## 2 Sources of Tanshinones, Preparative and Analytical Methodologies
### Biosynthesis of Tanshinones in S. miltiorrhiza  
Terpenes or terpenoids in higher plants are synthesized through at least two distinct pathways, the mexulonic acid (MVA) pathway occurring in the cytosol and the non-MVA or 2-\(C\)-methyl-d-erythnitol-4-phosphate (MEP) pathway occurring in the plastids [42]. Tanshinones are generally considered to be synthesized via the MEP pathways, which involve and generate the monoterpenes, certain sesquiterpenes, diterpenes, carotenoids, and the side chains of chlorophylls and plastoquinone. The MVA pathway is responsible for the synthesis of sterols, certain sesquiterpenes, and the side chain of ubiquinone [43; 44; 45]. However, the MVA pathway might also play a role in the synthesis of tanshinones for the extensive crosstalk between the two pathways has been reported [46; 47]. In MEP pathways, 1-deoxy-d-xylulose-5-phosphate synthase (DXS) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) have been identified as the first two enzymes [49; 50]. DXS  
Figure 2: Proposed biosynthetic pathways of tanshinones in _Salvia miltiorrhiza_[48]. Modified based on [PERSON] & [PERSON] 2010. By permission of [PERSON]. catalyzed pyruvate and glyceraldehyde-3-phosphate (GA-3P) into 1-deoxy-d-xylulose-5-phosphate (DXP), which is consecutively converted into MEP through the action of DXR. Then MEP undergoes a series of conversion and results in the formation of isopentenyldiphosphate (IPP) and dimethylallyldiphosphate (DMAPP), the universal precursors of more than 30,000 terpen [33,51]. The next step after the synthesis of IPP and DMAPP is chain elongation. By reacting with carbonium ion, IPP leads to the production of geranyldiphosphate (GPP). GPP, with an active allylic phosphate group, would reversely react with IPP to produce farnesyl pyrophosphate (FPP) [52]. Then, geranylgeranyldiphosphate synthase (GGPPS) catalyzes the consecutive condensation of three IPP molecules with DMAPP to give a C\({}_{20}\) compound, geranylgeranyldiphosphate (GGPP), an essential linear precursor for the biosynthesis of diterpenes [53] (Figure 2). GGPP could be transformed into labdedienyl/copalydiphosphate (CPP) through CPP synthase, followed by the conversion of CPP to miltiradiene catalyzed by CPP-specific class I diterpene synthase termed as kaurene synthase-like (KSL) [54]. [PERSON] and [PERSON] proposed a hypothetical biosynthetic pathway in which miltiradiene was a key intermediate (Figure 2) [48]. Aromatization and hydroxylation, due to the unstable cyclohexan-1,4-dienes on C ring of miltiradiene, led to ferruginol, which sequentially produced miltirone and neocryptotanshinone by the installation of other groups. Neocryptotanshinone would stepwisely convert into CT, TIIA, TIIB and TI, and probably DH-TI (Figure 2). _2.3 Botanical and Alternative Sources of Tanshinones_  
To date, more than 40 tanshinones or structurally related compounds have been identified from _S. miltiorrhiza_. Many of them have also been identified from related _Salvia_ species, which include _S. argentea_, _S. aerea_, _S. bowleyana_, _S. bulleyana_, _S. campanulata_, _S. castanea_, _S. columboriae_, _S. constanea_, _S._ deserta_, _S. dabieshanensis_, _S. evansiana_, _S. flava_, _S. honania_, _S. digitaloides_, _S. kiaometiensis_, _S. kiangsisensis_, _S. meiliensis_, _S. paramilitorhiza_, _S. plectranthoides_, _S. sinica_, _S. trijuga_, _S. prionitis_, _S. przewalskii_, _S. vasta_, and _S. yunnanensis_[55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66]. Although _S. miltiorrhiza_ was scribed the only official source of Danshen, 17 other _Salvia_ species, such as _S. przewalskii_, _S. yunnanensis_, _S. bowleyana_ and _S. trijuga_, have been collected, traded, and used in China herbal markets as substitutes [67,68]. _S. miltiorrhiza_ grows in the hilly area of China, Mongolia, Korea, and Japan. Because wild _S. miltiorrhiza_ became in short supply due to rapidly growing demanding and over-harvesting, it has been extensively cultivated since the 1970s [69].