Systematic Separation, Purification And Characterization Of The Chemical Constituents Of Tibetan Medicine

Lycium ruthenicum Murray is a kind of widespread nutritional food in salinized desert of Qinghai-Tibet Plateau. It is a famous traditional herb recorded in Tibetan medical classic "Jing Zhu Ben Cao". L. ruthenicum has been applied to treat many diseases such as heart disease, abnormal menstruation and menopause. Reportedly, L. ruthenicum contains abundant anthocyanins, which have aroused great interest to many researchers. Up to now, the chemical compositions of this plant are still unclear, which has been a great obstacle for the development of L. ruthenicum.Anthocyanins, a group of phenolic compounds, confer the characteristic colors to most fruits and vegetables. Increased evidences have proved their possible heath benefits in preventing chronic and degradative diseases including heart disease and cancer. High performace liquid chromatography (HPLC) has been widely used to isoalte anthocyanins in natural plants. However, the current HPLC method for the purification of anthocyanins suffers from limited separation selectivity. The separation of anthocyanins with HPLC remains to be further improved.In this paper, we aim to systematically investigate the chemical components of L. ruthenicum. Firstly, based on the novel stationary phases, serverals methods have been developed for the selective isolation of anthocyanins in L. ruthenicum. Secondly, the alkaloid and acidic/neutral compositions in this plant were efficiently purified using multidimensional liquid chromatography technique. Finally, the bioactivities of the isolated compounds were explored with cellular label-free integrative pharmacology profiling (CLIP).A mixed-mode chromatography method was developed to separate anthocyanin isomers from L. ruthenicum, based on a mixed-mode reversed phase/strong anion-exchange column (named XCharge C8SAX). The XCharge C8SAX column performed improved selectivity and remarkable column efficiency for the isomers. Six pure anthocyanins, including two pairs of cis-trans isomeric anthocyanins with one new anthocyanin, were purified from L. ruthenicum. This method was an effective way to separate anthocyanins, especially for cis-trans isomers.A hydrophilic interaction chromatography method was established to efficiently purify a challenging anthocyanin in Lycium ruthenicum Murray, based on a zwitterionic Click XIon column. This method solved the coelution problem of the anthocyanin and basic non-anthocyanins in one-dimensional HPLC, benefiting from different separation selectivity.Systematic characterization of anthocyanins in L. ruthenicum was carried out by HPLC-DAD/QTOF-MS/MS. A spectral rule was first proposed from the UV/Vis spectra of two pairs of cis-trans anthocyanin isomers. And this rule provided important information on identification of the isomers. Sixteen anthocyanins in L. ruthenicum were tentatively characterized, and seven of them were reported in this plant for the first time.In total, ten non-anthocyanins were obtained, including seven amide alkaloids, two phenolic acids and one flavonoid. These compounds were all separated from L. ruthenicum for the very first time. Besides, it was found that petunidin-3-cis-p-coumaroylrutinoside-5-glucoside had superior bioactivity, compared to other isolated anthocyanins. We speculated that the structure of anthocyanins had significant impact on the activity.