Phytochemical Composition, Antioxidant, Anti- Helicobacter pylori, and Enzyme Inhibitory Evaluations of Cleistocalyx operculatus Flower Bud and Leaf Fractions

Abstract

Six solvent fractions isolated from flower bud and leaf ethanolic extracts of Cleistocalyx operculatus were analyzed for their phytochemical contents, including phenolics, flavonoids, saponins, tannins, and alkaloids. Antioxidant activities were measured using the ABTS, DPPH, and FRAP assays. The results showed that the flower bud aqueous fraction (BAF) and the leaf aqueous fraction (LAF) rich in phenolic content (768.18 and 490.74 mg GAE/g dry extract, respectively) exhibited significantly higher antioxidant activities than the other fractions. The flower bud hexane fraction (BHF) had remarkably high flavonoid and saponin contents (134.77 mg QE/g and 153.33 mg OA/g dry extract, respectively), followed by that of the leaf hexane fraction (LHF) (76.54 mg QE/g and 88.25 mg OA/g dry extract, respectively). The BHF and LHF were found to have extremely high antibacterial activity against two H. pylori strains, ATCC 51932 and 43504 (MICs of 125 µg/mL). Interestingly, DMC (2',4'-Dihydroxy-6'-methoxy-3',5'-dimethylchalcone) isolated from the BHF displayed greater antibacterial activity against the bacterial strains (MICs of 25-50 µg/mL) than those of the fractions. In addition, DMC presented potent inhibitory effects on H. pylori urease (IC₅₀ of 3.2 µg/mL) and α-amylase (IC₅₀ of 83.80 µg/mL), but no inhibition against α-glucosidase. It was also demonstrated that DMC showed pronounced inhibitory effects on the urease activity and biofilm formation of H. pylori, and could increase the membrane permeability of the bacterial cells. Scanning electron micrographs depicted that the BHF and DMC had strong effects on the cell shape and significantly induced the distortion and damage of the cell membrane. The fractions and DMC showed no significant toxicity to four tested human cell lines. Efforts to reduce antibiotic use indicate the need for further studies of the flower buds and DMC as potential products to prevent or treat gastric H. pylori infections.

Keywords: Cleistocalyx operculatus; anti-H. pylori activity; antibiofilm formation; antioxidant effect; membrane permeability; morphological transformation.

Figure 1

Effect of C. operculatus fractions and DMC at sub-MICs on H. pylori biofilm formation 48 h post-treatment. Data are reported as means ± SD.

Figure 2

SEM micrographs of H. pylori ATCC 43504 depicting untreated cells (A,B) and cells treated with 125 µg/mL of BHF (C,D) and 50 µg/mL of DMC (E,F); bars 5 and 1 µm, respectively.

Figure 3

Effect of C. operculatus fractions and DMC at sub-MICs on the uptake of crystal violet by H. pylori ATCC 43504 after 2 h of treatment. Data are reported as means ± SD (n = 3).