Additional Minor Diterpene Glycosides from Stevia rebaudiana Bertoni

Two additional novel minor diterpene glycosides were isolated from the commercial extract of the leaves of Stevia rebaudiana Bertoni. The structures of the new compounds were identified as 13-{β-D-glucopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-oxy} ent-kaur-16-en-19-oic acid {β-D-xylopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)]-O-β-D-glucupyranosyl-ester} (1), and 13-{β-D-6-deoxy-glucopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-oxy} ent-kaur-16-en-19-oic acid {β-D-glucopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)-β-D-gluco-pyranosyl-ester} (2), on the basis of extensive 1D (1H- and 13C-) 2D NMR (COSY, HSQC and HMBC) and MS spectroscopic data as well as chemical studies.


Introduction
The major constituents isolated from the leaves of a perennial shrub Stevia rebaudiana Bertoni (family: Asteraceae) are the potently sweet diterpenoid glycosides stevioside and rebaudioside A. Extracts of the leaves of S. rebaudiana native to Brazil and Paraguay have been used for centuries

OPEN ACCESS
to sweeten food and beverages in Japan, South America and China. Due to the increase in demand for the major constituents in the leaves of S. rebaudiana which are the potently sweet diterpenoid glycosides stevioside, rebaudiosides A and D, and dulcoside A, it is now grown commercially in a number of countries, particularly in China, Japan, Taiwan, Korea, Thailand and Indonesia [1,2]. The compounds isolated from S. rebaudiana known as stevia sweeteners are the glycosides of the diterpene steviol, ent-13-hydroxykaur-16-en-19-oic acid [3].
In our continuing research to discover novel natural sweeteners, we have isolated several new steviol glycosides from the commercial extracts of the leaves of S. rebaudiana [4][5][6][7]. Apart from isolation and structural characterization of novel compounds from S. rebaudiana, and their possible utilization as natural sweeteners or sweetness enhancers, we have also studied the stability of many steviol glycosides in various systems of interest and characterized their degradation products using a number of spectroscopic methods [8]. We are also engaged in the synthesis of selected steviol diterpene glycosides using naturally occurring starting materials [9][10][11].

Results and Discussion
Purification of the commercial extract of rebaudioside M (also known as rebaudioside X) obtained from the leaves of S. rebaudiana from Lot No: PT01021 of Pure Circle, Malaysia resulted in the isolation of the two new diterpenoid glycosides 13-   Compound 1 was obtained as a white powder and its molecular formula was deduced as C 55 H 88 O 32 from its HRESI mass spectrum in the positive mode which showed a (M+H) + ion at m/z 1261.5353; this was supported by the 13 C-NMR spectral data. The 1 H-NMR spectrum of 1 showed the presence of two methyl signals resonating at δ 1.29 and 1.36 as singlets, two olefinic proton singlets at δ 4.89 and 5.69 corresponding to an exocyclic double bond, nine methylene and two methine protons between δ 0.75-2.70, characteristic for the ent-kaurane diterpenoids isolated earlier from the genus Stevia [6][7][8][9]. The basic skeleton of ent-kaurane diterpenoids was supported by the key 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 correlations: H-1/C-2, C-10; H-3/C-2, C-4, C-5, C-18, C-19; H-5/C-4, C-6, C-10, C-18, C-19, C-20; H-9/C-8, C-10, C-11; H-14/C-8, C-9, C-13, C-15, C-16 and H-17/C-13, C-15, C-16) correlations [4][5][6][7][8][9][10][11]. The presence of six sugar units in its structure was evident by the presence of the anomeric protons resonating at δ 5.33, 5.45, 5.46, 5.48, 5.62, and 6.38 in its 1 H-NMR spectrum. The presence of these six sugar units was further supported by the MS/MS spectrum of 1 in the positive ESI mode that showed fragment ions at m/z 1129, 967, 805, 643, 481 and 319; suggesting the presence of a pentose and five hexose moieties in its structure. Acid hydrolysis of 1 with 5% H 2 SO 4 afforded the sugars D-glucose and D-xylose, which were identified by direct comparison with authentic samples by TLC [12][13][14]. The anomeric proton observed at δ 6.38 showed an HMBC correlation to C-19 which indicated that it corresponds to the anomeric proton of Sugar I. Similarly, the anomeric proton observed at δ 5.45 showed an HMBC correlation to C-13 allowing it to be assigned as the anomeric proton of Sugar II. The sequence in sugar units and the assignments for C-2 through C-6 of Glc V and Glc VI were made using the 1 H-NMR, COSY and HSQC data. Further, the identification of sugars present in 1 and their configurations were achieved by preparing their thiocarbamoyl-thiazolidine carboxylate derivatives with L-cysteine methyl ester and O-tolyl isothiocyanate, and comparison of their retention times with the standard sugars as described in the literature; suggesting the sugar moieties present as β-D-glucopyranosyl and β-D-xylopyranosyl units [15]. Enzymatic hydrolysis of 1 furnished an aglycone which was identified as steviol (3) by comparison of 1 H-NMR and co-TLC with a standard [16]. The large coupling constants observed for the six anomeric protons of the sugar moieties at δ 5.33 (d, J = 8.1 Hz), 5.45 (d, J = 7.8 Hz), 5.46 (d, J = 7.5 Hz), 5.48 (d, J = 7.9 Hz), 5.62 (d, J = 7.8 Hz), and 6.38 (d, J = 8.4 Hz), suggested their β-orientation, as reported for steviol glycosides [4][5][6][7][8][9][10][11]. The 1 H-and 13 C-NMR values for all the proton and carbons were assigned on the basis of 2D NMR spectral data (COSY, HSQC and HMBC) and are given in Table 1.
From the above NMR spectral data and hydrolysis studies, it was inferred that there are five β-D-glucopyranosyl units and a β-D-xylopyranosyl unit in the structure of 1 connected to the aglycone steviol. Comparison of the 1 H-and 13 C-NMR values of 1 with rebaudioside D [4,17] suggested the presence of a steviol aglycone moiety with a 2,3-branched β-D-glucotriosyl substituent and another β-D-glucosyl moiety in the form of an ester at C-19, leaving the assignment of the additional β-D-glucosyl and β-D-xylosyl moieties. The downfield shift for both the 1 H and 13 C chemical shifts at C-2' and C-3' of sugar I (C-19 position) suggested that the additional β-D-xylosyl and β-D-glucosyl moieties are attached at thse positions respectively. This was confirmed by the key HMBC correlations as shown in Figure 2. Based on the results from chemical and spectral studies, 1 was assigned as 13-{β-D-  The molecular formula of compound 2 was determined to be C 56 H 90 O 32 by the [M+H] + ion at m/z 1,275.5485 in the positive ESI mass spectrum which was further supported by the 13 C-NMR spectral data. The 1 H-NMR spectrum of 2 also showed the presence of two methyl singlets, nine methylene and two methine protons as in 1. The positive ESI MS/MS spectrum of 2 showed the fragment ions at m/z 1,129, 967, 805, 643, 481, and 319 corresponding to the successive loss of a deoxyhexose and five hexose units from its [M+H] + ion, which was supported by the six anomeric protons observed at δ 5.30, 5.39, 5.48, 5.49, 5.81, 6.41 in its 1 H-NMR spectral data. Acid hydrolysis of 2 performed using the same procedure described as in 1 afforded D-glucose and D-deoxyglucose confirming the presence of a D-deoxypyranosyl and five D-glucopyranosyl units in its molecular structure. The two sugars present in 2 were identified as β-D-glucopyranosyl and β-D-6-deoxyglucopyranosyl units by preparing their thiocarbamoyl-thiazolidine carboxylate derivatives with L-cysteine methyl ester and O-tolyl isothiocyanate, as in 1. The large coupling constants observed for the six anomeric protons suggested their β-orientation, as reported for steviol glycosides such as in the case of 1.
Enzymatic hydrolysis of 2 furnished a compound which was found identical to steviol (3)

General
NMR spectra were acquired on a Bruker Avance DRX 500 MHz instrument with a 5 mm inverse detection probe using standard pulse sequences. The spectrum was referenced to the residual solvent signal (δ H 8.71, δ C 149.9 for pyridine-d 5 ), chemical shifts are given in δ (ppm), and coupling constants are reported in Hz. High Resolution Mass Spectral (HRMS) and MS/MS fragmentation pattern data were generated in the positive-ion mode using a mass spectrometer (Waters Premier Quadrupole Time-of-Flight) equipped with an electrospray ionization source. Samples were diluted with water: acetonitrile (1:1) containing 0.1% formic acid and introduced via infusion using the onboard syringe pump.

Plant Material
The commercial rebaudioside M with Lot No: PT01021 was obtained from Pure Circle, Malaysia. A voucher specimen is deposited at The Coca-Cola Company, No. VSPC-2973-6B.