# The Potential Role of Timosaponin-AIII in Cancer Prevention and Treatment
## 2 Chemical Structure, Biotransformation, and Structure-Activity Relationship of TSAIII  
TSAIII (Figure 1) has an IUPAC name: (ES3,R4S,S5,6R)-2-{(2R,3R,4S,5R,6R)- 4,5-dihydroxy-6-(hydroxymethyl)}-2-{(1R,2S,4S,5S,6R,7S,8R,9S,12S,13S,16S,18R)-5^7,7,9,13-tetramethylspiro [5-oxapentacyclo [10.8.0.02,9.04.8,013,18]icosane-6,2^-oxane]-16-yl] oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol), which is a molecular formula of C39H64O13, and it has a molecular weight of 740.92 g/mol. TSAIII is a white to off-white solid with various pharmacological activities and is soluble in methanol, butanol, 80% ethanol, and aqueous pentanol; however, it is insoluble in water.  
It has been reported that the content of TSAIII in the rhizomes of _A. asphalodeloides_ is approximately 0.19-0.28% [17]. However, another study reported that the content of TSAIII is too low to be detected in a Zhimu/Baihe formula [18]. Interestingly, TSBII has a much higher level and can be transformed into TSAIII. \(\beta\)-D-glycosidase, as a key enzyme, has been used to produce TSAIII by hydrolyzing TSBII (Figure 2) from the crude extract liquid of the rhizomes of _A. asphalodeloides_. Potentially, approximately 7 g of TSAIII can be isolated from 1 kg of the rhizomes of _A. asphalodeloides_ by five-step preparation. The optimal conditions for this include pH4.0, 55 \({}^{\circ}\)C, 2 h, and 600 U/g of \(\beta\)-D-glycosidase [17]. TSBII can be hydrolyzed and isomerized by the fungus _Colletotrichum gloeosporoides_ to generate TSAIII, and TSAIII can also be produced by _Aspergillus niger_[19]. However, TSAIII could be further transformed into sarsasapogenin by removing the sugar chain at the C-3 position  
Figure 1: A picture of _Anemarhena asphalodeloides_ Bunge and the chemical structure of TSAIII.  
(Figure 2); this could induce a low yield of TSAIII [9; 20]. Thus, an efficient industrial process for TSAIII production is essential.  
The sugar chain at the C-3 position in TSAIII is indispensable to its pharmacological effects. The IC\({}_{50}\) values of TSAIII and sarsasapogenin in lowing A\(\beta_{42}\) production in N2A-APPswe cells are 2.3 \(\upmu\)M and 53 \(\upmu\)M, respectively. In addition, disaccharide TSAIII has shown higher activity in reducing the production of A\(\beta_{42}\) compared with monosaccharide TSAI (IC\({}_{50}\) = 6.1 \(\upmu\)M). However, trisaccharide TSAV (IC\({}_{50}\) = 4.2 \(\upmu\)M) exhibits a lower activity than TSAIII [21]. Interestingly, an intact F ring is essential for TSAIII and sarsasapogenin to reduce A\(\beta_{42}\) production [21]. One study reported that an \(\upalpha\)-D-1,4-glucopyranosyl group at the C-4-OH of the glucosyl group in TSAIII could decrease the cytotoxicity of HL60 cells [22]. A structure-activity relationship study showed that the substituents at the C-3 and C-26 positions played a key role in selective anticancer activity [23; 24]. Specifically, linking an amine (N-methyl piperazinyl, piperidyl, piperzinyl, pyrrolidylinyl, N,N-dimethylamino, and N,N-diethylamino) to the C-3 or C-26 position and presenting a methoxyl group at the C-3 position could increase the selective cytotoxic activity of sarsasapogenin. In addition, the opening of the F ring in sarsasapogenin might enhance the antiproliferative activity [23].