Phytochemical characterization of major constituents in Thai traditional formulation for knee poultice (Ya-Pok-Dud-Pid) and their inhibition of nitric oxide production activity

Abstract

Importance of the work: The Thai traditional formulation for a knee poultice (Ya-Pok-Dud-Pid) has
been used for the treatment of knee inflammation. Basic information is required for quality assessment
of this traditional approach, regarding its major active components with anti-inflammatory activities.
Objectives: To isolate and identify the major components of a Ya-Pok-Dud-Pid formulation from
the plant ingredients and to investigate the inhibitory effects on the lipopolysaccharide-induced nitric
oxide release of its ingredients and isolated compounds.
Materials and Methods: The tincture of the Ya-Pok-Dud-Pid formulation and methanolic extracts
of each plant sample were subjected to analysis using ultra-high performance liquid chromatography
coupled with diode array detection, as well as using thin-layer chromatography. The major components
were isolated and their chemical structures were elucidated. Inhibitory effects on lipopolysaccharideinduced nitric oxide release were investigated using the murine macrophage RAW 264.7 cell line.
Results: The phytochemical investigation resulted in the identification of (E)-4-(3’,4’-dimethoxyphenyl)
but-3-en-1-ol (compound D, 1), (E)-4-(3’,4’-dimethoxyphenyl)but-3-en-1-yl acetate (D-acetate,
2), (E)-1-(3’,4’-dimethoxyphenyl)butadiene (DMPBD, 3), piperine (4), zerumbone (5), zerumbone
epoxide (6), 6’,7’-dihydroxybergamottin (7), oxypeucedanin hydrate (8), curcumin (9), β-asarone
(10), colchicine (11), plumbagin (12) and 6-gingerol (13). The Ya-Pok-Dud-Pid extract was antiinflammatory, with a half maximal inhibitory concentration (IC50) of 59.86 µg/mL. Based on the
crude 40% ethanolic extracts, Zingiber zerumbet had the highest activity with an IC50 of 15.45 µg/mL,
followed by Crinum asiaticum, Curcuma aromatica, Zingiber officinale, Zingiber cassumunar and
Globba variabilis. Furthermore, zerumbone (5) had the highest activity with an IC50 of 0.88 µg/mL
followed by plumbagin (12), curcumin (9), zerumbone epoxide (6), colchicine (11), DMPBD (3),
piperine (4), D-acetate (2) and compound D (1), while the NG-methyl-L-arginine acetate salt (positive
control) had an IC50 of 17.16 µg/mL.
Main findings: The active components were identified in the Ya-Pok-Dud-Pid formulation, which
could provide chemical markers for quality assessment and contribute valuable information for further
pharmaceutical development.