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Minor diterpenoid glycosides from the leaves of Stevia rebaudiana
Phytochemistry Letters 4 (2011) 209–212
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Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Minor diterpenoid glycosides from the leaves of Stevia rebaudiana Venkata Sai Prakash Chaturvedula a, John F. Clos a, Joshua Rhea b, Dennis Milanowski b, Ulla Mocek b, Grant E. DuBois a, Indra Prakash a,* a b
The Coca-Cola Company, Research and Technology, One Coca-Cola Plaza, Atlanta, GA 30313, USA AMRI, Bothell Research Center, 22215 26th Ave SE, Bothell, WA 98021, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 September 2010 Received in revised form 18 January 2011 Accepted 24 January 2011 Available online 17 February 2011
From the commercial extract of the leaves of Stevia rebaudiana, three new diterpenoid glycosides were isolated besides eight known steviol glycosides including stevioside, rebaudiosides A–F and dulcoside A. The structures of the three compounds were identified as 13-[(2-O-b-D-glucopyranosyl-b-Dglucopyranosyl) oxy]-kaur-16-en-18-oic acid-(6-O-b-D-xylopyranosyl-b-D-glucopyranosyl) ester (1), 13-[(2-O-b-D-glucopyranosyl-b-D-glucopyranosyl) oxy]-17-hydroxy-kaur-15-en-18-oic acid b-D-glucopyranosyl ester (2), and 13-[(2-O-b-D-glucopyranosyl-b-D-glucopyranosyl) oxy]-17-oxo-kaur-15-en18-oic acid b-D-glucopyranosyl ester (3) on the basis of extensive NMR and MS spectral studies. Another known diterpenoid glycoside, 13-[(2-O-b-D-glucopyranosyl-b-D-glucopyranosyl) oxy]-kaur-15-en-18oic acid b-D-glucopyranosyl ester (4) was also isolated and its complete NMR spectral assignments were made on the basis of COSY, HSQC and HMBC spectral data. ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Stevia rebaudiana Compositae Asteraceae Diterpenoid glycosides NMR MS
1. Introduction Stevia rebaudiana (Bertoni) Bertoni is a perennial shrub of the Asteraceae (Compositae) family native to certain regions of South America (Paraguay and Brazil). It is often referred to as ‘‘The sweet herb of Paraguay’’. The major constituents in the leaves of S. rebaudiana are the potently sweet diterpenoid glycosides stevioside, rebaudiosides A and C, and dulcoside A. These compounds are all glycosides of the diterpene steviol, ent-13-hydroxykaur-16-en18-oic acid (Brandle et al., 1998). As a part of our continuing research to discover natural sweeteners, we have obtained the commercial extract of S. rebaudiana (SG-95) from Pure Circle (Kuala Lumpur, Malaysia). The isolation and structure elucidation of the three new diterpenoid glycosides 1–3 as well as the isolation and complete NMR assignment of previously reported 4, the endocyclic isomer of stevioside (Clos et al., 2008) from SG-95 (Figs. 1 and 2). 2. Results and discussion Purification of the commercial extract obtained from the leaves of S. rebaudiana resulted in the isolation of three new diterpenoid glycosides 1–3, and the known steviol glycosides 13-[(2-O-b-Dglucopyranosyl-b-D-glucopyranosyl) oxy]-kaur-15-en-18-oic acid b-D-glucopyranosyl ester (4), stevioside, rebaudiosides A–F and
* Corresponding author. Tel.: +1 404 676 3007; fax: +1 404 598 3007. E-mail address: [email protected] (I. Prakash).
dulcoside A. The structures of all the known compounds were identified by comparison of their spectral data with those reported in the literature (Clos et al., 2008; Kohda et al., 1976; Kobayashi et al., 1977; Starratt et al., 2002; Sakamoto et al., 1977a,b). Compound 1 was isolated as a colorless oil and its molecular formula has been deduced as C43H68O22 on the basis of its positive ESI mass spectrum which showed an [M+H]+ ion at m/z 937.4316 together with [M+NH4]+ and [M+Na]+ adducts at m/z 954.4581 and 959.4135, respectively; this composition was supported by 13C NMR spectral data. The 1H NMR spectrum of 1 showed the presence of two methyl singlets at d 0.97 and 1.20, two olefinic protons as singlets at d 4.84 and 5.18 of an exocyclic double bond, nine methylene and two methine protons between d 0.85 and 2.26 characteristic for the ent-kaurene diterpenoids isolated earlier from the genus Stevia (Starratt et al., 2002). The basic skeleton of ent-kaurene 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/C2, 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, C10, C-18, C-19, C-20; C-9, C-8, C-11, C-12, C-14, C-15; H-14/C-8, C9, C-13, C-15, C-16 and H-17/C-13, C-15, C-16) correlations (Ohtani et al., 1992). The 1H NMR spectrum of 1 also indicated the presence of four anomeric protons at d 4.30, 4.58, 4.60, and 5.34 suggesting the presence of four sugar molecules in the structure. This was supported by the fragment ions observed in the positive ESI mode MS/MS spectrum of 1. Loss of a pentose moiety resulted in a fragment ion observed at m/z 805.3878; the additional successive loss of three hexose moieties were observed at m/z 643.3336, 481.2797, and 319.2271.
1874-3900/$ – see front matter ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2011.01.002
V.S.P. Chaturvedula et al. / Phytochemistry Letters 4 (2011) 209–212
210
J = 7.0 Hz), 4.58 (d, J = 7.8 Hz), 4.60 (d, J = 7.0 Hz), and 5.34 (d, J = 8.2 Hz) suggested the b-orientation as reported for steviol glycosides (Sakamoto et al., 1977a, b). Based on the results from chemical and spectral studies, 1 was assigned as 13-[(2-O-b-Dglucopyranosyl-b-D-glucopyranosyl) oxy]-kaur-16-en-18-oic acid-(6-O-b-D-xylopyranosyl-b-D-glucopyranosyl) ester. Compound 2 was also obtained as a colorless oil, and its molecular formula was assigned as C38H60O19 from the HRESI mass spectrum. The 1H NMR spectrum of 2 showed the presence of two methyl singlets at d 1.00 and 1.21, eight methylene and two methine protons between d 0.86 and 2.32, an oxymethylene group as a doublet of doublets at d 4.11 (J = 1.5, 14.8 Hz) and 4.29 (J = 1.5,
Table 1 1 H NMR spectral data for 1–4 isolated from Stevia rebaudiana recorded in CD3OD (500 MHz).a Position 1 1 2 3
Fig. 1. Structure of 1.
The 13C NMR values for all the carbons were assigned on the basis of HSQC and HMBC correlations (Table 2). A close comparison of the 1H and 13C NMR spectrum of 1 with stevioside suggested that compound 1 is also a steviol glycoside which has two glucose residues that are attached at the C-13 hydroxyl and another glucose moiety in the form of an ester at C-18 leaving the assignment of an additional pentose. An HMBC correlation was observed between the anomeric proton at d 4.30 and an oxymethylene proton of the glucose ester moiety suggesting the presence of the additional pentose at the C-6 position of the glucose moiety. Enzymatic hydrolysis of 1 furnished an aglycone which was identified as steviol by comparison of 1H NMR (Ohtani et al., 1992). Acid hydrolysis of 1 with 5% sulfuric acid (H2SO4) afforded D-glucose and D-xylose which were identified by direct comparison with authentic samples by TLC (Huan et al., 1998; Bedir et al., 2001; Chaturvedula et al., 2003). The large coupling constants observed for the four anomeric protons at d 4.30 (d, OR2 20 CH3
1 2
10
H 5
4 19 H3C
R3 17
13
11 9
14
16
8
O
R1
R2 2
2
glc
glc
3
glc
glc
4
2
2
glc
8 9 10 11 12 13 14 15 16 17 18 19 20 10 20 30 40 50 60 10 0 20 0 30 0 40 0 50 0 60 0
OR1
Compound
7
15
H 18
4 5 6
glc
glc = β-D-glucopyranosyl Fig. 2. Structures of 2–4.
R3 1
glc 1
glc 1
glc
CH2OH CHO CH3
10 0 0 20 0 0 30 0 0 40 0 0 50 0 0 60 0 0 10 0 0 0 20 0 0 0 30 0 0 0 40 0 0 0 50 0 0 0 a
2
3
4
0.85 1.88 1.41 1.94 1.05 2.15
m m m m m m
0.86 1.86 1.41 1.96 1.06 2.14
m m m m m d 12.6
0.89 1.88 1.42 1.95 1.07 2.17
m m m m m m
0.86 1.85 1.41 1.96 1.06 2.14
m m m m m d 12.2
1.12 1.83 2.03 1.44 1.55
d12.0 m m m m
1.12 1.83 2.00 1.55 1.65
d 12.0 m m m m
1.18 1.88 2.05 1.66 1.75
m m m m m
1.10 1.81 1.99 1.48 1.61
d 12.0 m m m m
0.97 m
0.90 d 8.2
1.00 m
0.85 d 8.2
1.63 1.80 1.54 1.99
m m m m
1.63 1.72 1.69 1.81
1.77 1.82 1.74 1.87
1.51 1.69 1.64 1.69
1.51 2.26 2.04 2.14
m d 11.9 d 15.9 m
1.73 m 2.32 d 10.4 5.36 s
4.84 s 5.18 s 1.20 0.97 5.34 3.35 3.42 3.39 3.52 3.76 4.06 4.60 3.45 3.54 3.25 3.25 3.62 3.83 4.58 3.27 3.36 3.29 3.24 3.62 3.83 4.30 3.58 3.53 3.79 3.50 3.84
m m m m
m m m m
1.95 m 1.67 m 2.32 d 11.9 2.25 d 10.0 6.57 s 5.10 s
4.11 dd 1.5, 14.8 9.61 s 4.29 dd 1.5, 14.8
s 1.21 s s 1.00 s d 8.2 5.37 d 8.2 m 3.35 m m 3.42 m m 3.36 m m 3.36 m dd 1.9, 11.5 3.68 m dd 5.2, 11.5 3.82 m d 7.0 4.66 d 7.8 m 3.42 m m 3.55 t 8.8 m 3.26 m m 3.24 m m 3.63 m m 3.86 m d 7.8 4.58 d 7.8 m 3.25 m m 3.35 m m 3.31 m m 3.25 m m 3.65 m m 3.84 m d 7.0 m m m m m
m m m m
1.22 1.02 5.37 3.36 3.41 3.36 3.36 3.68 3.82 4.75 3.45 3.53 3.23 3.24 3.62 3.83 4.58 3.28 3.36 3.28 3.24 3.62 3.83
s s d 8.2 m m m m m m d 7.8 m m m m m m d 7.8 m m m m m m
1.71 s
1.20 1.00 5.37 3.35 3.43 3.36 3.36 3.68 3.82 4.63 3.43 3.54 3.26 3.24 3.62 3.84 4.55 3.29 3.36 3.29 3.25 3.63 3.82
s s d 8.5 m m m m dd 3.7, 11.8 m d 7.8 m t 9.3 m m dd 5.9, 11.9 m d 7.8 m m m m dd 6.3, 12.0 m
Assignments made on the basis of COSY, HSQC and HMBC correlations.
V.S.P. Chaturvedula et al. / Phytochemistry Letters 4 (2011) 209–212
14.8 Hz) and a trisubstituted olefinic proton at d 5.36 (s). In addition, three anomeric protons were observed as doublets at d 4.58 (J = 7.8, 1H), 4.66 (J = 7.8, 1H), and 5.37 (J = 8.2, 1H). The NMR data for 2 were very similar to stevioside, except for the absence of an exocyclic double bond and a methylene group. A close comparison of the 1H and 13C NMR values of 2 with stevioside suggested the migration of the exocyclic double bond from C-16/C17 to C-15/C-16 with an additional hydroxyl group present at the C-17 position in 2. This was supported by the 13C NMR values for the oxymethylene group at C-17 which appeared at d 59.2 and a trisubstituted double bond between C-15 and C-16 which were observed at d 136.6 and 146.9, respectively, and also from the HMBC correlations (H-12/C-9, C-11, C-13, C-14, C-16; H-15/C-8, C9, C-14, C-16, C-17 and H-17/C-13, C-15, C-16). Acid hydrolysis of 2 with 5% H2SO4 afforded D-glucose that was identified by comparison with an authentic sample. The large coupling constants observed for the three glucose anomeric protons suggested the b-orientation similar to 1 and stevioside. Thus, structure of 2 was established as 13-[(2-O-b-D-glucopyranosyl-bD glucopyranosyl) oxy]-17-hydroxy-kaur-15-en-18-oic acid b-Dglucopyranosyl ester. Compound 3 was obtained as a colorless oil, and determined to have the molecular formula C38H58O19 by its HRESI mass spectrum. Its 1H NMR spectrum was very similar to that of 2 (Table 1), except Table 2 13 C NMR spectral data for 1–4 isolated from Stevia rebaudiana recorded in CD3OD (125 MHz).a Position
1
2
3
4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 10 20 30 40 50 60 100 200 300 400 500 600 10 0 0 20 0 0 30 0 0 40 0 0 50 0 0 60 0 0 10 0 0 0 20 0 0 0 30 0 0 0 40 0 0 0 50 0 0 0
41.5 19.8 38.7 44.8 58.2 22.8 42.4 43.3 55.0 40.7 21.0 37.9 88.0 45.0 48.3 153.7 105.2 178.5 28.6 16.0 95.4 73.7 78.2 70.9 77.6 68.8 97.4 82.4 77.9 71.6 77.9 62.6 105.1 77.8 77.9 71.4 77.7 62.6 104.6 72.2 74.0 69.1 66.3
41.8 19.8 38.8 44.7 58.0 21.5 40.2 49.5 47.7 40.1 21.8 30.4 90.2 49.6 136.6 146.9 59.2 178.4 28.6 15.8 95.4 73.7 78.2 70.8 78.4 62.3 96.7 82.5 78.0 71.4 78.0 62.5 104.9 77.7 77.8 71.1 77.7 62.5
41.7 19.7 38.6 44.3 57.6 21.1 39.0 50.6 46.1 40.5 20.6 31.4 88.5 47.7 158.0 147.7 191.4 178.0 28.5 16.0 95.5 73.8 78.6 70.9 78.4 62.2 96.8 82.5 77.9 71.7 77.6 62.5 105.1 77.8 77.9 71.4 77.7 62.6
41.7 19.8 38.8 44.6 58.0 21.5 40.4 49.1 48.1 40.1 21.7 29.9 90.9 48.5 136.6 143.8 12.2 178.1 28.5 15.8 95.4 73.7 78.3 70.7 78.4 62.1 96.8 82.3 77.9 71.6 77.5 62.8 104.9 75.8 77.6 71.3 78.1 62.6
a
Assignments made on the basis of HSQC and HMBC correlations.
211
for the presence of a singlet at d 9.61 in place of the C-17 hydroxymethyl group in 2. The 13C NMR spectrum of 3 showed the presence of an a,b-unsaturated carbonyl group at d 191.4 suggesting the oxymethylene group in 2 at the C-17 position was replaced with an aldehyde. This was supported by the HMBC correlations: (H-12/C-9, C-11, C-13, C-14, C-16; H-15/C-8, C-9, C14, C-16, C-17 and H-17/C-13, C-15, C-16). Furthermore, reduction of 3 with sodium borohydride provided 2. The 13C NMR values were assigned on the basis of HSQC and HMBC correlations and given in Table 2. On the basis of the above spectral and chemical data, 3 was assigned as 13-[(2-O-b-D-glucopyranosyl-b-D-glucopyranosyl) oxy]-17-oxo-kaur-15-en-18-oic acid b-D-glucopyranosyl ester. The molecular formula of compound 4 was established as C38H60O18 from its HRESI mass spectrum and 13C NMR spectral data. The 1H and 13C NMR data of compound 4 (Tables 1 and 2) were very similar to those of 2, except for the lack of signals for the oxymethylene group at the C-17 position. The presence of a methyl group in place of the oxymethylene group was supported by the observation of a singlet at d 1.71 and a signal at d 12.2 in the 1H and 13 C NMR spectra, respectively. These data indicated that 4 was identical to a previously described degradant of 5 (Clos et al., 2008). The complete 1H and 13C NMR assignment of 4 was made on the basis of HSQC and HMBC correlations (Tables 1 and 2). The large coupling constants observed for the three anomeric glucose protons suggested the b-orientation similar to 2. The structure of 4 was confirmed as 13-[(2-O-b-D-glucopyranosyl-b-D-glucopyranosyl) oxy]-kaur-15-en-18-oic acid b-D-glucopyranosyl ester. 3. Conclusions Twelve diterpenoid glycosides, including three new compounds (1–3) were isolated from the leaves of S. rebaudiana. Among them, 2–4 are ent-kaurene diterpenoids with a 15,16-double bond whereas 1 has the 16,17-double bond common to steviol glycosides. The discovery of these compounds is an important addition in expanding our understanding of the diversity of the diterpenoid glycosides present in the S. rebaudiana. 4. Experimental 4.1. General experimental procedures NMR spectra were acquired on a Bruker Avance DRX 500 MHz instrument with a 5 mm inverse detection probe using standard pulse sequences. The spectra were referenced to the residual solvent signal (dH 3.30, dC 49.0 for CD3OD), chemical shifts are given in d (ppm), and coupling constants are reported in Hz. MS and MS/MS data were generated with a Waters Premier Quadrupole Time-of-Flight (Q-Tof) mass spectrometer equipped with an electrospray ionization source operated in the positiveion mode. Samples were diluted with water:acetonitrile (1:1) containing 0.1% formic acid and introduced via infusion using the onboard syringe pump. Preparative HPLC was performed on an Agilent 1100 system using a Phenomenex Prodigy ODS (3) column (250 mm 21.2 mm, 5 mm). Semi-preparative HPLC was carried out with a Waters 600E multisolvent delivery system using a Phenomenex Synergi Hydro RP column (250 mm 10 mm, 4 mm) or Phenomenex Gemini C18 (250 mm 10 mm, 5 mm) column. 4.2. Plant material Stevia extract SG95, the commercial sample consisting of a mixture of diterpenoid glycosides from the leaves of S. rebaudiana was obtained from Pure Circle (Kuala Lumpur, Malaysia). A
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voucher specimen is deposited at The Coca-Cola Company, No. VSPC-3166-002.
4.5. 13-[(2-O-b-D-Glucopyranosyl-b-D-glucopyranosyl) oxy]-17hydroxy-kaur-15-en-18-oic acid b-D-glucopyranosyl ester (2)
4.3. Isolation
Colorless film; 1H NMR (500 MHz, CD3OD, d ppm) and 13C NMR (125 MHz, CD3OD, d ppm) spectroscopic data see Tables 1 and 2; +ESI TOFMS m/z 821.3817 (calcd. for C38H61O19: 821.3807). Acid hydrolysis of 2: Hydrolysis of 2 (250 mg) as described above furnished D-glucose.
Preliminary separation of the crude stevioside extract was carried out using a preparative HPLC method employing a water/acetonitrile gradient (25% B for 8.5 min, 25–29% B over 1.5 min, 29–30% B over 5.5 min, 30–34% B over 2.0 min, 34% B for 6 min, 34–52% B over 2.0 min, 52% B for 3.0 min, 52–70% B over 1.0 min, 70% B for 5.5 min) at 20 ml/min. The baseline fraction from 0 to 11 min from the preliminary separation was further fractionated on a Synergi Hydro RP column by semipreparative HPLC using a gradient of water (0.01156% acetic acid, 0.02844% ammonium acetate) in acetonitrile (25% B for 8.5 min, 25–100% B over 0.1 min, 100% B for 6.0 min) to yield 2 (tR 6.3 min, 3 mg). The peak eluting at 15.5 min in the preliminary separation provided rebaudioside D and was further purified on a Synergi Hydro RP column by semi-preparative HPLC using a gradient of water in acetonitrile (25% B for 8.5 min, 25–29% B over 1.5 min, 29% B for 3.0 min) to yield 3 (tR 8.6 min, 0.6 mg). The baseline fraction which eluted from 22 to 24 min in the preliminary separation was further fractionated on a Gemini C18 column by semi-preparative HPLC using a gradient of water (0.05% trifluoroacetic acid) in acetonitrile (25% B for 8.5 min, 25– 29% B over 1.5 min, 29–30% B over 6.5 min, 30–34% B over 2.0 min, 34% B for 6.0 min) to yield 4 (tR 21.2 min, 4.2 mg). Compounds 1 (tR 18.9 min, 2.10 mg, rebaudioside C (tR 24.6 min, 7.6 mg), rebaudioside D (tR 15.5 min, 11.5 mg), rebaudioside E (tR 11.7 min, 5.1 mg), rebaudioside F (tR 24.0 min, 2.2 mg), and dulcoside A (tR 25.2 min, 0.7 mg) were isolated directly by preparative HPLC. Compounds stevioside (tR 22.9 min), rebaudioside A (tR 22.1 min) and rebaudioside B (tR 28.7 min) were identified using LC–MS in comparison with authentic standards as described previously (Method 1, Clos et al., 2008). 4.4. 13-[(2-O-b-D-Glucopyranosyl-b-D-glucopyranosyl) oxy]-kaur16-en-18-oic acid (6-O-b-D-xylopyranosyl-b-D-glucopyranosyl) ester (1) Colorless film; 1H NMR (500 MHz, CD3OD, d ppm) and 13C NMR (125 MHz, CD3OD, d ppm) spectroscopic data see Tables 1 and 2; +ESI TOFMS m/z 937.4316 (calcd. for C43H69O22: 937.4280). Enzymatic hydrolysis of 1: A solution of 1 (250 mg) was dissolved in 2.5 ml of 0.1 M sodium acetate buffer, pH 4.5 and crude pectinase from Aspergillus niger (50 ml, Sigma–Aldrich, P2736) was added. The mixture was stirred at 50 8C for 48 h. The product precipitated out during the reaction and was filtered and then crystallized from methanol (MeOH). The resulting steviol was identical to an authentic sample by TLC and 1H NMR. Acid hydrolysis of 1: To a solution of 1 (250 mg) in MeOH (1 ml) was added 1 ml of 5% H2SO4 and the mixture was refluxed for 8 h. The reaction mixture was then neutralized with saturated sodium carbonate and extracted with ethyl acetate (EtOAc) (2 5 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) (Bedir et al., 2001; Huan et al., 1998); the two sugars were identified as Dxylose and D-glucose.
4.6. 13-[(2-O-b-D-Glucopyranosyl-b-D-glucopyranosyl) oxy]-17oxo-kaur-15-en-18-oic acid b-D-glucopyranosyl ester (3) Colorless film; 1H NMR (500 MHz, CD3OD, d ppm) and 13C NMR (125 MHz, CD3OD, d ppm) spectroscopic data see Tables 1 and 2; +ESI TOFMS m/z 819.3668 (calcd. for C38H59O19: 819.3651). Sodium borohydride (NaBH4) reduction of 3: A small aliquot (100 mg) of 3 was treated with NaBH4 in MeOH (120 ml, 1 mg/ml) for 16 h at rt. The reaction was stopped by the addition of 4.0% aqueous TFA (5 ml). Analysis of the reaction mixture by LC–MS (Method 1, Clos et al., 2008) indicated that the product formed was identical to 2. 4.7. 13-[(2-O-b-D-Glucopyranosyl-b-D-glucopyranosyl) oxy] kaur15-en-18-oic acid, b-D-glucopyranosyl ester (4) Colorless film; 1H NMR (500 MHz, CD3OD, d ppm) and 13C NMR (125 MHz, CD3OD, d ppm) spectroscopic data see Tables 1 and 2; +ESI TOFMS m/z 805.3897 (calcd. for C38H61O18: 805.3858).
Acknowledgement We wish to thank Pure Circle (Kuala Lumpur, Malaysia) for providing the stevia extract SG-95.
References Bedir, E., Toyang, N.J., Khan, I.A., Walker, L.A., Clark, A.M., 2001. A new dammarane type triterpene glycoside from Polyscias fulva. J. Nat. Prod. 64, 95–97. Brandle, J.E., Starrratt, A.N., Gijen, M., 1998. Stevia rebaudiana: its agricultural, biological and chemical properties. Can. J. Plant Sci. 78, 527–536. Chaturvedula, V.S.P., Schilling, J.K., Miller, J.S., Andriantsiferana, R., Rasamison, V.E., Kingston, D.G.I., 2003. New cytotoxic oleanane saponins from the infructescences of Polyscias amplifolia from the Madagascar rainforest. Planta Med. 69, 440–444. Clos, J.F., DuBois, G.E., Prakash, I., 2008. Photostability of rebaudioside A and stevioside in beverages. J. Agric. Food Chem. 56, 8507–8513. Huan, V.D., Yamamura, S., Ohtani, K., Yamasaki, R., Nham, K.N.T., 1998. Oleanane saponins from Polyscias fructicosa. Phytochemistry 47, 451–457. Kohda, H., Kasai, R., Yamsaki, K., Murakami, K., Tanaka., 1976. New sweet diterpene glucosides from Stevia rebaudiana. Phytochemistry 15, 981–983. Kobayashi, M., Horikawa, S., Degrandi, I.H., Ueno, J., Mitsuhashi, H., 1977. Dulcosides A and B, new diterpene glycosides from Stevia rebaudiana. Phytochemistry 16, 1405–1408. Ohtani, K., Aikawa, Y., Kasai, R., Chou, W., Yamasaki, K., Tanaka, O., 1992. Minor diterpene glycosides from sweet leaves of Rubus suavissimus. Phytochemistry 31, 1553–1559. Sakamoto, I., Yamasaki, K., Tanaka, O., 1977a. Application of 13C NMR spectroscopy to the chemistry of natural glycosides: rebaudioside C, a new sweet diterpene glycoside of Stevia rebaudiana. Chem. Pharm. Bull. 25, 844–846. Sakamoto, I., Yamasaki, K., Tanaka, O., 1977b. Application of 13C NMR spectroscopy to chemistry of plant glycosides: rebaudiosides D and E, new sweet diterpene glucosides of Stevia rebaudiana Bertoni. Chem. Pharm. Bull. 25, 3437–3439. Starratt, A.N., Kirby, C.W., Pocs, R., Brandle, J.E., 2002. Rebaudioside F, a diterpene glycoside from Stevia rebaudiana. Phytochemistry 59, 367–370.
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Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Minor diterpenoid glycosides from the leaves of Stevia rebaudiana Venkata Sai Prakash Chaturvedula a, John F. Clos a, Joshua Rhea b, Dennis Milanowski b, Ulla Mocek b, Grant E. DuBois a, Indra Prakash a,* a b
The Coca-Cola Company, Research and Technology, One Coca-Cola Plaza, Atlanta, GA 30313, USA AMRI, Bothell Research Center, 22215 26th Ave SE, Bothell, WA 98021, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 September 2010 Received in revised form 18 January 2011 Accepted 24 January 2011 Available online 17 February 2011
From the commercial extract of the leaves of Stevia rebaudiana, three new diterpenoid glycosides were isolated besides eight known steviol glycosides including stevioside, rebaudiosides A–F and dulcoside A. The structures of the three compounds were identified as 13-[(2-O-b-D-glucopyranosyl-b-Dglucopyranosyl) oxy]-kaur-16-en-18-oic acid-(6-O-b-D-xylopyranosyl-b-D-glucopyranosyl) ester (1), 13-[(2-O-b-D-glucopyranosyl-b-D-glucopyranosyl) oxy]-17-hydroxy-kaur-15-en-18-oic acid b-D-glucopyranosyl ester (2), and 13-[(2-O-b-D-glucopyranosyl-b-D-glucopyranosyl) oxy]-17-oxo-kaur-15-en18-oic acid b-D-glucopyranosyl ester (3) on the basis of extensive NMR and MS spectral studies. Another known diterpenoid glycoside, 13-[(2-O-b-D-glucopyranosyl-b-D-glucopyranosyl) oxy]-kaur-15-en-18oic acid b-D-glucopyranosyl ester (4) was also isolated and its complete NMR spectral assignments were made on the basis of COSY, HSQC and HMBC spectral data. ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Stevia rebaudiana Compositae Asteraceae Diterpenoid glycosides NMR MS
1. Introduction Stevia rebaudiana (Bertoni) Bertoni is a perennial shrub of the Asteraceae (Compositae) family native to certain regions of South America (Paraguay and Brazil). It is often referred to as ‘‘The sweet herb of Paraguay’’. The major constituents in the leaves of S. rebaudiana are the potently sweet diterpenoid glycosides stevioside, rebaudiosides A and C, and dulcoside A. These compounds are all glycosides of the diterpene steviol, ent-13-hydroxykaur-16-en18-oic acid (Brandle et al., 1998). As a part of our continuing research to discover natural sweeteners, we have obtained the commercial extract of S. rebaudiana (SG-95) from Pure Circle (Kuala Lumpur, Malaysia). The isolation and structure elucidation of the three new diterpenoid glycosides 1–3 as well as the isolation and complete NMR assignment of previously reported 4, the endocyclic isomer of stevioside (Clos et al., 2008) from SG-95 (Figs. 1 and 2). 2. Results and discussion Purification of the commercial extract obtained from the leaves of S. rebaudiana resulted in the isolation of three new diterpenoid glycosides 1–3, and the known steviol glycosides 13-[(2-O-b-Dglucopyranosyl-b-D-glucopyranosyl) oxy]-kaur-15-en-18-oic acid b-D-glucopyranosyl ester (4), stevioside, rebaudiosides A–F and
* Corresponding author. Tel.: +1 404 676 3007; fax: +1 404 598 3007. E-mail address: [email protected] (I. Prakash).
dulcoside A. The structures of all the known compounds were identified by comparison of their spectral data with those reported in the literature (Clos et al., 2008; Kohda et al., 1976; Kobayashi et al., 1977; Starratt et al., 2002; Sakamoto et al., 1977a,b). Compound 1 was isolated as a colorless oil and its molecular formula has been deduced as C43H68O22 on the basis of its positive ESI mass spectrum which showed an [M+H]+ ion at m/z 937.4316 together with [M+NH4]+ and [M+Na]+ adducts at m/z 954.4581 and 959.4135, respectively; this composition was supported by 13C NMR spectral data. The 1H NMR spectrum of 1 showed the presence of two methyl singlets at d 0.97 and 1.20, two olefinic protons as singlets at d 4.84 and 5.18 of an exocyclic double bond, nine methylene and two methine protons between d 0.85 and 2.26 characteristic for the ent-kaurene diterpenoids isolated earlier from the genus Stevia (Starratt et al., 2002). The basic skeleton of ent-kaurene 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/C2, 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, C10, C-18, C-19, C-20; C-9, C-8, C-11, C-12, C-14, C-15; H-14/C-8, C9, C-13, C-15, C-16 and H-17/C-13, C-15, C-16) correlations (Ohtani et al., 1992). The 1H NMR spectrum of 1 also indicated the presence of four anomeric protons at d 4.30, 4.58, 4.60, and 5.34 suggesting the presence of four sugar molecules in the structure. This was supported by the fragment ions observed in the positive ESI mode MS/MS spectrum of 1. Loss of a pentose moiety resulted in a fragment ion observed at m/z 805.3878; the additional successive loss of three hexose moieties were observed at m/z 643.3336, 481.2797, and 319.2271.
1874-3900/$ – see front matter ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2011.01.002
V.S.P. Chaturvedula et al. / Phytochemistry Letters 4 (2011) 209–212
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J = 7.0 Hz), 4.58 (d, J = 7.8 Hz), 4.60 (d, J = 7.0 Hz), and 5.34 (d, J = 8.2 Hz) suggested the b-orientation as reported for steviol glycosides (Sakamoto et al., 1977a, b). Based on the results from chemical and spectral studies, 1 was assigned as 13-[(2-O-b-Dglucopyranosyl-b-D-glucopyranosyl) oxy]-kaur-16-en-18-oic acid-(6-O-b-D-xylopyranosyl-b-D-glucopyranosyl) ester. Compound 2 was also obtained as a colorless oil, and its molecular formula was assigned as C38H60O19 from the HRESI mass spectrum. The 1H NMR spectrum of 2 showed the presence of two methyl singlets at d 1.00 and 1.21, eight methylene and two methine protons between d 0.86 and 2.32, an oxymethylene group as a doublet of doublets at d 4.11 (J = 1.5, 14.8 Hz) and 4.29 (J = 1.5,
Table 1 1 H NMR spectral data for 1–4 isolated from Stevia rebaudiana recorded in CD3OD (500 MHz).a Position 1 1 2 3
Fig. 1. Structure of 1.
The 13C NMR values for all the carbons were assigned on the basis of HSQC and HMBC correlations (Table 2). A close comparison of the 1H and 13C NMR spectrum of 1 with stevioside suggested that compound 1 is also a steviol glycoside which has two glucose residues that are attached at the C-13 hydroxyl and another glucose moiety in the form of an ester at C-18 leaving the assignment of an additional pentose. An HMBC correlation was observed between the anomeric proton at d 4.30 and an oxymethylene proton of the glucose ester moiety suggesting the presence of the additional pentose at the C-6 position of the glucose moiety. Enzymatic hydrolysis of 1 furnished an aglycone which was identified as steviol by comparison of 1H NMR (Ohtani et al., 1992). Acid hydrolysis of 1 with 5% sulfuric acid (H2SO4) afforded D-glucose and D-xylose which were identified by direct comparison with authentic samples by TLC (Huan et al., 1998; Bedir et al., 2001; Chaturvedula et al., 2003). The large coupling constants observed for the four anomeric protons at d 4.30 (d, OR2 20 CH3
1 2
10
H 5
4 19 H3C
R3 17
13
11 9
14
16
8
O
R1
R2 2
2
glc
glc
3
glc
glc
4
2
2
glc
8 9 10 11 12 13 14 15 16 17 18 19 20 10 20 30 40 50 60 10 0 20 0 30 0 40 0 50 0 60 0
OR1
Compound
7
15
H 18
4 5 6
glc
glc = β-D-glucopyranosyl Fig. 2. Structures of 2–4.
R3 1
glc 1
glc 1
glc
CH2OH CHO CH3
10 0 0 20 0 0 30 0 0 40 0 0 50 0 0 60 0 0 10 0 0 0 20 0 0 0 30 0 0 0 40 0 0 0 50 0 0 0 a
2
3
4
0.85 1.88 1.41 1.94 1.05 2.15
m m m m m m
0.86 1.86 1.41 1.96 1.06 2.14
m m m m m d 12.6
0.89 1.88 1.42 1.95 1.07 2.17
m m m m m m
0.86 1.85 1.41 1.96 1.06 2.14
m m m m m d 12.2
1.12 1.83 2.03 1.44 1.55
d12.0 m m m m
1.12 1.83 2.00 1.55 1.65
d 12.0 m m m m
1.18 1.88 2.05 1.66 1.75
m m m m m
1.10 1.81 1.99 1.48 1.61
d 12.0 m m m m
0.97 m
0.90 d 8.2
1.00 m
0.85 d 8.2
1.63 1.80 1.54 1.99
m m m m
1.63 1.72 1.69 1.81
1.77 1.82 1.74 1.87
1.51 1.69 1.64 1.69
1.51 2.26 2.04 2.14
m d 11.9 d 15.9 m
1.73 m 2.32 d 10.4 5.36 s
4.84 s 5.18 s 1.20 0.97 5.34 3.35 3.42 3.39 3.52 3.76 4.06 4.60 3.45 3.54 3.25 3.25 3.62 3.83 4.58 3.27 3.36 3.29 3.24 3.62 3.83 4.30 3.58 3.53 3.79 3.50 3.84
m m m m
m m m m
1.95 m 1.67 m 2.32 d 11.9 2.25 d 10.0 6.57 s 5.10 s
4.11 dd 1.5, 14.8 9.61 s 4.29 dd 1.5, 14.8
s 1.21 s s 1.00 s d 8.2 5.37 d 8.2 m 3.35 m m 3.42 m m 3.36 m m 3.36 m dd 1.9, 11.5 3.68 m dd 5.2, 11.5 3.82 m d 7.0 4.66 d 7.8 m 3.42 m m 3.55 t 8.8 m 3.26 m m 3.24 m m 3.63 m m 3.86 m d 7.8 4.58 d 7.8 m 3.25 m m 3.35 m m 3.31 m m 3.25 m m 3.65 m m 3.84 m d 7.0 m m m m m
m m m m
1.22 1.02 5.37 3.36 3.41 3.36 3.36 3.68 3.82 4.75 3.45 3.53 3.23 3.24 3.62 3.83 4.58 3.28 3.36 3.28 3.24 3.62 3.83
s s d 8.2 m m m m m m d 7.8 m m m m m m d 7.8 m m m m m m
1.71 s
1.20 1.00 5.37 3.35 3.43 3.36 3.36 3.68 3.82 4.63 3.43 3.54 3.26 3.24 3.62 3.84 4.55 3.29 3.36 3.29 3.25 3.63 3.82
s s d 8.5 m m m m dd 3.7, 11.8 m d 7.8 m t 9.3 m m dd 5.9, 11.9 m d 7.8 m m m m dd 6.3, 12.0 m
Assignments made on the basis of COSY, HSQC and HMBC correlations.
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14.8 Hz) and a trisubstituted olefinic proton at d 5.36 (s). In addition, three anomeric protons were observed as doublets at d 4.58 (J = 7.8, 1H), 4.66 (J = 7.8, 1H), and 5.37 (J = 8.2, 1H). The NMR data for 2 were very similar to stevioside, except for the absence of an exocyclic double bond and a methylene group. A close comparison of the 1H and 13C NMR values of 2 with stevioside suggested the migration of the exocyclic double bond from C-16/C17 to C-15/C-16 with an additional hydroxyl group present at the C-17 position in 2. This was supported by the 13C NMR values for the oxymethylene group at C-17 which appeared at d 59.2 and a trisubstituted double bond between C-15 and C-16 which were observed at d 136.6 and 146.9, respectively, and also from the HMBC correlations (H-12/C-9, C-11, C-13, C-14, C-16; H-15/C-8, C9, C-14, C-16, C-17 and H-17/C-13, C-15, C-16). Acid hydrolysis of 2 with 5% H2SO4 afforded D-glucose that was identified by comparison with an authentic sample. The large coupling constants observed for the three glucose anomeric protons suggested the b-orientation similar to 1 and stevioside. Thus, structure of 2 was established as 13-[(2-O-b-D-glucopyranosyl-bD glucopyranosyl) oxy]-17-hydroxy-kaur-15-en-18-oic acid b-Dglucopyranosyl ester. Compound 3 was obtained as a colorless oil, and determined to have the molecular formula C38H58O19 by its HRESI mass spectrum. Its 1H NMR spectrum was very similar to that of 2 (Table 1), except Table 2 13 C NMR spectral data for 1–4 isolated from Stevia rebaudiana recorded in CD3OD (125 MHz).a Position
1
2
3
4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 10 20 30 40 50 60 100 200 300 400 500 600 10 0 0 20 0 0 30 0 0 40 0 0 50 0 0 60 0 0 10 0 0 0 20 0 0 0 30 0 0 0 40 0 0 0 50 0 0 0
41.5 19.8 38.7 44.8 58.2 22.8 42.4 43.3 55.0 40.7 21.0 37.9 88.0 45.0 48.3 153.7 105.2 178.5 28.6 16.0 95.4 73.7 78.2 70.9 77.6 68.8 97.4 82.4 77.9 71.6 77.9 62.6 105.1 77.8 77.9 71.4 77.7 62.6 104.6 72.2 74.0 69.1 66.3
41.8 19.8 38.8 44.7 58.0 21.5 40.2 49.5 47.7 40.1 21.8 30.4 90.2 49.6 136.6 146.9 59.2 178.4 28.6 15.8 95.4 73.7 78.2 70.8 78.4 62.3 96.7 82.5 78.0 71.4 78.0 62.5 104.9 77.7 77.8 71.1 77.7 62.5
41.7 19.7 38.6 44.3 57.6 21.1 39.0 50.6 46.1 40.5 20.6 31.4 88.5 47.7 158.0 147.7 191.4 178.0 28.5 16.0 95.5 73.8 78.6 70.9 78.4 62.2 96.8 82.5 77.9 71.7 77.6 62.5 105.1 77.8 77.9 71.4 77.7 62.6
41.7 19.8 38.8 44.6 58.0 21.5 40.4 49.1 48.1 40.1 21.7 29.9 90.9 48.5 136.6 143.8 12.2 178.1 28.5 15.8 95.4 73.7 78.3 70.7 78.4 62.1 96.8 82.3 77.9 71.6 77.5 62.8 104.9 75.8 77.6 71.3 78.1 62.6
a
Assignments made on the basis of HSQC and HMBC correlations.
211
for the presence of a singlet at d 9.61 in place of the C-17 hydroxymethyl group in 2. The 13C NMR spectrum of 3 showed the presence of an a,b-unsaturated carbonyl group at d 191.4 suggesting the oxymethylene group in 2 at the C-17 position was replaced with an aldehyde. This was supported by the HMBC correlations: (H-12/C-9, C-11, C-13, C-14, C-16; H-15/C-8, C-9, C14, C-16, C-17 and H-17/C-13, C-15, C-16). Furthermore, reduction of 3 with sodium borohydride provided 2. The 13C NMR values were assigned on the basis of HSQC and HMBC correlations and given in Table 2. On the basis of the above spectral and chemical data, 3 was assigned as 13-[(2-O-b-D-glucopyranosyl-b-D-glucopyranosyl) oxy]-17-oxo-kaur-15-en-18-oic acid b-D-glucopyranosyl ester. The molecular formula of compound 4 was established as C38H60O18 from its HRESI mass spectrum and 13C NMR spectral data. The 1H and 13C NMR data of compound 4 (Tables 1 and 2) were very similar to those of 2, except for the lack of signals for the oxymethylene group at the C-17 position. The presence of a methyl group in place of the oxymethylene group was supported by the observation of a singlet at d 1.71 and a signal at d 12.2 in the 1H and 13 C NMR spectra, respectively. These data indicated that 4 was identical to a previously described degradant of 5 (Clos et al., 2008). The complete 1H and 13C NMR assignment of 4 was made on the basis of HSQC and HMBC correlations (Tables 1 and 2). The large coupling constants observed for the three anomeric glucose protons suggested the b-orientation similar to 2. The structure of 4 was confirmed as 13-[(2-O-b-D-glucopyranosyl-b-D-glucopyranosyl) oxy]-kaur-15-en-18-oic acid b-D-glucopyranosyl ester. 3. Conclusions Twelve diterpenoid glycosides, including three new compounds (1–3) were isolated from the leaves of S. rebaudiana. Among them, 2–4 are ent-kaurene diterpenoids with a 15,16-double bond whereas 1 has the 16,17-double bond common to steviol glycosides. The discovery of these compounds is an important addition in expanding our understanding of the diversity of the diterpenoid glycosides present in the S. rebaudiana. 4. Experimental 4.1. General experimental procedures NMR spectra were acquired on a Bruker Avance DRX 500 MHz instrument with a 5 mm inverse detection probe using standard pulse sequences. The spectra were referenced to the residual solvent signal (dH 3.30, dC 49.0 for CD3OD), chemical shifts are given in d (ppm), and coupling constants are reported in Hz. MS and MS/MS data were generated with a Waters Premier Quadrupole Time-of-Flight (Q-Tof) mass spectrometer equipped with an electrospray ionization source operated in the positiveion mode. Samples were diluted with water:acetonitrile (1:1) containing 0.1% formic acid and introduced via infusion using the onboard syringe pump. Preparative HPLC was performed on an Agilent 1100 system using a Phenomenex Prodigy ODS (3) column (250 mm 21.2 mm, 5 mm). Semi-preparative HPLC was carried out with a Waters 600E multisolvent delivery system using a Phenomenex Synergi Hydro RP column (250 mm 10 mm, 4 mm) or Phenomenex Gemini C18 (250 mm 10 mm, 5 mm) column. 4.2. Plant material Stevia extract SG95, the commercial sample consisting of a mixture of diterpenoid glycosides from the leaves of S. rebaudiana was obtained from Pure Circle (Kuala Lumpur, Malaysia). A
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voucher specimen is deposited at The Coca-Cola Company, No. VSPC-3166-002.
4.5. 13-[(2-O-b-D-Glucopyranosyl-b-D-glucopyranosyl) oxy]-17hydroxy-kaur-15-en-18-oic acid b-D-glucopyranosyl ester (2)
4.3. Isolation
Colorless film; 1H NMR (500 MHz, CD3OD, d ppm) and 13C NMR (125 MHz, CD3OD, d ppm) spectroscopic data see Tables 1 and 2; +ESI TOFMS m/z 821.3817 (calcd. for C38H61O19: 821.3807). Acid hydrolysis of 2: Hydrolysis of 2 (250 mg) as described above furnished D-glucose.
Preliminary separation of the crude stevioside extract was carried out using a preparative HPLC method employing a water/acetonitrile gradient (25% B for 8.5 min, 25–29% B over 1.5 min, 29–30% B over 5.5 min, 30–34% B over 2.0 min, 34% B for 6 min, 34–52% B over 2.0 min, 52% B for 3.0 min, 52–70% B over 1.0 min, 70% B for 5.5 min) at 20 ml/min. The baseline fraction from 0 to 11 min from the preliminary separation was further fractionated on a Synergi Hydro RP column by semipreparative HPLC using a gradient of water (0.01156% acetic acid, 0.02844% ammonium acetate) in acetonitrile (25% B for 8.5 min, 25–100% B over 0.1 min, 100% B for 6.0 min) to yield 2 (tR 6.3 min, 3 mg). The peak eluting at 15.5 min in the preliminary separation provided rebaudioside D and was further purified on a Synergi Hydro RP column by semi-preparative HPLC using a gradient of water in acetonitrile (25% B for 8.5 min, 25–29% B over 1.5 min, 29% B for 3.0 min) to yield 3 (tR 8.6 min, 0.6 mg). The baseline fraction which eluted from 22 to 24 min in the preliminary separation was further fractionated on a Gemini C18 column by semi-preparative HPLC using a gradient of water (0.05% trifluoroacetic acid) in acetonitrile (25% B for 8.5 min, 25– 29% B over 1.5 min, 29–30% B over 6.5 min, 30–34% B over 2.0 min, 34% B for 6.0 min) to yield 4 (tR 21.2 min, 4.2 mg). Compounds 1 (tR 18.9 min, 2.10 mg, rebaudioside C (tR 24.6 min, 7.6 mg), rebaudioside D (tR 15.5 min, 11.5 mg), rebaudioside E (tR 11.7 min, 5.1 mg), rebaudioside F (tR 24.0 min, 2.2 mg), and dulcoside A (tR 25.2 min, 0.7 mg) were isolated directly by preparative HPLC. Compounds stevioside (tR 22.9 min), rebaudioside A (tR 22.1 min) and rebaudioside B (tR 28.7 min) were identified using LC–MS in comparison with authentic standards as described previously (Method 1, Clos et al., 2008). 4.4. 13-[(2-O-b-D-Glucopyranosyl-b-D-glucopyranosyl) oxy]-kaur16-en-18-oic acid (6-O-b-D-xylopyranosyl-b-D-glucopyranosyl) ester (1) Colorless film; 1H NMR (500 MHz, CD3OD, d ppm) and 13C NMR (125 MHz, CD3OD, d ppm) spectroscopic data see Tables 1 and 2; +ESI TOFMS m/z 937.4316 (calcd. for C43H69O22: 937.4280). Enzymatic hydrolysis of 1: A solution of 1 (250 mg) was dissolved in 2.5 ml of 0.1 M sodium acetate buffer, pH 4.5 and crude pectinase from Aspergillus niger (50 ml, Sigma–Aldrich, P2736) was added. The mixture was stirred at 50 8C for 48 h. The product precipitated out during the reaction and was filtered and then crystallized from methanol (MeOH). The resulting steviol was identical to an authentic sample by TLC and 1H NMR. Acid hydrolysis of 1: To a solution of 1 (250 mg) in MeOH (1 ml) was added 1 ml of 5% H2SO4 and the mixture was refluxed for 8 h. The reaction mixture was then neutralized with saturated sodium carbonate and extracted with ethyl acetate (EtOAc) (2 5 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) (Bedir et al., 2001; Huan et al., 1998); the two sugars were identified as Dxylose and D-glucose.
4.6. 13-[(2-O-b-D-Glucopyranosyl-b-D-glucopyranosyl) oxy]-17oxo-kaur-15-en-18-oic acid b-D-glucopyranosyl ester (3) Colorless film; 1H NMR (500 MHz, CD3OD, d ppm) and 13C NMR (125 MHz, CD3OD, d ppm) spectroscopic data see Tables 1 and 2; +ESI TOFMS m/z 819.3668 (calcd. for C38H59O19: 819.3651). Sodium borohydride (NaBH4) reduction of 3: A small aliquot (100 mg) of 3 was treated with NaBH4 in MeOH (120 ml, 1 mg/ml) for 16 h at rt. The reaction was stopped by the addition of 4.0% aqueous TFA (5 ml). Analysis of the reaction mixture by LC–MS (Method 1, Clos et al., 2008) indicated that the product formed was identical to 2. 4.7. 13-[(2-O-b-D-Glucopyranosyl-b-D-glucopyranosyl) oxy] kaur15-en-18-oic acid, b-D-glucopyranosyl ester (4) Colorless film; 1H NMR (500 MHz, CD3OD, d ppm) and 13C NMR (125 MHz, CD3OD, d ppm) spectroscopic data see Tables 1 and 2; +ESI TOFMS m/z 805.3897 (calcd. for C38H61O18: 805.3858).
Acknowledgement We wish to thank Pure Circle (Kuala Lumpur, Malaysia) for providing the stevia extract SG-95.
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