Preparation of highly purified timosaponin AIII from rhizoma anemarrhenae through an enzymatic method combined with preparative liquid chromatography

Abstract Timosaponin AIII (TAIII) exhibits extensive pharmacological activities and has been reported as a potent antitumour agent for various human cancers. In the present study, a potential industrial process for producing TAIII that involves biotransformation directly in the crude extract liquid of rhizoma anemarrhenae (RA) was developed. β-D-glycosidase was used to transform timosaponin BII (TBII) into TAIII, and monofactor experiments were conducted to optimise the enzymolysis conditions. In addition, AB-8 macroporous resin column chromatography, preparative liquid chromatography, and crystallisation technique were applied for yielding TAIII crystals with a purity > 97%. Approximately, 7 g of TAIII with a high purity of > 97% was obtained from 1 kg of RA through this five-step preparation method, which can be used to produce TAIII on a large scale.


Introduction
The rhizoma of Anemarrhena asphodeloides Bunge (family Liliaceae) (RA) is a well-known traditional Chinese medicinal herb and officially listed in the Pharmacopoeia of the People's ABSTRACT Timosaponin AIII (TAIII) exhibits extensive pharmacological activities and has been reported as a potent antitumour agent for various human cancers. In the present study, a potential industrial process for producing TAIII that involves biotransformation directly in the crude extract liquid of rhizoma anemarrhenae (RA) was developed. β-D-glycosidase was used to transform timosaponin BII (TBII) into TAIII, and monofactor experiments were conducted to optimise the enzymolysis conditions. In addition, AB-8 macroporous resin column chromatography, preparative liquid chromatography, and crystallisation technique were applied for yielding TAIII crystals with a purity > 97%. Approximately, 7 g of TAIII with a high purity of > 97% was obtained from 1 kg of RA through this five-step preparation method, which can be used to produce TAIII on a large scale. (National Pharmacopoeia Committee 2010). TAIII, as an important spirostanol saponin in RA, exhibits extensive pharmacological activities, such as anti-platelet aggregation (Li et al. 2006), antidepressive (Jiang et al. 2014), and antiinflammatory (Lim et al. 2015) activities as well as very potent anticancer activity (Tsai et al. 2013;Wang et al. 2013). With an increasing number of studies revealing the potential of TAIII as an antitumour agent, developing a method for the industrial production of TAIII to meet the requirement of its further application is crucial. However, the content of natural TAIII in RA is approximately 0.19-0.28% (Yuan et al. 2006). TBII was reported to be at much higher levels in RA (Liang et al. 2010), and it can be transformed into TAIII. Acid hydrolysis of TBII can produce TAIII and sarsasapogenin (SAR, Figure 1), causing a low yield of TAIII. In this study, β-D-glycosidase was applied to hydrolyse TBII to produce TAIII directly from the crude extract of RA, and a more rapid and efficient method for separating and purifying TAIII, including macroporous adsorption technique and preparative liquid chromatography has been applied.

Optimisation of enzymolysis factors through monofactor experiments
To investigate the optimal pH for β-D-glycosidase, the pH of the acetic acid-sodium acetate buffer solution was set at 3.5, 4.0, 4.5, and 5.0, while the other parameters were fixed, with the temperature being 50 °C, reaction time being 2 h, and β-D-glycosidase amount being 600 U/g. When the pH exceeded 4.0, the TAIII yield declined ( Figure S1A); therefore, pH 4.0 was considered optimal. An optimal temperature was determined among the values of 37, 45, 50, 55, 60, 70, and 80 °C, while the pH was set at 4.0, the reaction time was 2 h, and the β-D-glycosidase amount was 600 U/g. The highest yield of TAIII (72.8%) was obtained at 55 °C ( Figure S1B). When the temperature exceeded 70 °C, the TAIII yield substantially decreased; therefore, 55 °C was considered the optimal enzymolysis temperature. For optimising the reaction time among the values of 0.5, 1, 1.5, 2, and 3 h, the pH of the buffer solution, temperature, and β-glucosidase amount were fixed at 4.0, 55 °C, and 600 U/g, respectively. After extension of the reaction time from 0.5 to 2 h, the TAIII yield increased to 72.0% ( Figure S1C); thus, 2 h was set as the optimal reaction time. Finally, the β-D-glycosidase amount was determined among the values of 100, 200, 600, 1200, and 2400 U/g after setting the pH of the buffer solution, temperature, and reaction time at 4.0, 55 °C, and 2 h, respectively. The TAIII yield substantially increased with the increasing amount of β-D-glycosidase and the highest yield of TAIII was 71.3% at 600 U/g of β-D-glycosidase ( Figure S1D). Additional confirmatory experiments were performed in triplicate under the defined optimal conditions; the mean TAIII yield was 80.2% ± 1.81% (n = 3), which indicated that the optimal enzymolysis procedure showed acceptable stability and reproducibility. The HPLC analysis revealed a TAIII purity of 44.4% ± 0.73% (n = 3) ( Figure S2C). TBII was not detected in the enzymolysis hydrolysate, indicating complete TBII transformation into TAIII.

Purification of TAIII through AB-8 macroporous resin column chromatography and preparative liquid chromatography
Static and dynamic absorption-desorption experiments were conducted using AB-8 columns to investigate the optimal parameters, such as the ethanol percentage in solutions and the volume of the eluents. Finally, the ratio of corresponding enzymolysis products to the dry mass of the resins was set to 15 mg/g, and water and 20, 50, and 80% ethanol were used as eluents at a bed volume of 10 and a flow rate of 2 mL/min. Under the aforementioned optimal conditions, the TAIII purity reached 60.8% (60.8% ± 2.95%, n = 3) with a recovery rate of 77.7% (77.7% ± 0.60%, n = 3). Figure S2D illustrates the efficient purification of TAIII by using macroporous adsorption resins. To obtain TAIII with higher quality, preparative liquid chromatography was performed. The complete separation procedure took 75 min, and three peaks were observed as the ratio of methanol increased from 80 to 85%. Fractions corresponding to the third peak were collected and confirmed as TAIII through analytical HPLC ( Figure S2e), indicating successful separation. Consequently, TAIII with a purity of 90.1% (90.1% ± 0.93%, n = 3) was obtained with a recovery rate of 56.4% (56.4% ± 5.45%, n = 3). Finally, the crystallisation technique was applied for obtaining TAIII crystals with a purity > 97% (97.4% ± 0.31%, n = 3) ( Figure S2F) and a recovery rate of 60.6% (60.6% ± 5.85%, n = 3).

Structural identification
The TAIII structure was confirmed by comparing its IR, eSI-MS, 1 H NMR, and 13 C NMR spectra with a reference. The analysis results are listed as follows. IR: 980.82,925.31,895.38,849.23; Table S1. These data were in agreement with those of the TAIII analytical results reported in the literature (Ouyang et al. 2011). The 1 H NMR and 13 C NMR spectra are shown in Figures  S3, S4.

Conclusion
A novel method for producing TAIII from RA was established using β-D-glycosidase. The biotransformation of TBII into TAIII was conducted directly in the crude extract liquid of RA, representing a completely different process from the conventional process (Zhou et al. 2010), which requires pure TBII as a raw material. Furthermore, multistage purification methods, namely AB-8 macroporous resin column and flash chromatography and crystallisation technique, were employed to yield pure compounds. Finally, approximately 7 g of TAIII, with a high purity of > 97% was obtained from 1 kg of RA slices through a five-step preparation. This study demonstrated a potential process for the large-scale industrial production of TAIII with satisfactory quality.