A new diterpene glycoside from Stevia rebaudiana

Article abstract of DOI:10.3390/molecules16042937

From the commercial extract of the leaves of Stevia rebaudiana, a new diterpene glycoside was isolated besides the known steviol glycosides including stevioside, rebaudiosides A-F, rubusoside and dulcoside A. The new compound was identified as 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-β-D-glucopyranosyl) ester (1) on the basis of extensive spectroscopic (NMR and MS) and chemical studies.

Full text of DOI:10.3390/molecules16042937

Molecules 2011, 16, 2937-2943; doi:10.3390/molecules16042937
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
A New Diterpene Glycoside from Stevia rebaudiana
Venkata Sai Prakash Chaturvedula * and Indra Prakash
The Coca-Cola Company, Organic Chemistry Department, Research and Technology, One Coca-Cola
Plaza, Atlanta, GA 30313, USA
* Author to whom correspondence should be addressed; E-Mail: vchaturvedula@na.ko.com;
Tel.: +1-404-676-9257; Fax: +1-404-598-9257.
Received: 18 February 2011; in revised form: 29 March 2011 / Accepted: 1 April 2011 /
Published: 4 April 2011
Abstract: From the commercial extract of the leaves of Stevia rebaudiana, a new diterpene
glycoside was isolated besides the known steviol glycosides including stevioside,
rebaudiosides A-F, rubusoside and dulcoside A. The new compound was identified as 13-
[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-
en-19-oic acid-(2-O--L-rhamnopyranosyl-β-D-glucopyranosyl) ester (1) on the basis of
extensive spectroscopic (NMR and MS) and chemical studies.
Keywords: Stevia rebaudiana; Compositae; Asteraceae; diterpenoid glycosides;
spectral data
1. 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”. The major
constituents in the leaves of S. rebaudiana are the potently sweet diterpenoid glycosides stevioside,
rebaudiosides A and D, and dulcoside A. These compounds, which are known as Stevia sweeteners,
are glycosides of the diterpene steviol (ent-13-hydroxykaur-16-en-19-oic acid) [1]. As a part of our
continuing research to discover natural sweeteners [2,3], we have reported several diterpene glycosides
from the commercial extract of S. rebaudiana. In this article, we present the isolation and structure
elucidation based on extensive spectroscopic (NMR and MS) and chemical studies of the new
diterpenoid glycoside 1, identified from the stevia extract (SG-95) obtained from Pure Circle (Kuala
Lumpur, Malaysia).

Molecules 2011, 16
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2. Results and Discussion
Purification of the commercial extract SG-95 obtained from the leaves of S. rebaudiana resulted in
the isolation of the new diterpenoid glycoside 1, and the known steviol glycosides, stevioside,
rebaudioside A (2), rebaudiosides B-F, rubusoside and dulcoside A (Figure 1). The structures of all the
known compounds were identified by comparison of their retention times with authentic standards
using the HPLC-MS method reported earlier [4] and the spectral data reported in the literature [4-11].
Figure 1. Structure of 1 and other compounds.
HO
HO
HO
O
O
HO
HO
sugar IV
O
O
sugar II
OH
O
HO
O
HO
HO
OH
20
sugar III
OH
13
14
CH2
17
11
9
CH3
1
CH2
16
CH3
2
10
8
H
15
H
4
5
18
H3C
O
HO
19
O
H3C
O
O
HO
HO
OH
OR
sugar I
3
1: R = -L-rha
2: R = H
Compound 1 was isolated as a colorless oil and its molecular formula was deduced as C50H80O27 on
the basis of its positive ESI mass spectrum, which showed an [M+H]+ ion at m/z 1,113.4977, together
with [M+NH4]+ and [M+Na]+ adducts at m/z 1,130.5243 and 1,135.4805, respectively. This
1
composition was supported by 13C-NMR spectral data. The H-NMR spectrum of 1 showed the
presence of two methyl singlets at δ 0.94 and 1.26, two olefinic protons of an exocyclic double bond as
singlets at δ 4.87 and 5.25, nine methylene and two methine protons between δ 0.85–2.27
characteristic for the ent-kaurane diterpenoids isolated earlier from the genus Stevia [7-9]. The basic
ent-kaurane diterpenoid skeleton 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 positive mode ESI MS/MS spectrum of 1
showed fragment ions at m/z 951, 789, 627 and 465, suggesting the presence of four hexose moieties.
The fragment ion observed at m/z 951 was further fragmented to an ion at m/z 805, suggesting an
additional deoxyhexose unit in its structure. The presence of five sugar units in its structure was

Molecules 2011, 16
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1
supported by the H-NMR spectrum, which showed the presence of anomeric protons at δ 4.62, 4.66,
4.86, 5.31, and 5.62.
Table 1. 1H- and 13C-NMR spectral data (chemical shifts and coupling constants) for 1 in CD3OD. a
Position
1
1H NMR
0.85 (m, 1H), 1.88 (m, 1H)
1.41 (m, 1H), 1.94 (m, 1H)
1.06 (m, 1H), 2.27 (m, 1H)
13C NMR
41.5
20.0
38.5
45.1
58.7
22.6
42.5
43.0
54.8
40.5
21.0
38.4
88.5
45.2
48.4
153.4
105.4
29.4
176.8
16.3
93.8
82.6
77.9
71.4
78.2
62.3
97.2
79.6
87.5
70.0
77.1
62.7
103.5
76.1
77.8
72.2
78.1
63.1
104.0
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
1′
1.10 (m, 1H)
1.88 (m, 1H), 1.94 (m, 1H)
1.43 (m, 1H), 1.55 (m, 1H)
1.00 (m, 1H)
1.65 (m, 1H), 1.80 (m, 1H)
1.53 (m, 1H), 1.95 (m, 1H)
1.53 (m, 1H), 2.25 (d, J = 12.2, 1H)
2.05 (m, 1H), 2.14 (d, J = 17.4, 1H)
4.87 (s, 1H), 5.25 (s, 1H)
1.26 (s, 3H)
0.94 (s, 3H)
5.62 (d, J = 7.4, 1H )
3.59 (m, 1H)
2′
3′
4′
5′
3.44 (m, 1H)
3.34 (m, 1H)
3.36 (m, 1H)
6′
3.60 (m, 1H), 3.82 (m, 1H)
4.62 (d, J = 7.8, 1H)
3.63 (m, 1H)
1′′
2′′
3′′
4′′
5′′
6′′
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
1′′′′
3.72 (m, 1H)
3.38 (m, 1H)
3.30 (m, 1H)
3.60 (m, 1H), 3.82 (m, 1H)
4.86 (d, J = 7.6, 1H)
3.18 (t, J = 8.2, 1H)
3.32 (m, 1H)
3.13 (m, 1H)
3.44 (m, 1H)
3.56 (m, 1H), 3.81 (m, 1H)
4.66 (d, J = 7.8, 1H)

Molecules 2011, 16
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Table 1. Cont.
2′′′′
3′′′′
4′′′′
5′′′′
6′′′′
1′′′′′
2′′′′′
3′′′′′
4′′′′′
5′′′′′
3.26 (m, 1H)
3.42 (m, 1H)
3.32 (m, 1H)
75.1
78.6
71.1
77.8
62.4
101.4
71.8
72.0
73.6
70.0
18.0
3.36 (m, 1H)
3.62 (m, 1H), 3.80 (m, 1H)
5.31 (d, J=1.5, 1H)
3.89 (m, 1H)
3.63 (m, 1H)
3.36 (m, 1H)
3.75 (m, 1H)
6′′′′′
1.24 (d, J = 6.3, 3H)
a
b
assignments made on the basis of COSY, HSQC and HMBC correlations; Coupling constants
are in Hz; c Chemical shift values are in δ (ppm).
Figure 2. Key COSY and HMBC correlations of 1.
HO
HO
sugar II
O
HO
O
HO
O
O
sugar IV
HO
OH
O
HO
HO
O
HO
sugar III
OH
20
CH3
COSY
13
14
CH2
17
11
9
1
16
HMBC
2
10
8
H
15
4
5
18
H3C
O
HO
19
O
sugar I
O
HO
HO
O
O
OH
sugar V
HO
OH
Enzymatic hydrolysis of 1 furnished an aglycone which was identified as steviol (3) by comparison
of 1H-NMR [10] and co-TLC with standard compound. Acid hydrolysis of 1 with 5% H2SO4 afforded
D-glucose and L-rhamnose, which were identified by direct comparison with authentic samples by
1
TLC [12-14]. The H- and 13C-NMR values for all the carbons in 1 were assigned on the basis of
COSY, HSQC and HMBC correlations (Table 1). Based on the results from NMR spectral data and
hydrolysis experiments of 1, it was concluded that there are four D-glucose and one L-rhamnose
moieties in its structure. A close comparison of the 1H- and 13C-NMR spectrum of 1 with rebaudioside

Molecules 2011, 16
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A (2) suggested that compound 1 is also a steviol glycoside which has three glucose residues that are
attached at the C-13 hydroxyl as a 2,3-branched β-D-glucotriosyl substituent and another glucose
moiety in the form of an ester at C-19 leaving the assignment of the additional rhamnose moiety. The
downfield shift for both the 1H and 13C chemical shifts at C-2′ suggested that the additional rhamnsoe
is attached at this position. This was confirmed by the key HMBC correlations: H-2′/C-1′, C-3′, C-1′′′′′
and H-1′′′′′/C-2′, C-2′′′′′, C-3′′′′′ (Figure 2).
The anomeric proton of the rhamnose residue was observed at δ 5.31 and had a coupling constant of
1.5 Hz, confirming that it has an -configuration, similar to dulcosides A and B [6]. The large coupling
constants observed for the four anomeric protons of the glucose moieties at δ 4.62 (d, J = 7.8 Hz), 4.66
(d, J = 7.8 Hz), 4.86 (d, J = 7.6 Hz), and 5.62 (d, J = 7.4 Hz), suggested their β-orientation as reported
for steviol glycosides [5-7]. Based on the results from chemical and spectral studies, 1 was assigned as
13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-en-19-
oic acid-(2-O--L-rhamnopyranosyl-β-D-glucopyranosyl) ester.
3. Experimental
3.1. General
NMR spectra were acquired on Bruker Avance DRX 500 MHz and Varian Unity Plus 600 MHz
instruments using standard pulse sequences. The spectra were referenced to the residual solvent signal
(H 3.30, C 49.0 for CD3OD), chemical shifts are given in  (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
positive-ion mode and ThermoFisher Discovery OrbiTrap in the positive mode electrospray. 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 × 21.2 mm, 5 m). Semi-preparative HPLC was carried
out with a Waters 600E multisolvent delivery system using a Phenomenex Synergi Hydro RP column
(250 × 10 mm, 4 m) column.
3.2. Plant Material
Stevia extract SG-95, 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 voucher
specimen is deposited at The Coca-Cola Company, No. VSPC-3166-002.
3.3. Isolation
Preliminary separation of the crude stevioside extract was carried out using a preparative HPLC
method employing a water/acetonitrile (B) gradient (25% B for 8.5 min, 25 to 29% B over 1.5 min, 29
to 30% B over 5.5 min, 30 to 34% B over 2.0 min, 34% B for 6 min, 34 to 52% B over 2.0 min, 52% B
for 3.0 min, 52 to 70% B over 1.0 min, 70% B for 5.5 min) at 20 ml/min. The baseline fraction at
16.8 min was collected and dried by rotary evaporation under reduced pressure as the crude impurity
fraction. Final fractionation was then performed using HPLC method by injecting the crude impurity

Molecules 2011, 16
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fraction (over several runs) on a Synergi Hydro RP column by semi-preparative HPLC using a gradient
of water (0.01156% acetic acid, 0.02844% ammonium acetate) in acetonitrile (25% B for 8.5 min,25 to
29% B over 1.5 min, 29 to 30% B over 5.5 min, 30 to 34% B over 2.0 min, 34% B for 6 min, 34 to
52% B over 2.0 min, 52% B for 3.0 min, 52 to 70% B over 1.0 min, 70% B for 5.5 min) at 5 mL/min
to yield 1 (tR 10.6 min, 1.6 mg). All the known compounds were identified in comparison of their
retention times with authentic standards using the HPLC-MS method as described previously [4] and
the spectral data that were reported in the literature [4-11].
13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-en-19-
oic acid-(2-O--L-rhamnopyranosyl-β-D-glucopyranosyl) ester (1). Colorless film; 1H-NMR (CD3OD,
δ ppm) and 13C-NMR (CD3OD, δ ppm) spectroscopic data, see Table 1; +ESI TOFMS m/z 1,113.4977
(calcd. for C50H81O27: 1,113.4965).
Enzymatic hydrolysis of 1. A solution of 1 (250 g) was dissolved in 0.1 M sodium acetate buffer, pH
4.5 (2.5 mL) and crude pectinase from Aspergillus niger (50 µL, Sigma-Aldrich, P2736) was added.
The mixture was stirred at 50o C for 48 hr. The product precipitated out during the reaction and was
filtered and then crystallized from methanol (MeOH). The resulting steviol (3) was identical to an
authentic sample by TLC and 1H-NMR.
Acid Hydrolysis of 1. To a solution of 1 (250 μg) in MeOH (1 mL) was added 5% H2SO4 (1 mL) and
the mixture was refluxed for 8 hours. 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 system EtOAc/n-butanol/water (2:7:1) and
CH2Cl2/MeOH/water (10:6:1) [12-14]; the two sugars were identified as L-rhamnose and D-glucose.
4. Conclusions
A new diterpenoid glycoside 1, as well as nine known steviol glycosides – stevioside, rebaudiosides
A-F, rubusoside and dulcoside A – were isolated from the commercial extract obtained from the leaves
of S. rebaudiana obtained from Pure Circle (Kuala Lumpur, Malaysia). The new compound was
identified as 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-
kaur-16-en-19-oic acid-(2-O--L-rhamnopyranosyl-β-D-glucopyranosyl) ester on the basis of 2D NMR
and +EI TOF MS, as well as chemical studies.
Acknowledgements
We wish to thank Pure Circle (Kuala Lumpur, Malaysia) for providing the stevia extract SG-95.
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Sample Availability: Samples of the steviol glycosides stevioside, rebaudiosides A-F, rubusoside and
dulcoside A are available from the authors.
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