The complete proton and carbon NMR spectral assignments of a diterpene glycoside isolated from the commercial extract of the leaves of Stevia rebaudiana Bertoni, 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] entkaur-16-en-19-oic acid-[(2-O-α-L-rhamnopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester] (1); also known as rebaudioside N, was achieved by the extensive 1D and 2D NMR (1H and 13C, COSY, HMQC, HMBC) as well as mass spectral data. Further, hydrolysis studies were performed on rebaudioside N using acid and enzymatic studies to identify aglycone and sugar residues in its structure.
Stevia rebaudiana (Bertoni) is a perennial shrub belonging to the family of Asteraceae (Compositae) native to Brazil and Paraguay, but now grown commercially in a number of areas, particularly in Japan, Taiwan, Korea, Mainland China, 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 from S. rebaudiana Morita including rebaudioside N [
HPLC analysis was performed using a Dionex UPLC ultimate 3000 system (Sunnyvale, CA), including a quaternary pump, a temperature controlled column compartment, an auto sampler and a UV absorbance detector. Phenomenex Luna NH2 with guard column, 150 × 3.0 mm, 3 µm (100A) were used for the characterization of rebaudioside N (1). 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 C5D5N; chemical shifts are given in d (ppm), and coupling constants are reported in Hz. The spectral data was referenced to the residual solvent signal (dH 7.19, and dC 123.5 for pyridine-d5). 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.
Compound 1 was purified by repeated isocratic elution (72% acetonitrile in water) of the commercial extract of Stevia rebaudiana Bertoni using Dionex UPLC ultimate 3000 system with Phenomenex Luna NH2 guard column. Collected the peak eluting at tR 7.03 min; and dried the corresponding solution under nitrogen yielded 1.
White powder; IR νmax: 3317, 2945, 1725, 1063, 914 cm−1; 1H-NMR (600 MHz, C5D5N, δ ppm) and 13C-NMR (150 MHz, C5D5N, δ ppm) spectroscopic data see
To a solution of compound 1 (5 mg) in MeOH (10 ml) was added 3 ml of 5% H2SO4 and the mixture was refluxed for 24 hours. The reaction mixture was then neutralized with saturated sodium carbonate and extracted with ethyl acetate (EtOAc) (2 × 25 ml) to give an aqueous fraction containing sugars and an EtOAc fraction containing the aglycone part. The aqueous phase was concentrated and compared with standard sugars using the TLC systems EtOAc/n-butanol/water (2:7:1) and CH2Cl2/MeOH/water (10:6:1) [6-8]; the sugars were identified as D-glucose and L-rhamnose.
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 (50 uL, Sigma-Aldrich, P2736) was added. The mixture was stirred at 50˚C for 96 hr. 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 (4) by comparison of its co-TLC with standard compound and 1H NMR spectral data [
Compound 1 was isolated as an crystalline material and its molecular formula has been deduced as C56H90O32 on the basis of its positive ESI TOF mass spectrum which showed [M + H]+ ion at m/z 1275.5493, and this composition was supported by 13C NMR spectral data. The 1H NMR spectrum of 1 showed the presence of two methyl singlets at δ 1.17 and 1.51, two olefinic protons as singlets at δ 5.05 and 5.69 of an exocyclic double bond, nine methylene and two methine protons between δ 0.72 - 2.60, characteristic for the ent-kaurane diterpenoids isolated earlier from the genus Stevia [10-12]. The basic skeleton of ent-kaurane diterpenoids was supported by COSY (H-1/H-2; H-2/H-3; H-5/H-6; H-6/H-7; H-9/H-11; H-11/H-12) and HMBC (H-1/C-2, C-10; H-3/C-1, C-2, C-4, C-5, C-18, C-19; H-5/C-4, C-6, C-7, C-9, C-10, C-18, C-19, C-20; H-9/C-8, C-10, C-11, C-12, C-14, C-15; H-14/C-8, C-9, C-13, C-15, C-16 and H-17/C-13, C-15, C-16) correlations. The 1H NMR spectrum of 1 showed the presence of six sugar units in its structure by the presence of the anomeric protons resonating at δ 5.03, 5.06, 5.37, 5.58, 6.22, and 6.32; which was further supported by the MS/MS spectrum of 1 in the positive ESI mode showed the fragment ions at m/z 1113, 951, 805, 643, 481 and 319. Acid hydrolysis of
1 with 5% H2SO4 afforded the sugars D-glucose and L-rhamnose, which were identified by direct comparison with authentic samples by TLC [6-8]. Enzymatic hydrolysis of 1 furnished an aglycone which was identified as steviol (4) by comparison of 1H NMR and co-TLC with standard compound [
Based on the results from NMR spectral data and hydrolysis experiments of 1, it was concluded that there are five β-D-glucosyl units and an α-L-rhamnosyl unit in its structure connected to the aglycone steviol. A close comparison of the 1H and 13C NMR values of 1 with rebaudioside M (2) and rebaudioside A (3) [
To the best of our knowledge, this is the first report of the isolation of rebaudioside N (1) from S. rebaudiana Bertoni. Though partial NMR spectral data has been reported earlier for rebaudioside N (1) by Ohta et al. [
is the first report of complete 1H and 13C NMR spectral assignments based on 1D (1H and 13C) and 2D (COSY, HMQC and HMBC) NMR as well as high resolution mass spectroscopic data which was supported by enzymatic and acid hydrolysis studies.
We are herewith reporting the isolation, complete 1H and 13C NMR spectral assignments for 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]-ent-kaur-16-en-19-oic-acid-[(2-O-α-L-rhamnopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)
ester], also known as rebaudioside N (1) on the basis of extensive 1D and 2D NMR as well as high resolution mass spectral data. Further, acid hydrolysis furnished D-glucose suggesting the presence of two sugar units that were identified as β-D-glucose and α-L-rhamnose; and enzymatic hydrolysis furnished steviol.
We wish to thank Dr. Shaoxiong Wu, and Dr. Bing Wang of Emory University, Atlanta, USA for obtaining some selected spectral data and other chemistry related help.