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Journal of Thai Traditional & Alternative Medicine Vol. 8 No. 2 May-August (Supplement) 2010 ÚÒ

OP-4

Chondroprotective potential of active compounds of Zingiber cassumunar:

In vitro assay from cartilage explant model to molecular biology

1 1 1 2
Rujirek Chaiwongsa , Siriwan Ongchai , Prachya Kongtawelert , Vichai Reutrakul
1
Thailand Excellence Center for Tissue Engineering, Department of Biochemistry and Center of Excellence for Innova-
tion in Chemistry, Faculty of Medicine, Chiang Mai University, Thailand.
2
Departmen of Chemistry and Center of Excellence for Innovation in Chemistry, Mahidol University, Bangkok, Thailand.

Rationale: Osteoarthritis (OA) and rheumatoid arthritis (RA) are degenerative joint diseases characterized
by progressive loss of articular cartilage, result in joint pain and disability. Zingiber cassumunar has long been
used in Thai traditional medicine for anti-inflammation and anti-pain of joint diseases. Scientific evidence of
activities in reduction of inflammation and pain of the active compounds from Z. cassumunar has been re-
ported. It is therefore interesting to explore whether active compounds of Z. cassumunar contain chondroprotective
activity.
Objective: To investigate the in vitro protective effects of active compounds of Z. cassumunar, (E)-1-
(3',4'-dimethoxyphenyl) butadiene (DMPBD), cis-3-(2',4',5'-Trimethoxyphenyl)-4-{(E)-2ûûû,4ûûû,5ûûû-
trimethoxystyryl}cyclohex-1-ene (CpC) and terpinene-4-ol, on experimentally-induced cartilage destruction us-
ing various approach techniques from cartilage explant model to molecular level of cartilage cells.
Methodology: Articular cartilage was freshly dissected for preparation of cartilage explants and chondrocytes
culture. Cartilage explants or chondrocytes were cultivated in a septic technique for 1-21 days in with or
without IL-1β (7 ng/ml) and the three active compounds at concentration 10 μM. The culture medium was
harvested for analysis of cartilage degradation markers such as sulfated glycosaminoglycan (s-GAG) and hyaluronan
(HA) by Dye binding assay and ELISA technique. The remaining of uronic acid and collagen in cartilage tissues
were analyzed by colorimetric assays. The chondrocytes treated with or without IL-1β 0.5 ng/ml) and DMPBD
(1-100 μM) were analyzed for gene expression of MMP-13, the key enzyme which causes cartilage degradation
in OA and RA, including the intracellular signaling pathway by RT-PCR and Western blotting, respectively.
Enzyme activity in the culture media was analyzed by fluorogenic assay.
Results: The three active compounds showed the protective effects against cartilage explants degrada-
tion induced by IL-1β. They significant preserved content of cartilage matrix biomolecules such as collagen and
UA within the cartilage explants, in concomitance with reduction of s-GAG and HA level in the culture medium
(p<0.05). DMPBD was found the most effective compared to terpinen-4-ol and CpC. In molecular analysis,
DMPBD significant inhibited gene expression of MMP-13 from 24.7% (p<0.05). Activity of this enzyme in the
culture medium was found to reduce by 13.8%. It was found that DMPBD inhibited IL-1 beta-induced the
mitogen-activated protein kinase (MAPK) by selectively reduction of JNK and ERK but not p38. Tissue viability
and cellular toxicity assays indicated that the three active compounds had no toxic effect on the explants and
chondrocytes at the concentration used in this study.
Conclusion: These data revealed chondroprotective potential of Z. cassumunar which indicated by the
inhibitory effects of the active compounds on IL-1beta-stimulated cartilage degradation and expression of
MMP-13, via MAPK pathway.