysis showed that 1 attenuated LPS-induced synthesis of iNOS protein in the macrophages. These results suggest that 1 could be potential anti-inflammatory agent by down-regulating iNOS expression. scoparone, were reported to be anti-inflammatory ingredients (Jang et al. 2005;Niu et al. 2012;Kim et al. 2014). This article describes the isolation and structure elucidation of a new coumarin, namely 3,5-dihydroxy-7-O-α-L-rhamno pyranosyl-2H-chromen-2-one (1) from S. glabra. In addition, the inhibitory effect of 1 on NO production and iNOS expression in lipopolysaccharide (LPS)-activated macrophage RAW264.7 cells were evaluated.

Structural elucidation of compound 1
Compound 1 was obtained as a white amorphous powder. The uV spectrum showed maximum absorption at 220, 251 and 299 nm ( Figure S1). The HR-eSI-MS exhibited a quasimolecular ion at m/z 339.07199 [M-H] − ( Figure S7), corresponding to the molecular formula of C 15 H 16 O 9 . The 13 C-NMR, dePT spectrum resolved 15 carbon signals ( Figures S3 and S4), which were classified by chemical shifts and HSQC spectrum ( Figure S5 The anti-inflammatory activity of 1 was determined in lipopolysaccharide (LPS)-stimulated murine RAW 264.7 macrophages. As shown in Figure 2(a), 1 significantly inhibited LPSinduced NO production in a dose-dependent manner. The potential cytotoxicity of 1 was then measured by the MTT assay after incubating cells for 24 h in the presence of LPS to examine whether inhibitory effect resulted from cellular toxicity. The result showed that 1 had no effect on cell viability up to a concentration of 200 µg/ml (Figure 2  Coumarins (e.g. isofraxidin), caffeoylquinic acids (e.g. chlorogenic acid), flavonoids (e.g. astilbin), sesquiterpenoids (atractylenolide IV) and dicaffeoyl derivatives (e.g. rosmarinic acid) were reported to representative constituents in S. glabra (Hu et al. 2013;Li et al. 2014), and they exerted anti-inflammatory effects through both identical and different mechanisms (Jiang et al. 2009;diao et al. 2014;Hwang et al. 2014;Liu et al. 2015). It is generally agreed that multiple components in traditional Chinese medicine (TCM) are responsible for therapeutic effects though exerting their synergistic effects at multiple targets and levels, and thus, 1 may be one of the anti-inflammatory contributing factors of this plant.

Plant material
The whole plant of S. glabra was supplied by Kangmei Pharmaceutical Co. Ltd with the origination from Sichuan province. A voucher specimen (No. 20081110) was deposited in the Center for Laboratory, Second Affiliated Hospital, Guangzhou university of Chinese Medicine.

Cell viability
Cell viability was evaluated by MTT assay. RAW 264.7 cells were plated at a density of 5 × 10 4 cells/well into 96-well plates containing 100 µl of new media. After the addition of 1 (25-200 µg/ml), the plates were incubated in a 37 °C, 5% CO 2 incubator for 2 h, followed by stimulation with 1 µg/ml LPS. After 24 h of LPS stimulation, 20 μl MTT (5 mg/ml) was added to each well, and the cells were further incubated for an additional 4 h. The supernatant was removed, and the formation of formazan was resolved with 150 μl/well of dMSO. The optical density was measured at 570 nm on a microplate reader.
Nitric oxide was determined by measuring the amount of nitrite. RAW264.7 cells (5 × 10 4 cells/well) were grown in 96-well plates for 24 h. After the addition of a range of concentrations of 1 for 2 h, then stimulation with LPS (1 µg/ml) for 24 h. Nitrite concentrations were determined according to the manufacturer′s instructions.

Total RNA extraction and RT-PCR
RAW264.7 macrophages were cultured at a density of 5 × 10 5 cells/ml in 6-well plates overnight. The cells were incubated for 2 h in combination with different concentrations of 1. The cells were further cultured for 12 h on treatment with LPS (final concentration 1 µg/ml).The cells were rinsed with cold PBS, and total RNA from RAW264.7 cells was extracted using a TRIzol Reagent kit according to instructions of the manufacturer. The RNA (1 μg) was reverse transcribed into cdNA using a Thermo RevertAid First Strand cdNA Synthesis Kit. The gene expression of iNOS was amplified from the synthesised cdNA. Real-time PCR was performed using Roche FastStart universal SYBR Green Master. The iNOS primers used for PCR amplification was TGAGTTCCGAAGCAAGCCAA (Forward); AGACCTCAA CAGAGCCCTCA (Reverse). GAdPH mRNA levels were used as internal controls. The PCRs were carried out as the following: 95 °C for 10 min; 40 cycles of 95 °C for 15 s, with a final extension at 60 °C for 1 min.

Western blot analysis
RAW 264.7 macrophages (2 × 10 5 cells/ml) were seeded in 6-well plates and incubated for 24 h, then pretreated with 50, 100, 150 and 200 μg/ml of 1 for 2 h. After LPS (1 μg/ml) stimulation for 8 h, the cells were collected and washed twice with cold PBS. The cells were lysed in RIPA buffer containing proteinase inhibitors. After lysis, the lysates were clarified by centrifugation at 12,000 × g for 20 min at 4 °C; the protein concentration in the supernatants was quantified with a BCA protein assay kit. Proteins (40 μg/lane) were resolved with SdSpolyacrylamide gel electrophoresis, and Western blot analysis was performed as described previously. Rabbit anti-iNOS was utilised as primary antibodies and HRP-goat anti-rabbit IgG was used as a secondary antibody, and detected with eCL reagent (Millipore, uSA).

Conclusion
Our study indicated that 1 as a potential anti-inflammatory agent, decreased NO production by LPS-stimulated RAW264.7 cells through down-regulating of iNOS expression both at mRNA and protein levels, which provide an important proof of principle for understanding the anti-inflammatory activities of 1.

Supplementary material
Supplementary data (uV, 1d, 2d NMR spectra and MS data of compound 1, Figure S1-S8) associated with this article are available online.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the National Nature Science Foundation of China under [grant number 81202888].