Isolation and establishment of major curcuminoids as reference standards from <i>Curcuma xanthorrhiza</i> rhizomes

Abstract Curcuma xanthorrhiza , or Java turmeric, is a medicinal plant from the Zingiberaceae family, widely grown in southern Vietnam and traditionally used to treat stomach pain, gastric ulcers, and hepatitis. Phytochemical investigation of the EtOAc extract of C. xanthorrhiza rhizomes was conducted, resulting in the isolation of five curcuminoids, namely curcumin ( 1 ), dihydrodemethoxycurcumin ( 2 ), demethoxycurcumin ( 3 ), letestuianin C ( 4 ), and dihydrobisdemethoxycurcumin ( 5 ). These isolated compounds exhibited various biological activities comprising of cytotoxicity to cancer cells, antibacterial, antioxidant, hypoglycemic, anti‐inflammatory, and triglyceride‐lowering effects. Due to the lack of reference standards for the quality control of C. xanthorrhiza rhizomes, this research effectively developed isolated curcuminoids from this species as reference standards, achieving purity levels between 96% and 98%, based on the standards of ISO and ICH Q2 (R2) guideline. An analytical method was validated for the simultaneous quantification of these compounds using an HPLC Waters Alliance 2998e system and a gradient mobile phase of acetonitrile‐formic acid 0.1%. The method's parameters for quantitative analysis, including system suitability, specificity, linearity, repeatability, and accuracy, were also determined. This study establishes a novel HPLC method for the simultaneous quantification of five curcuminoids from C. xanthorrhiza rhizomes, offering a reliable approach for quality control and product development.