Isolation, NMR Spectral Analysis and Hydrolysis Studies of a Hepta Pyranosyl Diterpene Glycoside from Stevia rebaudiana Bertoni

From the commercial extract of the leaves of Stevia rebaudiana Bertoni, a minor steviol glycoside, 13-[(2-O-β-d-glucopyranosyl-3-O-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(2-O-(3-O-β-d-glucopyranosyl-α-l-rhamnopyranosyl)-3-O-β-d-glucopyranosyl-β-d-glucopyranosyl) ester] (1); also known as rebaudioside O having seven sugar units has been isolated. Its structural characterization has been achieved by the extensive 1D (1H and 13C), and 2D NMR (COSY, HMQC, HMBC) as well as mass spectral data. Further, hydrolysis studies were performed on rebaudioside O using acid and enzymatic methods to identify aglycone and sugar residues in its structure as well as their configurations.


Introduction
Stevia rebaudiana (Bertoni) Bertoni is a perennial shrub of the Asteraceae (Compositae) family native to Brazil and Paraguay, which is often referred to as "The sweet herb of Paraguay". Currently, S. rebaudiana is being grown commercially in a number of countries, particularly in China, Japan, Taiwan, Korea, Thailand and Indonesia [1][2]. Extracts of the leaves of S. rebaudiana have been used for decades to sweeten food and beverages in Japan, South America and China. The major constituents in the leaves of S. rebaudiana are the potently sweet glycosides namely steviolbioside, stevioside, rebaudiosides A and E, dulcoside A and rubusoside; which are glycosides of the diterpene steviol, ent-13-hydroxykaur-16-en-19-oic acid [3][4]. These compounds are also known as Stevia sweeteners.
Recently, Ohta et al. have reported several minor steviol glycosides including rebaudioside O (1) from S. rebaudiana Morita, which was developed as a cultivar by selective breeding of S. rebaudiana Bertoni. However, they have not reported isolation or complete spectral assignments of pure rebaudioside O [5]. As a part of our research related to the discovery of natural sweeteners and sweetener enhancers, we are herewith describing the isolation, characterization and complete 1 H-and 13 C-NMR spectral assignments for the diterpene glycoside 13- (1), also known as rebaudioside O (Figure 1) isolated from the commercial extract of S. rebaudiana Bertoni. The complete NMR assignments were achieved on the basis of 1D ( 1 H and 13 C) and 2D (COSY, HMQC and HMBC) NMR as well as high resolution mass spectroscopic data. Acid and enzymatic hydrolysis studies on compound 1 were carried out to identify aglycone and sugar residues.
Based on the results from NMR spectral data and hydrolysis experiments of 1, it was concluded that there are six β-D-glucosyl units and an α-L-rhamnosyl unit in its structure connected to the aglycone steviol. A close comparison of the 1 H-and 13    This is the first report of the isolation of rebaudioside O (1) from S. rebaudiana Bertoni though it has been reported earlier from S. rebaudiana Morita [5]. Further, partial NMR spectral data has been reported earlier for rebaudioside O (1) [5], whereas we are herewith reporting its complete 1 H-and 13 C-NMR spectral assignments based on 1D ( 1 H and 13 C) and 2D (COSY, HMQC and HMBC) NMR as well as high resolution mass spectroscopic data which was supported by enzymatic and acid hydrolysis studies.

General
HPLC analysis was performed using a Dionex UPLC ultimate 3000 system (Sunnyvale, CA, USA), including a quaternary pump, a temperature controlled column compartment, an auto sampler and a UV absorbance detector. Phenomenex Luna NH 2 with guard column, 150 × 3.0 mm, 3 µm (100A) were used for the characterization of rebaudioside O (1). Analytical HPLC was carried out with a Waters 600E multisolvent delivery system using a Phenomenex Luna C18 (150 × 4.6 mm, 5 µm) column. NMR spectra were acquired on a Varian INOVA 600 MHz instrument with a 5 mm HCN probe using standard pulse sequences. The NMR spectra were performed in C 5 D 5 N; chemical shifts are given in  (ppm), and coupling constants are reported in Hz. The spectral data was referenced to the residual solvent signal ( H 7.19, and  C 123.5 for pyridine-d 5 ). IR spectral data was acquired using a Perkin Elmer 400 Fourier Transform Infrared (FT-IR) Spectrometer with Universal attenuated total reflectance (UATR) polarization accessory. MS and MS/MS data were generated with a Thermo LTQ-FTMS mass spectrometer (100,000 resolutions) equipped with a Nano spray ionization source. Samples were diluted with methanol and introduced via infusion using the onboard syringe pump.
Enzymatic hydrolysis of compound 1. Compound 1 (1 mg) was dissolved in 10 mL of 0.1 M sodium acetate buffer, pH 4.5 and crude pectinase from Aspergillus niger (100 uL, Sigma-Aldrich, P2736) was added. The mixture was stirred at 50 °C for 72 h. The product precipitated out during the reaction and was filtered and then crystallized. The resulting product obtained from the hydrolysis of 1 was identified as steviol (3) by comparison of its co-TLC with standard compound and 1 H NMR spectral data [14].

Conclusions
To the best of our knowledge, this is the first report of the isolation of rebaudioside O from S. rebaudiana Bertoni. Also, we are herewith reporting the complete 1 H-and 13