Chondroprotective and anti-inflammatory effects of ChondroT, a new complex herbal medication

Abstract

Background: Ganghwaljetongyeum (GHJTY) is a complex herbal decoction comprising 18 plants; it is used to treat arthritis. In order to develop a new anti-arthritic herbal medication, we selected 5 out of 18 GHJTY plants by using bioinformatics analysis. The new medication, called ChondroT, comprised water extracts of Osterici Radix, Lonicerae Folium, Angelicae Gigantis Radix, Clematidis Radix, and Phellodendri Cortex. This study was designed to investigate its chondroprotective and anti-inflammatory effects to develop an anti-arthritic herb medicine.

Methods: ChondroT was validated using a convenient and accurate high-performance liquid chromatography-photodiode array (HPLC-PDA) detection method for simultaneous determination of its seven reference components. The concentrations of the seven marker constituents were in the range of 0.81-5.46 mg/g. The chondroprotective effects were evaluated based on SW1353 chondrocytes and matrix metalloproteinase 1 (MMP1) expression. In addition, the anti-inflammatory effects of ChondroT were studied by Western blotting of pro-inflammatory enzymes and by enzyme-linked immunosorbent assay (ELISA) of inflammatory mediators in lipopolysaccharides (LPS)-induced RAW264.7 cells.

Results: ChondroT enhanced the growth of SW1353 chondrocytes and also significantly inhibited IL-1β-induced MMP-1 expression. However, ChondroT did not show any effects on the growth of HeLa and RAW264.7 cells. The expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) was induced by LPS in RAW264.7 cells, which was significantly decreased by pre-treatment with ChondroT. In addition, ChondroT reduced the activation of NF-kB and production of inflammatory mediators, such as IL-1β, IL-6, PGE2, and nitric oxide (NO) in LPS-induced RAW264.7 cells.

Conclusions: These results show that ChondroT exerted a chondroprotective effect and demonstrated multi-target mechanisms related to inflammation and arthritis. In addition, the suppressive effect was greater than that exhibited by GHJTY, suggesting that ChondroT, a new complex herbal medication, has therapeutic potential for the treatment of arthritis.

Keywords: Anti-arthritic drug; COX-2; ChondroT; Chondrocyte; Inflammatory cytokines; MMP-1; iNOS.

Fig. 1

Chemical structure of the seven marker compounds (a) and HPLC chromatogram of a standard solution (b) and ChondroT (c) with detection at 310 nm (I), 325 nm (II), 330 nm (III), 335 nm, and 340 nm (V). Chlorogenic acid (1), berberine Cl (2), nodakenin (3), isoferulic acid (4), oxypeucedanin hydrate (5), decursin (6), and decursinol angelate (7)

Fig. 2

Effects of ChondroT on the proliferation of SW1353 chondrocyte cells. a SW1353 cells were exposed to GHJTY or ChondroT for 2 days, and the cell proliferation was assayed by MTS. ChondroT increased the proliferation of SW1353 cells at concentrations of 0.1 ~ 1.0 mg/mL. HeLa cells (b) or RAW264.7 cells (c) were treated with GHJTY or ChondroT for 2 days, and the cell proliferation was assayed by MTS. ChondroT did not show any cytotoxic effect on HeLa and RAW264.7 cells. ^* P < 0.05 and ^*** P < 0.001 compared to the untreated group

Fig. 3

Effects of ChondroT on MMP1 expression in IL-1β- or PMA-activated SW1353 cells. SW 1353 cells were pretreated with or without ChondroT or GHJTY at a concentration of 0.3 mg/mL for 1 h and were then stimulated with PMA (a) or IL-1β (b). After 24 h, the MMP1 level was detected in cell culture supernatants using Western blot analysis. ChondroT reduced MMP1 expression in IL-1β- or PMA-activated SW1353 cells

Fig. 4

Effects of ChondroT on the induction of COX-2 and iNOS in LPS-activated RAW264.7 cells. RAW264.7 cells were pretreated with ChondroT for 2 h, and then LPS was added to the cells for 18 h. Beta actin was used as the control protein. The protein levels of COX-2 (a) and iNOS (b) were detected by Western blot analysis. Celecoxib (Cel) and ChondroT reduced the expression of COX-2 and iNOS in LPS-activated RAW264.7 cells

Fig. 5

Effects of ChondroT on the production of pro-inflammatory cytokines in LPS-activated RAW264.7 cells. RAW264.7 cells were pretreated with ChondroT for 2 h and then incubated with LPS for 24 h. Inflammatory mediators were measured in the cell supernatants by ELISA. ChondroT decreased the LPS-induced production of IL-1β (a), IL-6 (b), and PGE₂ (c). NO in the supernatant was detected by Griess reagent (d). LPS-induced NO production in RAW264.7 cells was decreased by ChondroT treatment. ⁺ P < 0.01 and ⁺⁺⁺ P < 0.001 compared with the untreated group. ^* P < 0.05, ^** P < 0.01, and ^*** P < 0.001 compared with the LPS-treated group

Fig. 6

Effects of ChondroT on PMA-induced NF-kB activation in 293T cells. The 293T cells were transfected with a reporter plasmid, pNF-kB-SEAP. PMA-induced NF- kB transcription, which was inhibited by ChondroT at a concentrations of 0.3 and 0.1 mg/mL. ^* P < 0.05 and ^** P < 0.01 compared with the PMA-treated group

Fig. 7

Anti-oxidant activity of ChondroT. ChondroT and other drugs dissolved in methanol were mixed with DPPH (0.15 mM in methanol) in 96-well plate for 30 min and then the absorbance was measured at 470 nm using an ELISA microplate reader. BHA and vitamin C were used as positive anti-oxidant drugs. DPPH free radicals were decreased by approximately 95 % at 0.3 mg/mL ChondroT, and the inhibitory effect was greater than that exhibited by GHJTY. ^*** P < 0.001 compared with the control group