| Summary: | Chemical profiling for quality monitoring and evaluation of medicinal plants is gaining attention. This study aims to develop an HPLC method followed by multivariate analysis to obtain HPLC profiles of five specific flavonoids, including rutin (1), hyperin (2), isoquercitrin (3), quercitrin (4), and quercetin (5) from <i>Houttuynia cordata</i> leaves and powder products and assess the quality of <i>H. cordata</i> samples. Eventually, we successfully established HPLC-based flavonoid profiles and quantified the contents of 32 <i>H. cordata</i> fresh leave samples and four powder products. The study also quantified the contents of those five essential flavonoids using an optimized RP-HPLC method. Peak areas of samples were then investigated with principal component analysis (PCA) and hierarchical cluster analysis (HCA) to evaluate the similarity and variance. Principal components in PCA strongly influenced by hyperin and quercetin showed that the samples were clustered into subgroups, demonstrating <i>H. cordata</i> samples’ quality. The results of HCA showed the similarity and divided the samples into seven subgroups. In conclusion, we have successfully developed a practical methodology that combined the HPLC-based flavonoid profiling and multivariate analysis for the quantification and quality control of <i>H. cordata</i> samples from fresh leaves and powder products. For further studies, we will consider various environmental factors, including climate and soil factors, to investigate their effects on the flavonoid contents of <i>H. cordata</i>.
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