Fruits of Hippophaë rhamnoides in human leukocytes and Caco-2 cell monolayer models—A question about their preventive role in lipopolysaccharide leakage and cytokine secretion in endotoxemia

Preparations from Hippophaë rhamnoides L. (sea buckthorn) have been traditionally used in the treatment of skin and digestive disorders, such as gastritis, gastric and duodenal ulcers, uterine erosions, as well as oral, rectal, and vaginal mucositis, in particular in the Himalayan and Eurasian regions. An influence of an aqueous extract from the fruits of H. rhamnoides (HR) on leakage of lipopolysaccharide (LPS) from Escherichia coli through gut epithelium developed from the human colorectal adenocarcinoma (Caco-2) monolayer in vitro and glucose transporter 2 (GLUT2) translocation were the principal objectives of the study. Additionally, the effect of HR on the production of pro- and anti-inflammatory cytokines (interleukins: IL-8, IL-1β, IL-10, IL-6; tumor necrosis factor: TNF-α) by the Caco-2 cell line, human neutrophils (PMN), and peripheral blood mononuclear cells (PBMC) was evaluated. The concentration of LPS on the apical and basolateral sides of the Caco-2 monolayer was evaluated with a Limulus Amebocyte Lysate (LAL) assay. GLUT2 translocation was evaluated using an immunostaining assay, whereas secretion of cytokines by cell cultures was established with an enzyme-linked immunosorbent (ELISA) assay. HR (500 μg/ml) significantly inhibited LPS leakage through epithelial monolayer in vitro in comparison with non-treated control. The treatment of Caco-2 cells with HR (50–100 μg/ml) showed GLUT2 expression similar to the non-treated control. HR decreased the secretion of most pro-inflammatory cytokines in all tested models. HR might prevent low-grade chronic inflammation caused by metabolic endotoxemia through the prevention of the absorption of LPS and decrease of chemotactic factors released by immune and epithelial cells, which support its use in metabolic disorders in traditional medicine.


Supplementary
. The scheme of isolation of compounds from the fruit of Hippophaë rhamnoides.

Chromatographic analysis
Chromatographic analysis was performed on a UHPLC-3000 RS system (Dionex, Germany) with DAD and an AmaZon SL ion trap mass spectrometer with an ESI interface (Bruker Daltonik GmbH, Germany). Separations were performed on a Kinetex XB-C18 column (150 × 2.1 mm, 1.7 μm) (Phenomenex, USA). The column oven temperature was 25 °C. For preliminary phytochemical analyses of the extracts and fractions, mobile phase A was 0.1% HCOOH in water, and mobile phase B was 0.1% HCOOH in acetonitrile. The gradient program was as follows: 0 -20 min. 4 -26% B; 20 -60 min. 26 -95% B. The flow rate was 0.2 mL/min. The column was equilibrated for 10 min. between injections. UV spectra were recorded in the range of 200 -800 nm, and chromatograms were acquired at 240, 280, 325 nm, or 520 nm. The LC eluate was introduced directly into the ESI interface without splitting. The nebulizer pressure was 40 psi; the dry gas flow was 9 L/min; the oven temperature was 300 °C, and the capillary voltage was 4.5 kV. Analyses were carried out scanning from m/z 200 to 2,200. Compounds were analyzed in negative and positive ion modes. The MS 2 fragmentation patterns were obtained for the most abundant ion (Świerczewska et al., 2019).

Identification of compounds
The structures of the isolated compounds were determined based on NMR spectral analysis. The identity of the aglycone and the absolute configuration of the sugars were confirmed based on the analysis of hydrolysates and sugar derivatization. NMR spectra were recorded on a Bruker DSX-400 spectrophotometer at the Laboratory of Nuclear Magnetic Resonance (NMR) Spectroscopy at the Department of Analytical Chemistry and Biomaterials, Medical University of Warsaw.

Hydrolysis
The accurately weighted 1-3 mg of glycoside was treated with 5% HCl in a water bath for 2 h at a temperature of 90 °C. Next, the reaction mixture was extracted with 3 × 3mL diethyl ether. The organic fraction was allowed to evaporate in a thermoblock at a temperature of 30 °C. A sample for HPLC analysis was prepared by dissolving in 250 µL of methanol. The aqueous fraction was concentrated under reduced pressure. The hydrolyzate was dissolved in 1 mL of distilled water, next 1 mL of methanolic (S)-(-)--methylbenzylamine solution (10 mg/mL), and 1 mL of methanolic NaBH3CN solution (3 mg/mL) were added. The reaction mixture was placed in a 40 °C water bath for 4h, then acidified to pH 3-4 with glacial acetic acid and evaporated under reduced pressure. The portion (1.2-1.4 mL) of acetic anhydride and pyridine (1:1, v/v) was added to the oily residue. After 24 h at room temperature, excess reagents were removed from the reaction mixture by washing it 5 times with portions of water (ca. 1 mL) and evaporated under reduced pressure. An aliquot of the oily residue was suspended in water (2 mL) and extracted with chloroform (3 mL) three times. The content of the vial was allowed to evaporate in a thermoblock at 50 °C. A sample for HPLC analysis was prepared by dissolving it in acetonitrile-water (20:80, v/v).

Isorhamnetin 3-O-glucoside
The structure of compound 11 was confirmed by a comparison of NMR data with available literature (Su et al., 2008).