Phenolic Compounds from the Rhizomes of Smilax china L. and Their Anti-Inflammatory Activity

A new triflavanoid, kandelin B-5 (1), was isolated from the rhizomes of Smilax china L., together with six known phenylpropanoid substituted flavan-3-ols (2–7), nine flavonoids (8–16), two stilbenoids (17, 18), and two other compounds (19, 20). The structure of compound 1 was determined on the basis of 1D, 2D NMR and HR-ESI-MS data, as well as chemical method. Compounds 2–5, 8–12, 15, 17, and 19 were evaluated for anti-inflammatory activity. Only compounds 10, 15 and 17 showed slightly IL-1β expression inhibitory activities on LPS induced THP-1 cells, with inhibition rate of 15.8%, 37.3%, and 35.8%, respectively, at concentration of 50 μg/mL.


Introduction
Smilax china L. (Liliaceae), a perennial climbing deciduous shrub, is widely distributed in Southern China, and Southeast Asian countries. The leaves of S. china are used as detoxication agent in folk China [1]; while the rhizomes of S. china, called "Jin Gang Teng", are collected by Chinese Pharmacopoeia with the efficacy of carminative, diaphoretic, and circulative [2]. Previous studies on the title species have disclosed the presence of steroidal saponins [3], flavonoids [4,5], phenylpropanoids [6], and stilbenoids [7,8]. These isolated compounds showed a wide spectrum of activities, such as immunosuppressive activity [9], anti-oxidative activities [10], anti-inflammatory activities [3,11].
The pharmaceutical preparations of S. china are widely used in clinic for the treatment of chronic pelvic inflammatory disease, a kind of chronic inflammation in the female genital organs, connective tissues and pelvic peritoneum. Recently, we reported five known flavonoids from the anti-chronic pelvic inflammation fraction of S. china using high speed counter current chromatography [12,13]. Further detail chemical investigation on the title species led to the isolation of a new triflavanoid (1) and 19 known phenolic compounds (2−20) (Figure 1). Compounds 2-5, 8-12, 15, 17, and 19 were evaluated for their anti-inflammatory activity. Herein we describe the isolation and structure elucidation of these compounds as well as their anti-inflammatory activities.

General Experimental Procedures
Optical rotation was measured with an MCP-500 polarimeter (Anton Paar). The UV spectrum was recorded with a TU-1810DSPC UV-Vis spectrometer (Puxi Tongyong). IR spectrum was measured on a IR Affinity-1 spectrophotometer (Shimadzu); CD was measuredon a Chirascan TM (Applied Photophysics Ltd., Leatherhead, UK). NMR was acquired on an AV-400 spectrometer (Bruker) with TMS as internal standard, J in Hz; HRESIMS spectra were measured on the Orbitrap Fusion high resolution mass spectrometer (Thermo) equipped with ESI source. Preparative or semi-preparative HPLC were performed on a HPLC system equipped with a Waters 1525 pump and  Interleukin-1β (IL-1β), an important mediator of the inflammatory response, is involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis. Compounds 2-5, 8-12, 15, 17, and 19 were evaluated for their IL-1β expression inhibitory activities on lipopolysaccharide (LPS) induced THP-1 cells. Compounds 10, 15, and 17 showed slightly inhibitory activities, with inhibition rate of 15.8%, 37.3%, and 35.8%, respectively, at concentration of 50 μg/mL. The other compounds showed no obvious activity at the same concentration. CCK-8 results revealed that these tested compounds showed no obvious cytotoxicities towards THP-1 cells at same concentration, indicating that the anti-inflammatory activities of compounds 10, 15, and 17 were not resulted from cytotoxic effects. Previous studies revealed the unique immunosuppressive activity of astilbin (9) [23]; While our results suggested that astilbin (9) and its isomers (10-12) didn't directly inhibit the production of proinflammatory cytokines IL-1β. (-)-Epicatechin and stilbene are reported to show anti-inflammatory activities [24]. Consistently, our results showed that (-)-Epicatechin (15) and stilbene dimer, scirpusin A (17), inhibited IL-1β expression on the LPS induced THP-1 cells.

General Experimental Procedures
Optical rotation was measured with an MCP-500 polarimeter (Anton Paar). The UV spectrum was recorded with a TU-1810DSPC UV-Vis spectrometer (Puxi Tongyong). IR spectrum was measured on a IR Affinity-1 spectrophotometer (Shimadzu); CD was measuredon a Chirascan TM (Applied Photophysics Ltd., Leatherhead, UK). NMR was acquired on an AV-400 spectrometer (Bruker) with TMS as internal standard, J in Hz; HRESIMS spectra were measured on the Orbitrap Fusion high resolution mass spectrometer (Thermo) equipped with ESI source. Preparative or semi-preparative HPLC were performed on a HPLC system equipped with a Waters 1525 pump and Interleukin-1β (IL-1β), an important mediator of the inflammatory response, is involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis. Compounds 2-5, 8-12, 15, 17, and 19 were evaluated for their IL-1β expression inhibitory activities on lipopolysaccharide (LPS) induced THP-1 cells. Compounds 10, 15, and 17 showed slightly inhibitory activities, with inhibition rate of 15.8%, 37.3%, and 35.8%, respectively, at concentration of 50 µg/mL. The other compounds showed no obvious activity at the same concentration. CCK-8 results revealed that these tested compounds showed no obvious cytotoxicities towards THP-1 cells at same concentration, indicating that the anti-inflammatory activities of compounds 10, 15, and 17 were not resulted from cytotoxic effects. Previous studies revealed the unique immunosuppressive activity of astilbin (9) [23]; While our results suggested that astilbin (9) and its isomers (10-12) didn't directly inhibit the production of proinflammatory cytokines IL-1β. (-)-Epicatechin and stilbene are reported to show anti-inflammatory activities [24]. Consistently, our results showed that (-)-Epicatechin (15) and stilbene dimer, scirpusin A (17), inhibited IL-1β expression on the LPS induced THP-1 cells.

General Experimental Procedures
Optical rotation was measured with an MCP-500 polarimeter (Anton Paar). The UV spectrum was recorded with a TU-1810DSPC UV-Vis spectrometer (Puxi Tongyong). IR spectrum was measured on a IR Affinity-1 spectrophotometer (Shimadzu); CD was measuredon a Chirascan TM (Applied Photophysics Ltd., Leatherhead, UK). NMR was acquired on an AV-400 spectrometer (Bruker) with TMS as internal standard, J in Hz; HRESIMS spectra were measured on the Orbitrap Fusion high resolution mass spectrometer (Thermo) equipped with ESI source. Preparative or semi-preparative HPLC were performed on a HPLC system equipped with a Waters 1525 pump and a Waters 2487 Dual Wavelength Detector using a Thermo hypersil C 18

Plant Materials
The rhizomes of S. china were purchased from Shenzhen Hongen Pharmaceutical Company, and were identified by one of the authors (L.B. Hou). The voucher specimen of this material (NO. SMU-NPC-201301) was deposited in Natural Product Chemistry Lab, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, China.

Extraction and Isolation
The rhizomes of S. china (6.0 kg) were refluxed with 95% ethanol at 75 • C for three hours, three times and filtered. The combined filtrate was concentrated to yield crude extracts (1.2 kg). The crude extracts were suspended in H 2 O, and then partitioned with EtOAc and n-BuOH consecutively.

Quantitation of Cytokine IL-1β
The human acute monocytic leukemia (THP-1) cells (Shanghai Cell Bank, C.A.S., Shanghai, China) were cultured in Dulbecco's Modefied Eagles Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) at 37 • C in a humidified atmosphere with 5% CO 2 . The cells were seeded into 96-well plates with density of 1 × 10 5 cells per milliliter. Thereafter, cells were treated with sample solutions diluted with cell sustainable medium. After incubation for 1 h, the cells were treated with 1 µg/mL of LPS for another 24 h. The cells were treated with LPS alone or LPS plus dexamethasone as blank control and positive control, respectively. The supernatant was collected by centrifugation (14,000 rpm, 4 • C). The quantity of IL-1β in culture supernatant was determined by using Enzyme Linked Immunosorbent Assay (ELISA) kit specific for IL-1β [25]. The inhibition rates were calculated according to the formula: where C is the average quantity of IL-1β of the blank control and T is the average quantity of IL-1β of the group with the test compound.

Cytotoxicity Assay
The effect of compounds 2-5, 8-12, 15, 17, and 19, on the viability of THP-1 cells were evaluated using the Cell Counting Kit-8 (CCK-8) according to the manufacturer's instructions. Briefly, THP-1 cells were seeded into 96-well plates at a density of 2.5 × 10 4 cells/well in the presence of sample solutions (50, 100, 200 µM, respectively) or absence of compounds for 48 h. Thereafter, 10 µL CCK-8 solutions were added to each well for further incubation at 37 • C in 4 h. The optical density was measured at a wavelength of 450 nm using a microplate reader [26]. Cell viability was expressed as a percentage of the control.