Phenolic compounds from the aerial parts of Clematis viticella L. and their in vitro anti-inflammatory activities

Phytochemical investigations on the EtOH extract of Clematis viticella led to the isolation of six flavonoid glycosides, isoorientin ( 1 ), isoorientin 3  - O -methyl ether ( 2 ), quercetin 7- O -α-L-rhamnopyranoside ( 3 ), quercetin 3,7-di- O -α-L-rhamnopyranoside ( 4 ), manghaslin ( 5 ) and chrysoeriol 7- O -  -D-glucopyranoside ( 6 ), one phenylethanol derivative, hydroxytyrosol ( 7 ), along with three phenolic acids, caffeic acid ( 8 ), ( E )- p -coumaric acid ( 9 ) and p hydroxybenzoic acid ( 10 ). The structures of the isolates were elucidated on the basis of NMR and HR-MS data. All compounds were isolated from C. viticella for the first time. Compounds 7 and 8 showed significant anti-inflammatory activity at 100  M by reducing the release of NO in LPS-stimulated macrophages comparable to positive control indomethacin. Compounds 3 and 7 exhibited anti-inflammatory activity through lowering the levels of TNF-α while 1 , 3 and 5 decreased the levels of neopterin better than the positive controls.


General
All NMR spectra were recorded on a Varian Mercury-Mx spectrometer (USA) at 600 MHz for 1 H NMR and at 150 MHz for 13

Plant material
The aerial parts of Clematis viticella Linaceus were collected from Sakarya, in June 2015.
The plant material was identified by Prof. Dr. E. Yeşilada. The voucher specimen (YEF 15009) has been deposited at the Herbarium of Faculty of Pharmacy, Yeditepe University, İstanbul.

Antiinflammatory activity assays
Cell Culture and Cell Viability Assay RAW264.7 murine macrophage cells (ATCC, USA) were maintained in DMEM, supplemented with 10% FBS and 1% streptomycin and penicillin at 37°C in 5% CO2. Cell viability was examined by using MTT assay. Plated RAW264.7 cells were treated with 100 µM of pure compounds and reference molecules, indomethacin and L-NAME. After 24 hours incubation process, MTT was added to each well at 0.5 mg/mL of concentration and incubated for an additional 2 hours at 37°C. After discarding all medium from plates, 100 μl of isopropanol was added to all wells. Absorbance of the blue formazan was determined at 540 nm by a UV-spectrophotometric plate reader. Viability was defined as the ratio (expressed as a percentage) of absorbance of treated cells to untreated cells and all measurements were done in triplicates.

Evaluation of Anti-inflammatory Activity by Griess Assay
Anti-inflammatory activities of the isolates were evaluated by measuring the stable nitric oxide (NO) metabolite, nitrite, with Griess reagent (Kiemer & Vollmar, 1997). Briefly, RAW264.7 cells were plated in a 48 well-plate and incubated for 24 hours at 37°C in 5% CO2. Plated cells were pre-treated with the isolated compounds and the reference molecules for 2 hours and then stimulated with 1 µg/mL of LPS for additional 22 hours. The culture supernatant (50 µL) was mixed with Griess reagent and incubated at room temperature for 10 min. The absorbance of the mixture was determined at 570 nm using a microplate reader (Multiskan Ascent, Finland). The amount of nitrite in the samples was calculated by using sodium nitrite standard curve. Indomethacin and L-NAME were used as positive controls at 100 M.

Measurement of Neopterin and TNF-α Levels
Neopterin and TNF-α concentrations were measured in cell culture supernatants by using a commercially available quantitative enzyme-linked immunosorbent assay (ELISA) system (IBL Rat Neopterin ELISA Kit, Germany; Abbkine Rat TNF-α ELISA Kit, China) according to manufacturer's instructions.

Statistical analysis
GraphPad Prism 6 was used for the all statistical analyses. Data related to cell viability, antiinflammatory activity, neopterin and TNF-α levels were analyzed by using one-way ANOVA following the post-hoc tests by Tukey. Differences were considered as significant a p < 0.05.