A quick and convenient 1H quantitative NMR method for determination of bioactive pyranocoumarins from Clausena excavata

Clausena excavata, a shrub in the Rutaceae family, has been used in traditional medicine to alleviate cold, malaria, abdominal pain, bacterial infection, and HIV infection. The major biologically active constituents in the rhizomes and roots of C. excavata are the three pyranocoumarins; clausenidin, dentatin, and nordentatin. Due to the medicinal values of this plant, we have developed a quick and convenient qHNMR method to quantify clausenidin, dentatin, and nordentatin from the rhizomes and roots of C. excavata collected from six geographical locations throughout Thailand. The dried and powdered samples were extracted with deuterated methanol. The parameters for 1H NMR measurements were optimized according to the measured T1 relaxation time of the protons from the bioactive pyranocoumarins and of 2,3,5,6-tetrachloronitrobenzene (TCNB), the internal standard. HPLC assays were also conducted to verify the results obtained from the qHNMR method. The amount of clausenidin, dentatin, and nordentatin varied from different geographical locations, and the results from the qHNMR method were comparable to those obtained from the HPLC assays. The reported qHNMR method was quick, more convenient, and more applicable to large-scale quality control or screening than conventional HPLC methodology. The method developed herein is beneficial to the pharmaceutical and health industries.