New compounds from the stems of Fissistigma acuminatissimum Merr. and their in vitro anti-inflammatory activity

Abstract Three new compounds, 4,5,6,7-tetramethoxy-3-benzoylbenzofuran (1), 4-hydroxy-3,5,6-trimethoxydihydrochalcone-2-O-β-D-glucopyranoside (2) and 2-hydroxy-3,4,5,6-tetramethoxyphenylethyl benzoate (3) along with five known flavonoids were isolated from the dichloromethane fraction of the stems of Fissistigma acuminatissimum Merr.’s ethanol extracts. The compounds were obtained by chromatographic methods and the structure elucidation was completed primarily on the basis of spectroscopic analyses, all of these compounds were isolated from F. acuminatissimum for the first time. All the fractions and compounds were evaluated for their anti-inflammatory activity against lipopolysaccharide (LPS)-stimulated tumor necrosis factor α (TNF-α) production in RAW264.7 cells in vitro. The dichloromethane fraction showed the most potent inhibition(38.2%) at 60 μg/mL, compound 1 (70.2%) and 3 (65.2%) showed significant inhibition at 10 μM. Graphical Abstract


Introduction
The genus Fissistigma, a large tribe of family Annonaceae, consists of more than 70 species distributing widely in tropics and subtropics regions in Africa, Oceania and Asia. In China Fissistigma contains 22 species and 1 variety (Flora of China Editorial Committee of Chinese Academy of Sciences. 1979), mainly growing from the southwest to south, such as Guangxi, Guangdong, Yunnan and Hainan province. In ethnic Yao medicine it was known as 'iron-drill' (Fu et al. 2015) with a great medicinal value. Previous phytochemical studies on this genus reported that these plants posses several compounds, including alkaloids , flavonoids (Ngoc et al. 2019), terpenoids (Wu et al. 2021) and so on. These plants perform extensive pharmacological effects on cardioprotection (Chang et al. 2004), anti-inflammatory (Hu et al. 2008), immunosuppression (Zhang et al. 2007), anti-tumor (Thuy et al. 2012), etc. Fissistigma acuminatissimum Merr. is a representative species which used to treat anemofrigid-damp arthralgia in Guangdong folk in China. Its chemical constituents is still in lack of further research. With only flavonoids (Van et al. 2007) and essential oils (Thang et al. 2016) reported, its therapeutic material basis has not been thoroughly studied up to now. Herein, it is necessary to calarify the constituents and biological activity of organic compounds contained in the plant. In this paper, we will report the isolation and structure elucidation of three new compounds, 4,5,6,7-tetramethoxy-3benzoylbenzofuran (1), 4-hydroxy-3,5,6-trimethoxydihydrochalcone-2-O-b-D-glucopyranoside (2) and 2-hydroxy-3,4,5,6-tetramethoxyphenylethyl benzoate (3) along with five known flavonoids from the stems of Fissistigma acuminatissimum Merr. The anti-inflammatory effects of all the fractions and compounds separated from this plant had been investigated in vitro.

Compounds isolated from Fissistigma acuminatissimum Merr
The CH 2 Cl 2 -soluble part partitioned from the ethanol extract of Fissistigma acuminatissimum Merr. was isolated by combination of chromatographic methods to yield eight compounds, including three new compound (1-3) and five known compound (4-8), they were shown in Figure 1.
Compound 1 was obtained as a yellow amorphous powder with its molecular formula established as C 19 H 18 O 6 with 11 degrees of unsaturation as deduced from its HR-ESI-MS spectrometry at m/z 341.1029 [M-H] -. The 1 H-NMR spectrum (Table S1) showed one olefinic proton (d H 8.39, 1H, s), a monosubstituted phenyl group (d H 7.55-7.89, 5H, m, overlap), four methoxy groups (d H 4.03, 3.89, 3.79, 3.59). The 13 C-NMR spectrum (Table S1) showed the presence of nineteen carbon atoms belonging to one carbonyl carbon (d C 188.8), fourteen aromatic and olefinic carbon atoms between d C 149.6 and 115.5. The above information suggested compound 1 had a skeleton of 3-benzoylbenzofuran, and the 1 H and 13 C NMR data closely resembles those of 6-methoxy-5,2 0 ,4 0 -trihydroxy-3-benzoylbenzofuran (Wang et al. 2014). However, the main differences between the 1 H-NMR of compound 1 and that of 6methoxy-5,2 0 ,4 0 -trihydroxy-3-benzoylbenzofuran was that the latter exhibited an ABX coupled system in A ring and two singlet aromatic protons in B ring, but compound 1 showed a characteristic monosubstituted benzene and a hexasubstituted benzene. It suggested four methoxy groups were located at B ring. It was also confirmed by the HMBC and NOESY spectra. In HMBC spectrum ( Figure S1), the olefinic proton (d H 8.39, H-2) showed cross-peaks with C-3 (d C 120.8), C-3a (d C 115.4) and C-7a (d C 143.4). In NOESY spectrum ( Figure S1), the methoxyl proton at d H 4.03 was correlated to H-2 (d H 8.39) that indicated the methoxyl was connected to C-7, the methoxyl proton at d H 3.89 was correlated to 7-OCH 3 (d H 4.03) that confirmed the methoxyl was connected to C-6, then 5-OCH 3 (d H 3.79) and 4-OCH 3 (d H 3.59) were successively identified by the correlations in NOESY spectrum. Based on the above data analysis, compound 1 was established as 4,5,6,7-tetramethoxy-3-benzoylbenzofuran. Compound 2 was obtained as yellow amorphous powder and its molecular formula was established as C 24 H 30 O 11 by HR-ESI-MS spectrum ([M-H] -, found: m/z 493.1681) showing 10 degrees of unsaturation. The 1 H-NMR spectrum (Table S2) showed a hydroxyl proton (d H 8.88, 1H, s), a monosubstituted phenyl group (d H 7.49-8.04, 5H, m, overlap), as well as three methoxy groups at d H 3.73, 3.72, 3.70, one a-carbonyl methylene at d H 3.03 (2H, m) and one benzylic methylene at d H 2.83 (2H, m). A signal of anomeric proton at d H 4.93 (d, J ¼ 7.36 Hz, H-1 0 ') indicated a sugar was existed which was further determined to be glucose by the typical carbon signals at d C 102.8 (C-1"), 73.9(C-2"), 76.3(C-3"), 70.0(C-4"), 77.2(C-5") and 60.9(C-6") observed in the 13 C-NMR spectrum (Table S2), the relative coupling value (J ¼ 7.36 Hz) of the anomeric proton was characteristic of the b configuration of the glucose (Hanh et al. 2020). The connectivity of glucose part was identified by HMBC correlations between the H-1 0 ' (d H 4.93) and the carbon d C 143.3. The 13 C-NMR spectrum also showed the presence of seventeen carbon atoms belonging to carbonyl carbon at d C 200.2, twelve aromatic carbon atoms between d C 146.9 and 117.9, three methoxy carbon between d C 60.6 and 60.1, as well as two methylene carbon atoms at d C 39.6 and 19.4. The above data of compound 2 showed similar features to those of 2-hydroxy-3,4,6-trimethoxydihydrochalcone (Lien et al. 2000). However, the main differences between two compounds were 2-hydroxy-3,4,6-trimethoxydihydrochalcone had an aromatic proton at d H 6.25 in ring B but compound 2 was fully substituted. The methylene proton H-7 (d H 2.83) show HMBC correlations ( Figure S1) with aromatic carbon at d C 117.9 (C-1), 143.3 (C-2), 146.9 (C-6), indicated the glucose was connected to C-2. The key HMBC correlations from d H 3.73 to d C 146.9 indicated that C-6 was connected to methoxy. Based on the NOESY spectrum ( Figure S1), 6-OCH 3 (d H 3.73) was correlated to protons at d H 3.72, which indicated the carbon's chemical shift of 5-OCH 3 was 60.5. H-1 0 ' (d H 4.93) was correlated to protons at d H 3.70 indicated 3-OCH 3 at d C 60.1. Based on the above data analysis, compound 2 was established as 4-hydroxy-3,5,6-trimethoxydihydrochalcone À2-O-b-Dglucopyranoside.
Compound 3 was obtained as colorless amorphous powder with its molecular formula established as C 19 H 22 O 7 , as deduced from its HR-ESI-MS spectrometry at m/z 361.1291 [M-H] -. The 1 H-NMR spectrum (Table S1) showed one hydroxyl proton (d H 8.83, 1H, s), a monosubstituted phenyl group (d H 7.49-7.93, 5H, m, overlap), two aliphatic proton signals due to -CH 2 CH 2 O-moiety at d H 2.97 and 4.35 (each 2H, t, J ¼ 7.0 Hz) (Ma et al. 2008), as well as four methoxy groups at d H 3.82, 3.74, 3.71, 3.69. The 13 C-NMR spectrum (Table S1) showed the presence of nineteen carbon atoms belonging to one carbonyl carbon at d C 165.7, twelve aromatic carbon atoms between d C 147.9 and 112.7, five carbon atoms which connected oxygen atom between d C 63.6 and 60.8, as well as one aliphatic at d C 23.2. Based on DEPT spectrum, five aromatic carbon atoms in d C 133.2, 129.1(2 C), 128.6(2 C) were carbon in monosubstituted phenyl, as well as d C 60.9, 60.8, 60.8, 60.8 were methoxy carbon atoms, d C 63.6 and 23.2 were methylene carbon atoms. H-2 0 ,6 0 (d H 7.93) show HMBC correlations with carbonyl carbon (d C 165.7) indicated the monosubstituted phenyl was connected with carbonyl directly. From the above data it was indicated compound 3 had a skeleton of phenylethyl benzoate (Ma et al. 2008;Zhang et al. 2019). Comparison of the ABX-pattern proton signals of reference compound 4 0 -methoxyphenethyl-4-hydroxy-3-methoxybenzoate, ring A of compound 3 was monosubstituted and ring B was fully substituted. The key HMBC correlations ( Figure S1) from H-7 (d H 2.97) to aromatic carbon d C 147.9 and 144.6, from d H 3.74 to d C 147.9 and d H 8.83 to d C 144.6 indicated C-6 (d C 147.9) was connected to methoxy and C-2 (d C 144.6) was connected to hydroxyl. The chemical shift of C-3 (d C 136.9), C-4 (d C 145.4), C-5 (d C 138.6) and 3-OCH 3 (d C 60.8), 4-OCH 3 (d C 60.8), 5-OCH 3 (d C 60.8) was indicated by conformational analyses which were carried out via random searching in the Sybyl-X 2.0 using the MMFF94S force field with an energy cutoff of 2.5 kcal/mol (Sybyl Software. 2013). Based on the above data analysis, compound 3 was established as 2-hydroxy-3,4,5,6-tetramethoxyphenylethyl benzoate.

The viability assay of RAW264.7 macrophages
The RAW264.7 macrophages were treated with different concentrations of tested fractions and compounds dissolved in DMSO for 24 h. The results of tested fractions were in Table S5, and compounds in 10 mM were not influenced the viability of cells at all. Based on the results, we selected 40 and 60 mg/mL of total extract, petroleum ether fraction, dichloromethane fraction, N-butanol fraction, 80 and 100 mg/mL of ethyl acetate fraction, 10 mM of each compound for further research.

Elisa assays
All the fractions and compounds were tested for the anti-inflammatory activity against TNF-a factor by ELISA kits. The results were in Table S6.The dichloromethane fraction showed the most potent inhibition (38.2%) at 60 lg/mL and compound 1 (70.2%) and 3 (65.2%) in this fraction showed significant inhibition at 10 lM.

General experimental procedures
NMR conditions：1D and 2 D-NMR spectra were obtained on Bruker AV-400 spectrometer with TMS as internal reference, and using DMSO-d 6 as solvents.
UHPLC-DAD-Q-TOF-MS conditions： The LC analyses were conducted on a Shimadzu UHPLC system (Kyoto, Japan) consisting of a LC-30AD solvent delivery system, a SIL-30AC autosampler, a CTO-30A column oven, a DGU-20A3 degasser and a CBM-20A controller. The separation was carried out on a ACE Excel II C 18 column (100 mm Ã 2.1 mm), water (A) and acetonitrile (B) was taken as mobile phase under a gradient program, flow rate at 0.3 mL/min, temperature at 25 C. The mass spectra were acquired using a TripleTOF TM 5600þ system with a Duo Spray source (AB SCIEX, Foster City, CA, USA) in negative and positive ESI mode. The data were analyzed by Peak View Software TM 1.2.

Cell culture and viability assay
After resuscitation, RAW264.7 cells were cultured in DMEM supplemented with high glucose and 10% heat inactivated fetal bovine serum at 37 C in an atmosphere of 5% CO 2 and 95% air. Cells were passage cultured when covered the bottom and grew to above 80% confluence. Using Cell Counting Kit-8 (CCK-8) method to detect the cell viability (Xiao et al. 2021). The RAW264.7 cell seeded in 96-well plates (each well contained cell nearly 2 Ã 10 4 ) and incubated for 24 h, then changed to DMEM supplemented with high glucose and 5% heat inactivated fetal bovine serum. Subsequently the cells were treated with different concentrations of tested fractions and compounds dissolved in DMSO for 24 h. The concentrations of tested fractions (including total extract, petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, N-butanol fraction) were 40, 60, 80, 100, 160 mg/mL respectively, as well as the concentrations of compounds were 10 mM. The cells were subsequently added 10 lL CCK-8 and co-incubated for 4 h. The cell viability was assessed by monitoring the absorbance at 450 nm with a microplate reader (Bio-Tek Instruments, Elx800, USA).

Elisa assays
The RAW264.7 cells seeded in 96-well plates (each well contained cell nearly 2 Ã 10 4 ) and incubated for 24 h, then changed to DMEM supplemented with high glucose and 5% heat inactivated fetal bovine serum. Subsequently the cells were treated with different concentrations of tested fractions and compounds after stimulation with 100 ng/mL LPS for 3 h. After 18 h, the supernatants were collected, TNF-a in the supernatants were detected by ELISA kits according to the manufacturer's manual (Jian et al. 2021).