Bioactive fractions and compound of Ardisia crispa roots exhibit anti-arthritic properties mediated via angiogenesis inhibition in vitro

Abstract

Background: Ardisia crispa (Thunb.) A.DC (Primulaceae), is a medicinal herb traditionally used by Asian people as remedies to cure inflammatory related diseases, including rheumatism. The plant roots possess various pharmacological activities including antipyretic, anti-inflammation and antitumor. Previous phytochemical studies of the plant roots have identified long chain alkyl-1,4-benzoquinones as major constituents, together with other phytochemicals. Hexane fraction of the plant roots (ACRH), was previously reported with anti-angiogenic and anti-arthritic properties, while its effect on their anti-arthritic in vitro, is yet unrevealed. Considering the significance of angiogenesis inhibition in developing new anti-arthritic agent, thus we investigated the anti-arthritic potential of Ardisia crispa roots by suppressing angiogenesis, in vitro.

Methods: Ardisia crispa roots hexane extract (ACRH) was prepared from the plant roots using absolute n-hexane. ACRH was fractionated into quinone-rich fraction (QRF) and further isolated to yield benzoquinonoid compound (BQ), respectively. In vitro experiments using VEGF-induced human umbilical vein endothelial cells (HUVECs) and IL-1β-induced human fibroblast-like synoviocytes for rheumatoid arthritis (HFLS-RA) were performed to evaluate the effects of these samples on VEGF-induced HUVECs proliferation and tube formation, and towards IL-1β-induced HFLS-RA proliferation, invasion, and apoptosis, respectively. Therapeutic concentrations (0.05, 0.5, and 5 μg/mL) tested in this study were predetermined based on the IC₅₀ values obtained from the MTT assay.

Results: ACRH, QRF, and BQ exerted concentration-independent antiproliferative effects on VEGF-induced HUVECs and IL-1β-induced HFLS-RA, with IC₅₀ values at 1.09 ± 0.18, 3.85 ± 0.26, and 1.34 ± 0.16 μg/mL in HUVECs; and 3.60 ± 1.38, 4.47 ± 0.34, and 1.09 ± 0.09 μg/mL in HFLS-RA, respectively. Anti-angiogenic properties of these samples were verified via significant inhibition on VEGF-induced HUVECs tube formation, in a concentration-independent manner. The invasiveness of IL-1β-induced HFLS-RA was also significantly inhibited in a concentration-independent manner by all samples. ACRH and BQ, but not QRF, significantly enhanced the apoptosis of IL-1β-induced HFLS-RA elicited at their highest concentration (5 μg/mL) (P < 0.05).

Conclusions: These findings highlight the bioactive fractions and compound from Ardisia crispa roots as potential anti-arthritic agents by inhibiting both HUVECs and HFLS-RA's cellular functions in vitro, possibly mediated via their anti-angiogenic effects.

Keywords: Angiogenesis; Ardisia crispa; Human fibroblast-like synoviocytes for rheumatoid arthritis; Human umbilical vein endothelial cells; Rheumatoid arthritis.

Fig. 1

Concentration-response graphs showing the antiproliferative effect of a ACRH, b QRF, and c BQ towards VEGF-induced human umbilical vein endothelial cells (HUVECs) after 24 h of incubation. Data were expressed as percentage mean ± SEM (n = 3) of viable cells normalized to 100% cell viability in the negative control and were analyzed using one-way ANOVA followed by Tukey HSD post-hoc test. Significant differences (P < 0.05) are indicated by different letters between different concentrations

Fig. 2

Concentration-response graphs showing the antiproliferative effect of a ACRH, b QRF, and c BQ, respectively towards IL-1β-induced human fibroblast-like synoviocytes for rheumatoid arthritis (HFLS-RA) after 24 h of incubation. Data were expressed as percentage mean ± SEM (n = 3) of viable cells normalized to 100% cell viability in the negative control and were analyzed using one-way ANOVA followed by Tukey HSD post-hoc test. Significant differences (P < 0.05) are indicated by different letters between different concentrations

Fig. 3

a Representative fluorescent images (40 × magnification) showing inhibitory activities of ACRH, QRF, and BQ on HUVECs tube formation after 16 h treatment. b Quantitative data of VEGF-induced HUVECs tube formation post treatments with all samples for 16 h. Data were represented as the percentage of tube lengths relative to negative control (normalized to 100% tubular formation) and expressed as mean ± SEM (n = 3). Data were analyzed using one-way ANOVA followed by Tukey HSD post-hoc test. Significant differences (P < 0.05) are indicated by different letters between different groups

Fig. 4

a Illustrated micrographs (10 × magnification) showing anti-invasive effects of ACRH, QRF, and BQ on HFLS-RA invasion. b Quantitative data of HFLS-RA cell invasion post treatments with all samples for 22 h. Data were represented as the percentage of invaded cells relative to negative control (at 100% invading cells) and expressed as mean ± SEM (n = 3). Data were analyzed using one-way ANOVA followed by Tukey HSD post-hoc test. Significant differences (P < 0.05) are indicated by different letters between different groups

Fig. 5

The dot plots showing the cell distribution of IL-1β-induced HFLS-RA as determined by Annexin V-PI staining. The cells were induced with IL-1β (10 ng/mL) and concurrently exposed to various concentrations (0.05, 0.5, and 5 μg/mL) of ACRH, QRF, and BQ respectively for 24 h