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Journal of Thai Traditional & Alternative Medicine Vol. 7 No. 2 May-August (Supplement) 2009 ÒÛ˘

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Cyclooxygenase assays and molecular modeling study of some chemical

constituents from Zingiber cassumunar

1 2 2 3
Prasan Tangyuenyongwatana , Nipa Jongkon , Chak Sangma , Wandee Gritsanapan
1
Faculty of Oriental Medicine, Rangsit University, Pathumtani 12000, Thailand.
2
Department of Chemistry, Faculty of Science, Kasetsart University Bangkok 10900, Thailand.
3
Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand.
Rationale: The rhizome of Zingiber cassumunar Roxb. has been used in Thai traditional preparations
especially Prasaplai, a preparation for treatment of dysmenorrhea and adjusting the menstrual cycle. The
chemical constituents of Z. cassumunar are isolated for a long time and some in vitro and in vivo studies have
been reported however few studies work on cyclooxygenase enzyme. In this study, we are interested in the
inhibition properties of some active anti-inflammatory constituents in Z. cassumunar hexane extract with
cyclooxygenase-1 and 2. The molecular modeling study has been carrying out with AutoDock 3.0.5.
Objective: To study cyclooxygenase-1 and 2 inhibition activity of some chemical constituents from Zingiber
cassumunar Roxb. and perform molecular modeling of each compound with AutoDock 3.0.5
Methodology: The immortalized mouse PGH-1 and PGH-2 null cells were used as the COX-1 and COX-2
deficient cell lines, respectively to investigate the inhibition activities and selectivity of (E)-4-(3û,4û-dimthoxyphenyl)
butadiene (DMPBD), (E)-4-(2û,4û,5û-trimethoxyphenyl) butadiene (TMPBD), and (E)-4-(3û,4û-dimethoxyphenyl) but-
o
3-en-1-ol (compound D). Cell were incubated at 37 C in humidified incubator with 5% CO2 for 72 h, then
washed with DMEM medium and preincubated for 30 min with 83 (l of serum free DMEM medium containing
test compound. Following the preincubation period, the medium was removed and the cells were immediately
treated with serum-free medium containing test compound and 20 μM arachinodic acid (AA) for 30 min.
Culture supernatant was then collected from wells and analyzed for PGE2 concentration by radioimmunoassay
(RIA). The molecular modeling performed by converting 2D structure of selected compounds to 3D structures
using CORINA program and the geometries optimization were carry out with GAMESS package. Crystal
structure of COX-1 (1-EQH) and COX-2 (1CX-2) were obtained from Brookhaven Protein Data Bank (www.rcsb.org).
Binding conformation of selected compounds with COX-1 and COX-2 were performed by AutoDock 3.0.5 (http/
/autodock.scripp.edu/).
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Results: DMPBD showed 76 % inhibition on COX-1 at concentration of 1 x 10 g / mL while the control
aspirin showed 78 % inhibition at the same concentration. For COX-2 inhibition, DMPBD showed 60 % inhibi-
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tion on COX-2 at concentration of 1 x 10 g / mL while the control aspirin showed 89 % inhibition at the same
concentration. From the results DMPBD has the selectivity ratio of COX-1 / COX-2 equal to 1.27. TMPBD
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showed selective inhibited on COX-1 which had 67 % inhibition at concentration of 1 x 10 g / mL and showed
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41 % COX-2 inhibition at concentration of 1 x 10 g / mL in which the potency less than 50 % inhibition is
suggest to be inactive for COX-2. The selectivity ratio of COX-1 / COX-2 is equal to 1.63. Compound D which
is expected to be a good COX-inhibitor showed inactive result on the assays. From COX inhibition assay,
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compound D showed 24 % inhibition on COX-1 at concentration of 1 x 10 g / mL and showed 25 % COX-2
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inhibition at concentration of 1 x 10 g / mL. The possible explanation for the incongruity result is compound
D may bind with the proteins on the surface of cell lines and only small amount of the substance enter into the
active site. The other possibility is compound D may exhibit the anti-inflammatory activity via other pathways
such as the NF- (B pathway or nitric oxide (NO) pathway. The molecular modeling of the first two compound
seemed to follow the above results while compound D showed a good alignment in COX-1 and COX-2 pockets
which were contradicted with the assay result.
Conclusion: DMPBD and TMPBD showed both COX-1 and COX-2 inhibitions with the ratio closed to 1
which is suggested to be non-selective anti-inflammatory agents. The molecular modeling results were also

supported. However, mechanism of compound D, may need further investigation.