Science based authentication of dietary supplements---studies on the chemical constituents, analytical method and biological activities of Scutellaria spp and Pfaffia paniculata Kuntze

The primary objective of this project is to explore the chemical constituents of American skullcap, (Scutellaria lateriflora), which will be used as reference markers to develop a validated chemical fingerprinting method for the differentiation of it from its adulterant, germander. From the MeOH extract of the aerial parts of this plant sixteen compounds, including three minor coumarins in which two are new, six flavonoids, four triterpenes, two steroids, and one fatty acid were isolated and characterized. In order to compare the similarity of chemical components between American skullcap and Chinese skullcap (S. baicalensis), also to obtain more marker compounds, the chemical components of Chinese skullcap, which is the most often used herb medicine in this genus, were also studied and seventeen flavonoids have been isolated. Based on the chemical constituents, a simple HPLC-PDA fingerprinting method was developed, which can be used to authenticate S. lateriflora species, to distinguish it from its potential adulterants, to compare the chemical profiles of related species in this genus, and to assess the quality of commercial products claiming to contain S. lateriflora. In the present study three classes of compounds, including eleven flavonoids isolated from two Scutellaria species mentioned above and two phenylpropanoids, as well as one diterpene isolated from T. canadense, were used as markers simultaneously to provide comprehensive analysis for the above mentioned purposes. As part of the efforts of this project, a HPLC/ESI-MSn method was established which allowed a fast characterization of thirteen flavonoids and one stilbene derivative from American skullcap without time-consuming isolation of compounds even if reference compounds are not available. The biological activities of the two skullcap extracts and thirteen flavonoids isolated from them were evaluated on a panel of bioassays, including cytotoxicity, antioxidant, anti-inflammatory, estrogenic activity and activations on peroxisome proliferator-activated receptor-alpha and -gamma (PARPalpha and PPARgamma). Neither skullcap extracts nor pure compounds tested showed cytotoxicity against a panel of mammalian normal and solid tumor cell lines at the tested concentrations. During the investigation of chemical constituents of American skullcap no neoclerodane diterpenes, which are responsible for the hepatotoxicity in germander (Kouzi et al., 1994), were found. Taken together, these evidences suggest that the reports of hepatotoxic reactions on American skullcap might very likely be due to its adulterant germander. In the present bioassay study, the most significant finding was the identification of PPARs activation of skullcap extracts as well as several characteristic flavonoids, such as baicalein, baicalin, wogonin, and chrysin etc. that provides some information on possible new application of skullcap, especially American skullcap, for the treatment of diabetic-related diseases. Also the potent activity of these flavonoids on PPARs suggests the potential of these flavonoids as leads for PPARalpha and PPARgamma agonists.;P. paniculata Kuntze, belonging to the family Amaranthaceae, is a large, rambling, shrubby ground vine with an intricate, deep, and extensive root system. It is the most employed species in commercial preparations in Brazil as "Brazilian ginseng" and has been commonly used for three centuries with the same indications as American and Asian ginseng. More than fifty preparations, which contained or were produced from plant P. paniculata are available on USA market, but no analytical method(s) existed to assess the quality of P. paniculata raw materials, and the products claiming to contain P. paniculata. In order to develop an appropriate validated quantitative analytical method for the quality control of this plant material and dosage form the detailed phytochemical investigation of P. paniculata was carried out. Two new nortriterpenoids pfaffine A and B, one new compound pfaffine C, along with eleven known compounds were isolated and identified from the roots of this plant. These compounds will be used as markers for quality control of raw materials of P. paniculata as well as products claiming to contain this species. (Abstract shortened by UMI.)...