## 2 Sources of Tanshinones, Preparative and Analytical Methodologies  
### Isolation, Purification and Analytical Methodologies  
Tanshinones were first isolated in 1934 from the intensely red rhizomes (roots) of _S. miltiorrhiza_[3]. Their isolation from this and other _Salvia_ species usually involved conventional methanolic extraction (MeOH) method. The crude extract was subjected to silica gel column chromatography, using CH\({}_{2}\)Cl\({}_{2}\)-MeOH mixture as elution solvent. Each fraction was re-chromatographed to enhance the concentration by using a gradient of C\({}_{6}\)H\({}_{6}\)-MeOH mixture as the mobile phase. Pigments from each fraction were further purified through recrystallization and preparative thin-layer chromatography [18, 19, 20]. Thin-layer chromatography (TLC) was reported as a rapid, sensitive, and accurate method in quantitative determination of both aqueous and lipophilic compounds of _S. miltiorrhiza_ and could be employed in quality control of Danshen production [21, 22, 23]. Figure 1: Chemical structures of major tanshinones and tanshinlactones. High-performance liquid chromatography (HPLC) has been used to simultaneously determine both the aqueous phenolic and non-polar diterpenoid constituents of Danshen products with diode array (DAD) and/or evaporative light scattering (ESL) detectors [10,24,25]. HPLC coupled with electrospray ionization quadrupole ion trap mass spectrometry (HPLC/ESI-IT-MS) or electrospray ionization time-of-flight mass spectrometry (HPLC/ESI-TOF-MS) has been used to provide complementary information for HPLC-DAD by differentiating the isotopic components in Danshen products [9,26]. More recent reports of ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC/qTOF-MS) showed better qualitative and quantitative analyses of diterpenoids from 12 _Salvia_ species regarding resolution, sensitivity, reproducibility and structural information to differentiate positional isomers [27]. Countercurrent chromatography (CCC) is a liquid-liquid partition chromatographic technique with a support-free liquid stationary phase [28, 29, 30]. Compared with the classical liquid chromatography (LC), CCC is advantageous for no loss due to irreversible solute absorption, no surface interference including contamination, surface silanol reaction and deactivation, no restriction to flow speed, and able to handle more sample quantities, making it an ideal technique for the separation and purification of natural products [30]. In 2000, [PERSON] and co-workers reported separation of TI, TIIA and CT through high-speed counter-current chromatography (HSCCC) in a single run by using stepwise elution [31]. Multidimensional HSCCC successfully isolated and purified a set of tanshinone analogs including TI, TIIA, DH-TI and CT [32]. Similar result was also observed by using HSCCC with two-phase solvent systems composed of \(n\)-hexane-ethanol-water to obtain relatively pure compounds including CT, TIIA, TI, DH-TI, methylenetanshiquinone and danshenxinkun B in a stepwise elution mode [33]. In a recent study, DH-TI, 1,2,15,16-tetrahydrotanshiquinone, CT, TI, TIIA, neo-przewaquinone A and miltirone were obtained in an one-step separation by HSCCC with light petroleum-ethyl acetate-methanol-water as the two phase solvent system [34]. Furthermore, a gradient elution model has been developed for CCC systems and was successfully used to separate DH-TI, CT, TI, TIIA and 1,2-dihydrotanshinquinone with a biphasic liquid system composed of hexane-ethyl acetate-ethanol-water [35]. Additionally, chemical fingerprints of _S. miltiorrhiza_ were developed to identify major active components by using HSCCC [7,36]. In recent years, the development and application of molecularly imprinted polymers (MIPs) have attracted increasing interest [37]. MIP is a synthetic polymer with molecular recognition sites, which is prepared using molecular imprinting approach.