We analysed four different Sudanese roselle samples for their potential as novel xanthine oxidase (XO) inhibitors. Phytochemical screening showed the presence of polyphenols, flavonoids, organic acids, saponins and sterols in all samples. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used to identify and characterise five anthocyanins in all samples: cyanidin-3-glucoside (cy-3-glu), delphinidin-3-sambubioside (dp-3-sam), cyanidin-3-rhamnoside (cy-3-rhm), delphinidin-3-rhamnoside (dp-3-rhm) and pelargonidin-3-glucoside (pg-3-glu). Identification of cy-3-rhm, dp-3-rhm and pg-3-glu confirmed the selectivity and sensitivity of LC-MS as a powerful technique for identifying anthocyanins. In silico studies of the identified anthocyanins were performed to explore their promising inhibitory activity toward XO. Interactions between the ligand and the enzyme were via the H-bond, and hydrophobic (π-alkyl, π-sigma and alkyl) and/or electrostatic (π-cation) bonds. Inhibition of the anthocyanins was compared with that of topiroxostat, a commercial drug for hyperuricaemia. Dp-3-rhm was the most active inhibitor with a binding energy of ca. -10.90 kcal/mol compared to topiroxostat’s binding energy of ca. -8.60 kcal/mol. “The good inhibition results obtained from anthocyanins toward XO suggest their application as a drug candidate to treat gout and other diseases related to the activity of XO.

Phytochemicals and in silico investigations of Sudanese roselle

**_Chemicals_**

All chemicals were of analytical grade except those used in LC-MS analysis which were HPLC-grade.”

_**Phytochemical screening**_

**Preparation of extracts**

The extract was prepared according to Wrolstad et al.’s18 alternative protocol with some modifications in the purification step. The purification was established by simple liquid-liquid extraction using an organic non-polar solvent (petroleum ether) and (0.01% (v/v) HCl) acidified methanol to remove water-insoluble impurities. The desired layer was reserved, the solvent was evaporated using a rotatory evaporator (Heidolph Laborota 4000 efficient HB digital, Germany) and allowed to air dry for 3–4 h. The attained gummy extract was used for all the phytochemical qualitative and quantitative analyses after dissolving in 1–2 cm3 methanol for each qualitative test.

**Preliminary screening**

Qualitative analysis was carried out according to methods described by Harborne10 and Sofowara19 via simple test tube tests. These are the test for polyphenols (FeCl3 test), test for flavonoids (AlCl3 test), test for sterols (H2SO4 test), test for saponins (Frothing test), and test for anthraquinones (KOH test).

**Thin-layer chromatography**

An amount of 10 mg from the gummy powdered extract of each of the samples was dissolved in 1 cm3 of methanol and, using a capillary tube, spots were made from each sample extract on an aluminium sheet of pre-coated silica gel thin-layer chromatography plates, then elution was carried out in a covered thin-layer chromatography chamber using the mobile phase of a mixture of chloroform and methanol (7:3 v/v) according to Harborne10. The procedure was repeated on two other plates.

After performing the elution, plates were examined immediately in daylight, then sprayed with chemical reagents and visualised under a UV lamp (at 365 nm). Bromocresol green was the spray reagent employed for detection of organic acids, while AlCl3 was used for detection of flavonoids.20

**Quantitative analysis**

Total phenolic content was measured using Folin–Ciocalteu’s reagent as described by Wolfe et al.21 Total flavonoid and total anthocyanin contents were measured according to Shanmukha et al.22 and Pacôme et al.6, respectively.

For total phenolic and flavonoid contents, absorbance was measured at 765 nm and 510 nm, respectively, against a reagent blank using a Jenway 7205 UV/Vis double-beam spectrophotometer. The concentrations of phenolics and flavonoids in the test sample were determined and expressed as gram equivalent of gallic acid (g GAE) and quercetin per gram of air-dried extract (g ADE), using a standard calibration plot for each individually. All measures were performed in duplicate.

Determination of total anthocyanins

Total anthocyanin content was measured at an absorbance of 530 nm using a Jenway 7205 UV/Vis spectrophotometer against the blank; total anthocyanin content was estimated as cyanidin-3-glucoside at 530 nm using a molar extinction coefficient of 26 900 L/mol/cm) and molar mass (449 g/mol) and was expressed as cyanidin-3-glucoside (g cy-3-glu)/(100 g of air-dried extract).

**LC-MS/MS identification**

Anthocyanin extraction was carried out using 250 cm3 acidified methanol by Soxhlet apparatus2 at a temperature of 50–55 °C. The extract was purified by simple liquid/liquid extraction after reducing the extract volume using the rotatory evaporator (Heidolph Laborota 4000 efficient HB digital, Germany).

Anthocyanins were identified in the purified sample extracts using LC-MS/MS with a UPLC-Q-TOF-MS (XEVO-G2 QTOF YCA119, Waters, using Acquity UPLC HS T3 (100 × 2.1 mm, 1.8 μm; Waters) column maintained at 45 °C in a column oven in the LC-MS Laboratory, Beijing University of Chemical Technology BUCT, China. The analysis was carried out according to the method described by Cahlíková et al.23 with some modifications. The conditions of the chromatographic run were as follows: 0–1 min: 90% A and 10% B, 1–5 min: 85% A and 15% B, 5–10 min: 40% A and 60% B, 10–13 min: 0% A and 100%B, 13–15 min: 90% A and 10% B. Gradient mobile phase composed of water acidified with 0.1% formic acid (solvent A) and acetonitrile (solvent B). The flow rate and sample injection volume were 0.4 cm3/min and 3.00 µL, respectively.

The ion source of the quadropole time-of-flight (Q-TOF) detector was set at the positive electrospray ionisation polarity mode. The optimum conditions of the Q-TOF system, performed in the resolution mode, were: capillary voltage: +3.000 kV, ion source temperature: 100 °C, extractor: ٤.٠ V; RF lens: ٠.٣ V, mass range m/z: 50 to 2000 Da, nitrogen as the desolvation gas at a flow rate of 800 L/h and at a temperature of 400 °C. Nitrogen was used also as the cone gas (٢٠ L/h), and argon as the collision gas. The cone voltage, collision energy, and well time were carefully optimised for each compound and transition individually (cone voltage set at 4/30 V, collision energy set finally at 6 eV). The software used for the MS control and data gathering was Mass Lynx 4.1.

**Molecular docking**

Molecular docking is a method used to screen for the ability of specific small compounds (ligands) to fit into a previously identified therapeutic target (the receptor; usually a protein enzyme).24,25 Several docking programs can be used: AutoDock Vina, GOLD, and MOE-Dock are top ranked with the best scores.25 We used the AutoDock Vina program26 for the docking of anthocyanin ligands to the active site of the XO enzyme, after performing the required preparations.

First, the protein crystal structure and ligand 3D structures were downloaded from RCSB Protein Data Bank27 (1FIQ PDB code) and PubChem online databases28, respectively. Then the suitable binding site of the protein was obtained using Chimera software29 and the ligands were converted via Open Babel software30 from SDF format to pdbqt. Thereafter, using autodock tools31, all water molecules in the protein pdb file were removed, hydrogen atoms were added, and the grid box dimensions were set, then the file saved as pdbqt. Ligands were prepared by detecting torsions for each structure and saved as a pdbqt file. The AutoDock Vina order was then created after preparing the configuration files. The process was repeated for each docking trial for all ligands.

**Data analysis**

The obtained (out.pdbqt) file for each ligand was visualised using discovery studio32 and pymol33 visualisation programs.

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