An Agilent (Wilmington, DE) 1100 HPLC System, including a quaternary pump, a temperature controlled column compartment with an additional 6 port switching valve, an auto sampler and VWD absorbance detector was used for analysis. The detector was set-up at UV 210 nm and the data acquisition was done using a Chemastation A 10.02 software. The column used for HPLC analysis was a reversed-phase C18 (2) 100 A Phenomenex (Torrance CA) (250 × 4.6 mm, 5 µm); pH was measured using meter Metler Toledo seven compact pH/ion S220 (Switzerland); Branson Ultrasonic Cleaner Model 2510 (Maplewood, NJ) was used for degassing HPLC solvents. NMR spectra were acquired on BrukerAvance DRX 500 MHz or Varian INOVA 600 MHz instrument instruments using standard pulse sequences. High Resolution Mass Spectral (HRMS) data were generated with a LTQ Orbitrap Discovery instrument with its resolution set to 30 k. The needle voltage was set to 4 kV; the other source conditions were sheath gas = 25, aux gas = 0, sweep gas = 5 (all gas flows in arbitrary units), capillary voltage = 30 V, capillary temperature = 300˚C, and tube lens voltage = 75. Sample was diluted with 2:2:1 CH3CN:MeOH:water (same as infusion eluent) and injected 50 microliters. TLC was performed on Baker Si- C18F plates with mobile phase H2O-MeOH (80:20). Identification of the spots on the TLC plate was carried out by spraying 10% H2SO4 in EtOH and heating the plate at about 80C.
The commercial sample of Stevia extract from the leaves of S. rebaudiana which is a mixture of diterpene glycosides was obtained from Sinochem Qingdao Co Ltd, China with Lot No: 20140611. The authenticity of the commercial extract was confirmed by performing its retention time (tR) comparison with the internal standard compounds of known JECFA steviol glycosides isolated from S. rebaudiana using the HPLC method as reported earlier [
2.3. Isolation and Purification of 13-[(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-(2-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-β-D-gluco- pyranosyl) ester (1)
Compound 1 was purified from the commercial Stevia extract obtained from Sinochem Qingdao Co. Ltd., China using an Agilent 1100 HPLC system with Phenomenex column (250 × 4.6 mm, 5 µm) by RP-HPLC in 3 stages. The first stage utilized an isocratic elution method using the mobile phase acetonitrile/phosphate buffer (20:80); flow rate: 2 mL/min; injection volume: 50 µL; detection: 210 nm. The eluent collected between tR 8.0 and 9.5 min has been combined over several runs; dried the corresponding solution under nitrogen yielded a mixture (12.4 mg), which on second stage of purification with an isocratic mobile phase acetonitrile/phosphate buffer (25:75); flow rate: 1 mL/min; injection volume: 10 µL; detection: 210 nm.. The eluent collected between tR 12.0 and 14.0 min has been combined over several runs; dried the corresponding solution under nitrogen yielded a mixture (8.4 mg), which on final stage of purification with an isocratic mobile phase acetonitrile/phosphate buffer (32:68); flow rate: 0.5 mL/min; injection volume: 10 µL; detection: 210 nm. The peak eluting at tR 16.24 min has been collected over multiple runs; dried the corresponding solution under nitrogen yielded 1 (2.4 mg).
Identification and spectroscopic data of Compound 1. White powder; 1H NMR (600 MHz, C5D5N, δ ppm) and 13C NMR (150 MHz, C5D5N, δ ppm) spectroscopic data see
| Position | 1H NMR | 13C NMR |
|---|---|---|
| 1 | 0.75 t (12.4), 1.72 m | 41.4 |
| 2 | 1.46 m, 2.21 m | 20.3 |
| 3 | 1.02 m, 2.40 d (12.3) | 38.6 |
| 4 | --- | 45.0 |
| 5 | 1.00 d (10.8) | 58.0 |
| 6 | 1.87 m, 2.10 m | 22.6 |
| 7 | 1.26 m, 134 m | 42.2 |
| 8 | --- | 43.2 |
| 9 | 0.86 d (7.2) | 54.7 |
| 10 | --- | 40.1 |
| 11 | 1.68 m | 21.2 |
| 12 | 1.96 m, 2.24 m | 37.9 |
| 13 | --- | 87.0 |
| 14 | 1.78 d (10.8), 2.67 d (11.3) | 44.7 |
| 15 | 1.34 m, 2.06 m | 48.4 |
| 16 | --- | 154.8 |
| 17 | 5.05 s, 5.66 s | 105.2 |
| 18 | 1.40 s | 28.8 |
| 19 | --- | 176.4 |
| 20 | 1.12 s | 17.1 |
| 1′ | 6.32 d (7.8) | 94.0 |
| 2′ | 4.34 m | 81.4 |
| 3′ | 4.18 m | 78.7 |
|---|---|---|
| 4′ | 4.26 m | 72.2 |
| 5′ | 4.08 m | 78.6 |
| 6′ | 4.42 m, 4.62 m | 70.2 |
| 1′′ | 5.14 d (7.4) | 98.4 |
| 2′′ | 4.18 m | 84.6 |
| 3′′ | 4.32 m | 78.8 |
| 4′′ | 4.24 m | 71.8 |
| 5′′ | 3.80 m | 78.9 |
| 6′′ | 4.26 m, 4.41 m | 63.0 |
| 1′′′ | 5.22 d (7.8) | 107.0 |
| 2′′′ | 4.14 t (8.4) | 77.6 |
| 3′′′ | 4.25 m | 78.7 |
| 4′′′ | 4.34 m | 72.4 |
| 5′′′ | 3.94 m | 79.1 |
| 6′′′ | 4.29 m, 4.45 m | 63.3 |
| 1′′′′ | 5.48 d (7.5) | 106.0 |
| 2′′′′ | 4.07 m | 77.2 |
| 3′′′′ | 4.18 m | 79.1 |
| 4′′′′ | 4.32 m | 71.5 |
| 5′′′′ | 3.96 m | 79.3 |
| 6′′′′ | 4.16 m, 4.38 m | 63.6 |
| 1′′′′′ | 5.06 d (7.5) | 106.4 |
| 2′′′′′ | 4.04 m | 77.2 |
| 3′′′′′ | 4.18 m | 78.8 |
| 4′′′′′ | 4.28 m | 72.0 |
| 5′′′′′ | 4.02 m | 79.2 |
| 6′′′′′ | 4.32 m, 4.43 m | 63.4 |
aAssignments made on the basis of TOCSY, HMQC and HMBC correlations; bChemical shift values are in δ (ppm); cCoupling constants are in Hz.
Acid Hydrolysis of 1. Compound 1 (500 μg) is dissolved in MeOH (3 ml) and added 5% H2SO4 (10 mL). The mixture was refluxed for 16 hours and then neutralized with saturated sodium carbonate after cooling to room temperature. The aqueous phase was extracted with ethyl acetate (EtOAc, 2 ´ 15 ml) to separate an EtOAc fraction containing the aglycone part. The aqueous layer was concentrated and compared with standard sugars using the TLC system EtOAc/n-butanol/water (2:7:1) and CH2Cl2/MeOH/water (10:6:1) [
Enzymatic hydrolysis of 1. Compound 1 (500 μg) was dissolved in 5.0 mL of 0.1 M sodium acetate buffer (pH 4.5) and crude pectinase from Aspergillusniger (250 µL, Sigma-Aldrich, P2736) was added. The mixture was stirred at 50˚C for 48 hr. The product precipitated out during the reaction was filtered and then purified using reversed-phase preparative TLC using water:MeOH (70:30) yielded a pure compound, which was identified as steviol by comparison with co-TLC and 1H-NMR of an authentic sample as well as from the spectral data from the literature [