Xanthones Isolated from Cratoxylum cochinchinensis Reduced Oxidative Stress in Periodontal Ligament Stem Cells

Abstract

Xanthone compounds from Cratoxylum cochinchinensis (C. cochinchinensis) have demonstrated antioxidant effects and potency in treating many inflammatory diseases. However, the efficiency of the three xanthone extracts isolated from the young fruit of this plant, i.e., two geranyloxy xanthones (F6, F8) and one 1,3,7-hydroxy xanthone (F137), as antioxidants and therapeutics for periodontal disease has not been evaluated. The aim of this study was to investigate the antioxidant effects of three xanthones isolated from C. cochinchinensis on periodontal ligament stem cells (PDLSCs) and their osteogenic differentiation. The antioxidant activity of the aqueous extracts was determined using a DPPH assay, and their cytotoxicity was evaluated using an MTT assay. H₂O₂ was used to induce intracellular stress, and the scavenging effect of the isolated compounds against reactive oxygen species (ROS) was analyzed with a fluorescence assay. The expression of nuclear factor-erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) was evaluated, and the effects of the three compounds on PDLSCs osteogenic differentiation were investigated. The isolated compounds reduced both extracellular and intracellular ROS in a dose-dependent manner and induced the expression of Nrf2 and HO-1 in PDLSCs. Under redox conditions, these compounds potentiated PDLSCs osteogenic differentiation. Our study demonstrated that the hydroxy xanthones from C. cochinchinensis had antioxidant effects on the Nrf2/HO-1 pathway and might be effective therapeutic substrates for damage prevention and the regeneration of damaged periodontal tissues in periodontitis patients.

Keywords: Cratoxylum cochinchinensis; antioxidant; periodontal ligament stem cells; xanthone compounds.

Figure 1

The chemical structure of the xanthones isolated from C. cochinchinensis. (1) 1,3,7-hydroxy xanthone, F137, and two geranyloxy xanthones, including (2) 7-geranyloxy-1,3-dihydroxy xanthone, F6, and (3) 3-geranyloxy-1, 7-dihydroxy xanthone, F8.

Figure 2

Radical scavenging effects of the C. cochinchinensis derivatives. F6 and F8 are geranyloxy xanthones, and F137 is a 1,3,7-hydroxy xanthone. Each experiment was performed three times.

Figure 3

Cytotoxicity and reactive oxygen species (ROS) production. (a) Hydrogen peroxide (H₂O₂) demonstrated cytotoxicity and (b) stimulated ROS production in periodontal ligament stem cells. (c) The three C. cochinchinensis derivatives, F6, F8, and F137, did not demonstrate cytotoxicity at concentrations below 50 µg/mL. F6 and F8 are geranyloxy xanthones, and F137 is a 1,3,7-hydroxy xanthone. Each experiment was performed three times. *, **, and *** indicate significance at p < 0.05, 0.001, and 0.0001, respectively.

Figure 4

Antioxidant effects of the C. cochinchinensis derivatives on oxidative stress-induced periodontal ligament stem cells. H₂O₂-induced oxidative stress resulted in increased intrinsic reactive oxygen species, while combining the treatment with the C. cochinchinensis derivatives, F6, F8, and F137, demonstrated the opposite effect. F6 and F8 are geranyloxy xanthones, and F137 is a 1,3,7-hydroxy xanthone. Each experiment was performed three times. **, and *** indicate significant differences at p < 0.05, 0.001, and 0.0001, respectively.

Figure 5

Effects of the C. cochinchinensis derivatives on HO-1 and Nrf2 expression in PDLSCs. (a) The three derivatives, F137, F8, and F6, at 25 µg/mL upregulated HO-1 and Nrf2 expression in PDLSCs. (b) HO-1 and Nrf2 were significantly increased in cells treated with F8. * and ** indicate significance at p < 0.05 and 0.001, respectively. F6 and F8 are geranyloxy xanthones, and F137 is a 1,3,7-hydroxy xanthone. Each experiment was performed three times.

Figure 6

PDLSCs osteogenic differentiation after being treated with H₂O₂ and the three C. cochinchinensis derivatives for 7 and 21 days. (a) ALP activity evaluation after 7 days demonstrated that H₂O₂ treatment decreased ALP activity and that the treatment with each derivative and an oxidizing agent, H₂O₂, enhanced ALP activity. (b) The mineralization assay after 21 days demonstrated that each derivative and the positive control exhibited significantly more calcified nodules compared with H₂O₂ treatment. (c) During calcification, H₂O₂ impeded the formation of calcified nodules, while the xanthone derivative treatments improved the osteogenic differentiation and mineralization of PDLSCs under H₂0₂-induced oxidative stress. Each experiment was performed three times. ALP activity was analyzed using the multiple t-test, and a p-value ≤ 0.05 was considered significant. Percent mineralization was analyzed with one-way analysis of variance (ANOVA) followed by Turkey’s multiple comparisons. *,**^/##, ***^/### indicate significance with H₂O₂ (*) or negative control (#) at p < 0.05, 0.001 and 0.0001, respectively.