Flavonoid Glycosides of Polygonum capitatum Protect against Inflammation Associated with Helicobacter pylori Infection

Abstract

The antibacterial and anti-inflammatory activities, and protective effects of extracts (flavonoid glycosides) of Polygonum capitatum were investigated to detect the evidence for the utilization of the herb in the clinical therapy of gastritis caused by H. pylori. A mouse gastritis model was established using H. pylori. According to treating methods, model mice were random assigned into a model group (MG group), a triple antibiotics group (TG group, clarithromycin, omeprazole and amoxicillin), low/middle/high concentrations of flavonoid glycosides groups (LF, MF and HF groups) and low/middle/high concentrations of flavonoid glycosides and amoxicillin groups (LFA, MFA and HFA groups). A group with pathogen-free mice was regarded as a control group (CG group). The eradicate rates of H. pylori were 100%, 93%, 89% in TG, MFA and HF groups. The serum levels of IFN-gamma and gastrin were higher in a MG group than those from all other groups (P < 0.05). The serum levels of IFN-gamma and gastrin were reduced significantly in LF, MF and HF groups (P < 0.05) while little changes were observed in LFA, MFA and HFA groups. In contrast, the serum levels of IL-4 were lower and higher in MG and CG groups compared with other groups (P<0.05). The serum levels of IL-4 were increased significantly in LF, MF and HF groups (P < 0.05) while little changes were found in LFA, MFA and HFA groups. According to pathological scores, flavonoid glycosides therapy showed better protection for gastric injuries than the combination of flavonoid glycoside and amoxicillin (P < 0.05). The results suggested that flavonoid glycoside has repairing functions for gastric injuries. The results suggest that the plant can treat gastritis and protect against gastric injuries. The flavonoid glycosides from Polygonum capitatum should be developed as a potential drug for the therapy of gastritis caused by H. pylori.

Fig 1. Sensitivity test of H. pylori to flavonoid glycoside.

A: blank control. B: 40 ug/mL flavonoid glycoside. C: 80 ug/mL flavonoid glycoside.

Fig 2. Identification of H. pylori.

A: Gram staining, Gram staining differentiates bacteria by detecting peptidoglycan, which exists in the cell walls of gram-positive bacteria. After a Gram stain test, gram-positive bacteria shows the crystal violet dye, while a counterstain (safranin) added, gives gram-negative bacteria a pink coloring. B, rapid urease test, is a rapid diagnostic test for H. pylori. H. pylori secrete the urease enzyme, which can catalyze the conversion of urea to NH3 and raises the pH of the medium. The medium contains urea and an indicator such as phenol red, and thus the raised pH changes the color of the medium from yellow (a blank control) to red (an experimental group). C, the oxidase test is used to determine if a bacterium produces certain cytochrome c oxidases. It uses disks impregnated with a reagent such as TMPD, which is a redox indicator. The reagent can be changed from a dark-blue to maroon color when it is oxidized. D, the catalase test is one of main tests to identify H. pylori. The presence of catalase enzyme in the species is detected via hydrogen peroxide. If the bacteria possess catalase, when bacterial isolate is added to hydrogen peroxide, bubbles of oxygen will be observed. If the mixture produces bubbles, the organism is regarded as 'catalase-positive'.

Fig 3. The comparision for histochemistry and histopathology between a control and a model group.

Giemsa staining (×400), Giemsa stain was used to observe the adherence of pathogenic bacteria to gastric cells. Microaerophilic culturing of H. pylori, the extracellular H. pylori are microaerophilic. Hematoxylin and Eosin staining of gastric tissues was used for the detection of H. pylori (HE×200). The staining of G cells of Gastric mucosa-associated lymphoid tissue (MALT) lymphoma (PV×200). The staining of D cells of Gastric MALT lymphoma (PV×200).

Fig 4. Hematoxylin and Eosin staining of gastric tissues (HE×200).

Normal mice (without H. pylori infection) were assigned as a control group (CG group) and mice models infected with H. pylori were divided into model group (MG group, only treated with saline solution), triple combination therapy group (TG group, the daily medicine intake is 0.5 ug clarithromycin, 0.02 ug omeprazole, and 1 ug amoxicillin), low/middle/high concentrations of flavonoid glycoside group (LF/MF/HF group, treated with one daily dose of Flavonoid glycoside at 32/64/128 ug), low/middle/high concentrations of flavonoid glycoside and common concentration of amoxicillin group (LFA/MFA/HFA group, treated with one daily dose of flavonoid glycoside at 32/64/128 ug and amoxicillin at 1 ug). Pathological score of mice gastric antrum in each group (±SD, n = 10). *P < 0.05 vs model group.

Fig 5. The staining of G cells of Gastric mucosa-associated lymphoid tissue (MALT) lymphoma (PV×200).

Normal mice (without H. pylori infection) were assigned as a control group (CG group) and mice models infected with H. pylori were divided into model group (MG group, only treated with saline solution), triple combination therapy group (TG group, the daily medicine intake is 0.5 ug clarithromycin, 0.02 ug omeprazole, and 1 ug amoxicillin), low/middle/high concentrations of flavonoid glycoside group (LF/MF/HF group, treated with one daily dose of Flavonoid glycoside at 32/64/128 ug), low/middle/high concentrations of flavonoid glycoside and common concentration of amoxicillin group (LFA/MFA/HFA group, treated with one daily dose of Flavonoid glycoside at 32/64/128 ug and amoxicillin at 1 ug). Comparison of G cells and gastrin gray value in each group (±s, n = 10). *P < 0.05 vs model group.

Fig 6. The staining of D cells of Gastric mucosa-associated lymphoid tissue (MALT) lymphoma (PV×200).

Normal mice (without H. pylori infection) were assigned as a control group (CG group) and mice models infected with H. pylori were divided into model group (MG group, only treated with saline solution), triple combination therapy group (TG group, the daily medicine intake is 0.5 ug clarithromycin, 0.02 ug omeprazole, and 1 ug amoxicillin), low/middle/high concentrations of flavonoid glycoside group (LF/MF/HF group, treated with one daily dose of flavonoid glycoside at 32/64/128 ug), low/middle/high concentrations of flavonoid glycoside and common concentration of amoxicillin group (LFA/MFA/HFA group, treated with one daily dose of Flavonoid glycoside at 32/64/128 ug and amoxicillin at 1 ug). Analysis of G cells and somatostatin gray value in each group (n = 10). *P < 0.05 vs model group.

Fig 7. The effects of flavonoid glycoside on the number of lymphocytes, protein levels of inflammatory biomarkers.

A, the effects of different concentrations of flavonoid glycoside on the number of lymphocytes. B, the effects of different concentrations of flavonoid glycoside on protein levels of INF-gamma. C, the effects of different concentrations of flavonoid glycoside on protein levels of IL-4. D, the serum levels of INF-gamma in different groups. E, the serum levels of IL-4 in different groups. F, the serum levels of gastrin in different groups. G, the serum levels of somatostatin in different groups. Normal mice (without H. pylori infection) were assigned as a control group (CG group) and mice models infected with H. pylori were divided into model group (MG group, only treated with saline solution), triple combination therapy group (TG group, the daily medicine intake is 0.5 ug clarithromycin, 0.02 ug omeprazole, and 1 ug amoxicillin), low/middle/high concentrations of flavonoid glycoside group (LF/MF/HF group, treated with one daily dose of flavonoid glycoside at 32/64/128 ug), low/middle/high concentrations of flavonoid glycoside and common concentration of amoxicillin group (LFA/MFA/HFA group, treated with one daily dose of flavonoid glycoside at 32/64/128 ug and amoxicillin at 1 ug). *P < 0.05 vs a model group (n = 10).

Fig 8. The effects of flavonoid glycoside on gastric cells infected by H. pylori.

A, the effects of different concentrations of flavonoid glycoside on the number of gastric cells MGC803. B, the effects of different concentrations of flavonoid glycoside on protein levels of INF-gamma. C, the effects of different concentrations of flavonoid glycoside on protein levels of IL-4. D, the effects of different concentrations of flavonoid glycoside on protein levels of gastrin. E, the effects of different concentrations of flavonoid glycoside on protein levels of somatostatin. *P < 0.05 vs a control group without flavonoid glycoside.

Fig 9. The flowchart of study.

Normal mice (without H. pylori infection) were assigned as a control group (CG group) and mice models infected with H. pylori were divided into model group (MG group, only treated with saline solution), triple combination therapy group (TG group, the daily medicine intake is 0.5 ug clarithromycin, 0.02 ug omeprazole, and 1 ug amoxicillin), low/middle/high concentrations of flavonoid glycoside group (LF/MF/HF group, treated with one daily dose of flavonoid glycoside at 32/64/128 ug), low/middle/high concentrations of flavonoid glycoside and common concentration of amoxicillin group (LFA/MFA/HFA group, treated with one daily dose of Flavonoid glycoside at 32/64/128 ug and amoxicillin at 1 ug). Gastrin, GAS; Somatostatin, SS.