Standardized Combined Plant Extract, RUG-com, Reduces Bacterial Levels and Suppresses Acute and Chronic Inflammation in Balb/c Mice Infected with CagA+ Helicobacter pylori

Abstract

Helicobacter pylori are etiological agents in the development of gastritis, gastroduodenal ulcers, gastric cancer, and mucosa-associated lymphoid tumors. Our previous investigations demonstrated that standardized combined plants extracts (Rubus crataegifolius and Ulmus macrocarpa) inhibit the growth of H. pylori in in vitro experiments. Also, we demonstrated that Gardenia jasminoides is effective in preventing gastritis and gastric ulcers in animal experiments. In the present work, we tested the standardized combined three plant extract (RUG-com) on the mouse model of H. pylori infectious disease to examine the effects of RUG-com on both the prevention and curing on the stomachs of infected mice. After the final administrations, biopsy samples of gastric mucus were assayed for bacterial numbers, biochemical analysis, inflammatory scores, and histology. Treatment with standardized plants extracts, single or combined, reduced the H. pylori load compared with the control. Treatment also significantly (P<0.05) reduced both acute and chronic mucosal and subacute inflammation, and epithelial cell degeneration and erosion induced by H. pylori infection. Further investigations demonstrated that H. pylori-induced inflammation was decreased by RUG-com extracts via down regulating cyclooxygenase-2 and inducible nitric oxide synthase pro-inflammatory gene expression. Our results suggest that RUG-com is useful to prevent H. pylori infection, H. pylori-induced inflammation and associated gastric damage.

Keywords: Helicobacter pylori; anti-inflammation; gastric lesion; gastritis; plant extracts RUG-com.

Fig. 1

High-performance liquid chromatography-diode-array detector chromatogram for sample standardization. (A) Ellagic acid standard at 254 nm, (B) Rubus crataegifolius extracts, (C) geniposide standard at 254 nm, (D) Gardenia jasminoides extracts, (E) catechin-7-O-β-D-apiofuranoside at 280 nm, and (F) Ulmus macracarpa extracts. The contents were standardized to contain 11.2 mg/g, 10.2 mg/g and 72.0 mg/g, respectively.

Fig. 2

Experimental schedule for in vivo evaluation of efficacy of plant extracts on anti-Helicobacter pylori activity. Time-points of H. pylori infection and plants extracts treatment in Balb/c mice. Balb/c mice were orally administered with H. pylori (2.0×10⁸ CFU) three times over 4 days. After 4 weeks, H. pylori treated mice were assessed for infection by sacrificing, followed histology analysis of H. pylori cultures. After confirming H. pylori infection, mice received plant extracts 4 weeks for recovery from H. pylori infection, as assessed by H. pylori counting and histology analysis. RF, Rubus crataegifolius; UL, Ulmus macrocarpa Hance; GJ, Gardenia jasminoides Ellis; OMP, omeprazole; AMX, ampicillin; CLM, clarithromycin; CFU, colony-forming unit.

Fig. 3

Inflammatory scores of the stomachs of Helicobacter pylori infected Balb/c mice treated with single or complex plant extracts. Mouse stomach sections were stained with hematoxylin and eosin and graded to determine the amount of lymphocyte infiltrations. (This work was carried out by Dr. Moon Hyung Bae at Wonkwang Medical School, Iksan, Korea). RF, Rubus crataegifolius; UL, Ulmus macrocarpa Hance; GJ, Gardenia jasminoides Ellis; AMX, ampicillin; CLM, clarithromycin; OMP, omeprazole. The results were compared by Student’s t-test (*P <0.05 and ***P <0.001). The error bars represent the SD.

Fig. 4

Histological analysis (magnification ×40). Helicobacter pylori-induced gastritis showed increased amounts of immune cells in the mouse stomachs (A. H. pylori infection). Treatment with and antibiotics (B) and plants extract (E and F) protected against inflammation. Stillen (C) and lactobacillus (D) were used as positive controls. In Giemsa staining, blue spots showed the presence of H. pylori. H&E, haematoxylin and eosin; RF, Rubus crataegifolius; UL, Ulmus macrocarpa Hance; GJ, Gardenia jasminoides Ellis.

Fig. 5

Effect of standardized combined three plant extract (RUG-com) administration on expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the stomachs of Helicobacter pylori infected Balb/c mice. The expression of iNOS proteins and COX-2 proteins were determined by Western blotting using specific antibodies. β-Actin was used as an internal control. NOR, control group; RUG, RUG-com treated group; ACO, triplet antibiotics treated group; H. pylory, H. pylori treated group. The results were compared by Student’s t-test (*P <0.05 and ***P <0.005). The error bars represent the SD.