A new phenylpropanoid-substituted epicatechin from the rhizome of Smilax china

Abstract A new phenylpropanoid-substituted epicatechin, (2 R,3S,9R)-methyl {2-(3,4-dihydroxyphenyl)-3,5,8a,4a-tetrahydroxy-3,4-dihydro-2H,12H-pyrano[2,3-α]xanthen-12-yl}acetate (1) was isolated from the rhizome of Smilax china, along with twelve known compounds (2 − 13), which were isolated from the Smilax genus for the first time. On the basis of chemical evidences and spectral data analysis (UV, ECD, 1 D and 2 D-NMR, HR-ESI-MS), the structures of compound 1 was elucidated. Furthermore, all compounds have been tested for their inhibitory effects on NO production in LPS-induced RAW 264.7 cells, and compounds 6, 7, 11 and 13 have obvious inhibitory effect, in which the IC50 value of compound 7 reached 11.63 ± 1.29 μM. Through target screening and molecular docking, we can speculate that compound 7 may exert its anti-inflammatory effect by binding to MAPKAP kinase 2 and Leukocyte Proteases Cathepsin G & Chymase.


Introduction
Natural products or modified natural products have been an important source for drugs, such as artemisinin and aspirin (Li et al. 2022). Finding potential natural drugs from plants is a recognized route. About 300 species in the Smilax genus (Liliaceae family) are distributed in tropical and subtropical regions of the world. Smilax china, known as Jingangteng, is a perennial deciduous climbing shrub of the Smilax genus in the Liliaceae family, which is widely distributed in East Asia and North America (National Pharmacopoeia Committee 2020). In China, it is mainly distributed in Anhui, Fujian, Guangdong and Hubei. S. china is often used as a traditional folk medicine to treat symptoms such as sore muscles and bones, dripping urination, excessive amount of discharge, and carbuncle . Clinically, it has a good effect on diseases such as gynecological pelvic inflammation.
Previous studies have found that the chemical components in the Smilax genus include steroidal saponins, flavonoids, phenolic acids and stilbenes (Soto-Hern andez et al. 2017;Hafiz et al. 2021). Many compounds have been isolated from S. china including steroidal saponins (Liu et al. 2017;Xie et al. 2018), flavans and phenolic acids (Cheng et al. 2017;Zheng et al. 2020), stilbene (Shao et al. 2007) and phenylpropanoids (Kuo et al. 2005), and they have been shown to have anti-inflammatory (Zheng et al. 2020), anti-cancer (Liu et al. 2017) and antidiabetic (Zhao et al. 2016) activities in further pharmacological studies. In continuation of phytochemical studies of S. china, herein we report the isolation and characterization of a new flavan-3-ol derivative (1) and 12 known compounds (2-13) ( Figure 1) from its rhizome and their anti-inflammation activities, and compounds 6, 7, 11 and 13 have obvious inhibitory effect, in which the IC 50 value of compound 7 reached 11.63 ± 1.29 lM. For compound 7 with better activity, we predicted its possible protein targets by molecular docking.

Result and discussion
Compound 1 was isolated as a pale yellow amorphous powder, and has the molecular formula C 25 H 22 O 10 , corresponding to 15 degrees of unsaturation, as deduced from the detailed analysis of NMR and HREIMS data, showing a molecular ion peak [M þ H] (Table S1) showed signals of ABX-type aromatic resonances at d H 6.84 (1H, d, J ¼ 1.6 Hz),6.78 (1H, d, J ¼ 8.0 Hz) and 6.73 (1H, dd, J ¼ 8.0, 1.6 Hz), and a methyl ester group at d H 3.61 (3H, s). In addition, 1 H NMR spectrum showed four characteristic signals of flavan-3-ol at d H 4.60 (1H, d, J ¼ 7.5 Hz, H-2), d H 4.00 (1H, m, H-3), d H 3.00 (1H,dd,J ¼ 16.4,5.6 Hz,m,. A singlet resonating at d H 6.10 suggested only one position on the A ring in 1 is unsubstituted. The 13 C-NMR spectrum (CD 3 OD) of compound 1 showed the presence of 25 carbon signals, including three characteristic carbon signals of flavan-3-ol at d C 83.0 (C-2), 68.6(C-3) and 28.5(C-4), a methyl ester carbon signal at d C 174.3, a methine at d C 31.7, a methylene at d C 44.5, and an additional 1,2,4,5-tetrasubstituted benzene ring with two hydroxy groups [d C 116.1, 146.5, 104.5, 146.0, 142.4, 115.3]. These spectral data showed strong similarities with that of catiguanin A, isolated from Trichilia catigua (Tang et al. 2007). It is found that compound 1 is different from the known catiguanin A at C-2, C-3 and C-4 of the C ring of flavan-3-ol. A C-2/C-3 trans relationship in the C ring was supported by a large coupling (J ¼ 7.5 Hz) between H-2 and H-3 in 1, while a negligible coupling between H-2 and H-3 was observed in catiguanin A. It is confirmed that the configuration of C-2 and C-3 of compound 1 is opposite to catiguanin A. In the HMBC spectrum ( Figure  S1), the correlations of d H 4.60 (H-2) with d C 68.6 (C-3), 132.0 (C-1 0 ) and 120.1 (C-6 0 ), and the correlations of d H 3.00 (H-4) with d C 83.0 (C-2), 68.6 (C-3), 100.8 (C-4a) and 155.1 (C-5) confirmed the presence of epicatechin group in the structure. Also, d H 4.45 (H-9) correlated with d C 105.5 (C-8), 152.5 (C-7), 154.9 (C-8a) and 115.3 (C-6 00 ), whereas d H 6.10 (H-6) correlated with d C 155.1 (C-5), 105.5 (C-8) and 100.8 (C-4a), indicating that the C-9 of the CH(9)CH 2 (10)COOMe unit was linked to between C-8 and C-1 00 . The absolute configuration at the chiral center C-9 in compound 1 was established by comparing its circular dichroism (CD) spectrum ( Figure S9) with that of catiguanin A. According to the literature data, Catiguanin A and Catiguanin B have the same plane structure, except for the configuration of C-9, and their De values of ECD data at about 290 nm and 275 nm showed opposite symbols (Tang et al. 2007). The both compounds exhibited a positive Cotton effect at 295 nm and a negative Cotton effects at 274 nm, suggesting that the absolute configurations at C-9 in 1 is R. In the NOESY spectrum ( Figure S2), H-10 showed crosspeak to H-2, and H-3 showed crosspeak to H-6 0 , indicating the b-orientation for H-2 and a-orientation for H-3. According to these evidences, the structure of compound 1 was determined as (2 R,3S,9R)-methylf2-(3,4dihydroxyphenyl)-3,5,8a,4a-tetrahydroxy-3,4-dihydro-2H,12H-pyrano[2,3-a]xanthen-12ylgacetate, and named as catiguanin C.
Naturally occurring furofuran lignans have been reported to possess typical cis-1,5fused mode (Lu et al. 2015). The chemical shift differences of H 2 -4 and H 2 -8 (Dd H 2 -8 ¼ 0.32; Dd H 2 -4 ¼ 0.51) of 1,5-H type furofuran lignans indicate that H-5/H-6 are trans and H-1/H-2 are cis relative configurations (Shao et al. 2018). Comparing the specific rotation data of compound 13 f½a 20 D : À35.2 (c ¼ 0.15, CH 3 OH)g with that of known compounds, indicated that the absolute configuration of compound 13 is consistent with the known compounds (Xu et al. 2020). The method for determining the relative configuration of compound 12 was the same as that of compound 13, and its optical rotation data was f½a 20 D : þ32.8 (c ¼ 0.17, CH 3 OH)g. Finally, it can be determined that its absolute configuration was the same as that of syringaresinol (Qin Wang et al. 2009).
All the compounds (1-13) were evaluated for their inhibitory effects on NO production in LPS-induced RAW 264.7 cells. As shown in Figure S11, compounds 6, 7, 11 and 13 showed significant inhibitory effect, and compounds 7 and 13 has activity equivalent to that of positive drug, with IC 50 values of 11.63 ± 1.29 lM and 18.88 ± 1.64 lM ( Figure S13), respectively.

Plant material
The dried rhizomes of S. china were picked from Fujian in July 2018, and identified by Prof. Jingmin Jia, Shenyang Pharmaceutical University. A voucher specimen (20180809) was deposited in the Key Laboratory of Structure-Based Drug Design & Discovery, Wuya College of Innovation, Shenyang Pharmaceutical University.

Extraction and isolation
The dried rhizomes of S. china (12.5 kg) were pulverized and extracted with 75% ethanol (60 L) for two times (3 h each time). The resulting solution was concentrated at 40 C under reduced pressure until no alcohol taste, and the extract (2.5 kg) was obtained. The crude extract was dispersed to 5 L with water and partitioned continuously with ethyl acetate (EtOAc), then concentrated in vacuum to obtain extracts of EtOAc (255.4 g) and water (1.5 kg).

Assay for anti-inflammatory activity
Cells were maintained in DMEM supplemented with 10% FBS, 100 units/mL penicillin and 100 mg/mL streptomycin in 10 cm diameter Petri dishes in a humidified atmosphere of 95% air and 5% CO 2 at 37 C. Cells were maintained in continuous passages by trypsinization of subconfluent cultures and supplied with fresh medium every 48 h. We adjusted the concentration of RAW264.7 cells to 3.5 Â 10 4 cell/well and put it into 96-well plate, and added 100 lL cell suspension into each well. In the experiment, control group (RAW264.7 cells, DMSO), model group (RAW264.7 cells, DMSO, 0.5 lg/mL LPS), positive drug group (RAW264.7 cells, dexamethasone, 0.5 lg/mL LPS) and drug group to be tested (RAW264.7, compounds, 0.5 lg/mL LPS) were set. Incubate in 5% CO 2 and 37 C constant temperature incubator for 24 hours, then suck 40 lL of cell supernatant into the enzyme label plate, add 40 lL of Griess reagent to each well to mix it with cell supernatant and react completely. After reaction at room temperature for 10 min, the absorbance of the solution in the well at 540 nm was detected by enzyme labeling instrument, and the inhibition rate formula was obtained: NO release inhibition rateð%Þ ¼ ½NO À 2 model group À½NO À 2 drug group=positive drug group ½NO À 2 model group À ½NO À 2 control group Â 100

Molecular docking simulation
The X-ray crystal structures of the enzyme MAPKAP kinase 2 (PDB:1ny3), HGST T2-2 (PDB:1ljr), Leukocyte Proteases Cathepsin G & Chymase (PDB:1t32) and PDE4 (PDB:1ptw) were obtained from the RCSB protein data bank. The protein was optimised by the Discovery Studio 4.3 program (Accelrys Inc., San Diego, USA) to add all hydrogen atoms and remove water molecules, and the structure was carried out for the docking calculations using the ICM-pro (MolSoft, California, USA).

Conclusion
In summary, a phytochemical investigation of the rhizome of S. china resulted in the isolation of 13 compounds including a new one, catiguanin C (1). Compounds 1-2, 13 were obtained from the genus Smilax for the first time. Compounds 6, 7, 11 and 13 showed significant inhibitory effects on NO production in LPS-induced RAW 264.7 cells, and the IC 50 value of compound 7 even reached 11.63 ± 1.29 lM. Through target screening and molecular docking, we can speculate that compound 7 may exert its anti-inflammatory effect by binding to MAPKAP kinase 2 and Leukocyte Proteases Cathepsin G & Chymase.
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