Phenolic components from the fruits of Solanum xanthocarpum with anti-inflammatory activity

Abstract Two new compounds (1 and 2), along with thirty-one known compounds (3-33) were isolated from the fruits of Solanum xanthocarpum. The structure of isolates was elucidated by analysis of spectroscopic data and the physicochemical methods. Meanwhile, the anti-inflammatory activity of isolates was determined using LPS-induced RAW 264.7 cells. The results of anti-inflammatory assays indicated that most isolated compounds (3, 4, 6, 8-14, 17-20, and 30) possessed significant nitric oxide (NO) production inhibition in lipopolysaccharide (LPS)-induced RAW 264.7 cells with IC50 values ranging from 14.33 to 48.55 μM. Graphical Abstract


Introduction
The genus Solanums belongs to the family Solanaceae, including comprises 90 genera and 2000-3000 species (Preet and Gupta 2018).It was noted that the Solanum xanthocarpum plays an important role in the Ayurvedic system of medicine (Velu et al. 2016;Parmar et al. 2017), has various interesting biological effects, such as antimicrobial activity (Pungle et al. 2023), antitumor activity (Kumar and Pandey 2014), and anti-inflammatory activity (More et al. 2013).However, there has been little research on the chemical constituents of their fruits.Therefore, in this study, a systematic chemical investigation of the fruits of Solanum xanthocarpum was conducted, resulting in the discovery of two new compounds (1 and 2) (Figure 1, Note: see Figure 1 in a separate file), along with thirty-one known compounds (3-33) (Figure S15).Meanwhile, the anti-inflammatory activity of isolates was determined using LPS-induced RAW 264.7 cells.
Compound 2 (Figure 1) was obtained as a white amorphous powder.The molecular formula was determined to be C 22 H 32 O 15 based on HRESIMS (Figure S14) at m/z 537.1801 [M + H] + (calculated for 537.1814) and m/z 554.2078 [M + NH4] + (calculated for 554.2079), accounting for seven degrees of unsaturation.The above 1 H and 13 C NMR features of 2 were related closely to those of seguinoside A, except that there is one more molecule of xylose in 2 than in seguinoside A (Zhong et al. 1998).The structure was supported by the key HMBC correlations (Figure S16) from xly-H-1 (δ H 4.31, d, J = 7.5 Hz) to glu-C-6 (δ C 69.8).The 1 H-1 H COSY (Figure S12) correlations of 2 The acid hydrolysis experiment of 2 gave D-glucose, D-xylose, and D-apiose identified by GC (Figure S21).Combining with DEPT, hydrogen and its directly connected carbon atoms were assigned by HSQC.Thus, the structure of compound 2 was determined assigned to be hydroxyphenyl-O-β-D-apiofuranosyl-(1→2)-β-D-xylopyranosyl-(1→6)-β-D-glucopyranoside by analysis of a combination of DEPT, 1 H-1 H COSY, HSQC, and HMBC spectra.

Anti-inflammatory effects
Increased NO production is associated with acute and chronic inflammatory diseases, and NO production inhibitors were considered an excellent candidate for the treatment of such diseases.Therefore, the determination of NO production of different phytochemicals using various in vitro cell models was considered an important anti-inflammatory estimation method (Aeberhard et al. 1995;Garzón-Porras et al. 2020)

Measurement
HRESIMS spectra were performed on an UHPLC-Orbitrap-MS instrument or an AB SCIEX Triple TOF 5600 instrument.NMR spectra were obtained on a Bruker DPX 600 instrument (600 MHz for 1 H NMR and 150 MHz for 13 C NMR) or a Bruker DPX 400 instrument (400 MHz for 1 H NMR and 100 MHz for 13 C NMR) with TMS as an internal standard.Optical rotations were measured by a JASCO P-2000 digital polarimeter.

Plant material
The fruits of Solanum xanthocarpum were collected from Guilin, Guangxi Province, China, in March 2016, and identified by Prof. Rui-Feng Fan.A voucher specimen (No. 20160327) was deposited in the laboratory at the School of Pharmacy, Heilongjiang University of Chinese Medicine.

Extraction and isolation
The air-dried fruits of Solanum xanthocarpum (9 kg) were crushed and extracted with 70% ethanol (54 L × 3, each for 2 h) under reflux.The extract was filtered and then concentrated in vacuo to get the crude extract (2473.6 g).Then the crude extract was suspended in water and subsequently partitioned with petroleum ether, EtOAc, and n-BuOH.
The isolation procedure for the known compounds is given in the Supporting Information (S1).

Acid hydrolysis and GC analysis
Compounds 1-2 (each 1.0 mg) were individually dissolved in 1 M HCl (1.0 mL) and hydrolyzed by refluxing for 4 h.After cooling, the reaction mixture was partitioned between ethyl acetate and H 2 O three times (1.0 mL each).The residue from the water part was dissolved in pyridine (1.0 mL), to which L-cysteine methyl ester hydrochloride (1.0 mg) was added.After reacting at 60 °C for 1 h, N-(trimethylsilyl) imidazole (0.1 mL) was added to the reaction mixture and kept at 60 °C for another 60 min.The residue was partitioned between H 2 O and n-hexane three times (1.0 mL each).The n-hexane layer was analyzed by GC [detector, flame ionization detector (FID); detector temperature, 280 °C; DB-210 capillary column, 30 m × 0.25 mm × 0.25 μm; and carrier gas, N 2 ].By comparison with the retention time of the authentic sample, the absolute configurations of sugar components were determined (D-glucose, 6.60; D-xylose, 11.48; D-apiose, 13.81).

Anti-inflammatory assays
The RAW 264.7 cells were cultured in the DMEM medium with 10% FBS and 100 U/ mL Penicillin/Streptomycin in 96-well plates (5 × 10 4 /well) for 24 h.Then, the RAW 264.7 cells were divided into four groups containing the control group, the model group (1 μg/mL LPS), the positive control group (1 μg/mL LPS, and indomethacin at final concentrations of 1.25, 2.5, 5, 10, 20 μM/mL), and the sample group (1 μg/mL LPS and the test compounds at final concentrations of 5, 10, 20, 30, 50 μM/mL) for 24 h.The NO concentration in the culture medium was measured by the Griess reagent at 540 nm (Xu et al. 2022).

Conclusion
In summary, 33 phenolic compounds were isolated from the fruits of Solanum xanthocarpum, including two new compounds 1 and 2, and 31 known compounds 3-33.In vitro, the anti-inflammatory assay revealed that compounds (3, 4, 6, 8-14, 17-20, and 30) can inhibit the excretion of NO in LPS-activated RAW 264.7 macrophages, especially 3 (IC 50 14.33 μM), 4 (IC 50 14.74 μM), and 20 (IC 50 15.23 μM).This study provided a theoretical basis for the important anti-inflammatory ingredients of the fruits of Solanum xanthocarpum, and provides a basis for the further development and utilization of Solanum xanthocarpum.

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

Table 1 .
anti-inflammatory activity of compounds 1-33.a a data are shown as the mean ± sd, n = 3