Diterpene glycosides from stevia rebaudiana

Article abstract of DOI:10.3390/molecules16053552

Three novel diterpene glycosides were isolated for the first time from the commercial extract of the leaves of Stevia rebaudiana, along with several known steviol glycosides, namely stevioside, rebaudiosides A-F, rubusoside and dulcoside A. The new compounds were identified as 13-[(2-O-β-D- glucopyranosyl-3-O-β-D-glucopyranosyl-β-Dglucopyranosyl) oxy] ent-kaur-15-en-19-oic acid (1), 13-[(2-O-β-D-glucopyranosyl-3-O-β- D-glucopyranosyl-β-D-glucopyranosyl)oxy]-16β-hydroxy-ent-kauran-19- oic acid (2) and 13-methyl-16-oxo-17-nor-ent-kauran-19-oic acid-β-D- glucopyranosyl ester (3) on the basis of extensive 2D NMR and MS spectroscopic data as well as chemical studies.

Full text of DOI:10.3390/molecules16053552

Molecules 2011, 16, 3552-3562; doi:10.3390/molecules16053552
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Diterpene Glycosides from Stevia rebaudiana
Venkata Sai Prakash Chaturvedula *, Mani Upreti 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: 24 March 2011; in revised form: 16 April 2011 / Accepted: 20 April 2011 /
Published: 28 April 2011
Abstract: Three novel diterpene glycosides were isolated for the first time from the
commercial extract of the leaves of Stevia rebaudiana, along with several known steviol
glycosides, namely stevioside, rebaudiosides A-F, rubusoside and dulcoside A. The new
compounds were identified as 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-
glucopyranosyl)oxy] ent-kaur-15-en-19-oic acid (1), 13-[(2-O-β-D-glucopyranosyl-3-O-β-
D-glucopyranosyl-β-D-glucopyranosyl)oxy]-16β-hydroxy-ent-kauran-19-oic acid (2) and
13-methyl-16-oxo-17-nor-ent-kauran-19-oic acid-β-D-glucopyranosyl ester (3) on the basis
of extensive 2D NMR and MS spectroscopic data as well as chemical studies.
Keywords: Stevia rebaudiana; Compositae; Asteraceae; diterpenoid glycosides; spectral
data; chemical studies
1. Introduction
Stevia rebaudiana (Bertoni) 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 countries,
particularly in 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 diterpenoid
glycosides stevioside, rebaudiosides A and D, and dulcoside A. These compounds, which are known as
Stevia sweeteners, are the glycosides of the diterpene steviol, ent-13-hydroxykaur-16-en-19-oic acid [3].

Molecules 2011, 16
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As a part of our continuing research to discover natural sweeteners [4-5], we have collected
commercial extracts of S. rebaudiana from various suppliers all over the World. This paper describes
the isolation of three diterpene glycosides 1-3 isolated for the first time from the crude extract of the
leaves of S. rebaudiana obtained from ShenZhen NII Natural Food Ingredient Co. Ltd, China and their
identification based on extensive spectroscopic (NMR and MS) and chemical studies.
2. Results and Discussion
Purification of the commercial extract of S. rebaudiana obtained as Lot No: 20071003 from
ShenZhen NII Natural Food Ingredient Co. Ltd, China, resulted in the isolation of three new
diterpenoid glycosides 1-3, and the known steviol glycosides, stevioside, rebaudiosides A-F,
rubusoside and dulcoside A (Figure 1). The structures of the known compounds were identified in
comparison of their retention times with authentic standards using the HPLC-MS method as described
previously [6] and the spectral data that were reported in the literature [7-13].
Figure 1. Structures of 1-3.
Compound 1 was obtained as a white powder and its molecular formula was assigned as C38H60O18
from its HRESI mass spectrum, which showed a (M + Na)+ ion at m/z 827.3661; this was supported by
the 13C-NMR spectral data. The 1H-NMR spectrum of 1 (Table 1) showed the presence of three methyl
singlets at δ 0.99, 1.17, and 1.72, eight methylene and two methine protons between δ 0.86-2.23, and a
trisubstituted olefinic proton at δ 5.14, similar to ent-13-hydroxykaur-15-en-19-oic acid [15]. The basic
skeleton of 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; H-14/C-8, C-9, C-13, C-15, C-16

Molecules 2011, 16
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and H-17/C-13, C-15, C-16) correlations (Figure 2). In addition, the 1H-NMR spectrum of 1 also showed
three anomeric protons as doublets at δ 4.66 (J = 7.8 Hz), 4.85 (J = 7.8 Hz), and 4.82 (J = 7.8 Hz)
suggesting the presence in its structure of three sugar units, which was supported by the fragment ions
observed at m/z 643, 481, and 319 in the ESI MS/MS spectrum corresponding to the successive loss of
three hexose moieties from its [M + H]+ ion. Enzymatic hydrolysis of 1 furnished a compound which
was found identical to ent-13-hydroxykaur-15-en-19-oic acid on the basis of NMR spectral data [13-14].
Acid hydrolysis of 1 afforded D-glucose that was identified by preparing the corresponding
thiocarbamoyl-thiazolidine carboxylate derivative with L-cysteine methyl ester and O-tolyl
isothiocyanate, and in comparison of its retention time with the standard sugars as described in the
literature comparison [15].
1
Table 1. H-NMR chemical shift values for 1–3 isolated from Stevia rebaudiana recorded
in CD3OD a.
Position
1
0.86 (m, 1H), 1.86 (m,
1H)
2
0.86 (m, 1H), 1.86 (m,
1H)
3
0.97 (m, 1H), 1.71 (m,
1H)
1
2
3
1.39 (m, 1H), 1.90 (m,
1H)
1.42 (m, 1H), 1.94 (m,
1H)
1.38 (m, 1H), 1.90 (m,
1H)
1.02 (m, 1H), 2.10 (d,
11.9, 1H)
1.00 (m, 1H), 2.12 (d,
12.6, 1H)
1.08 (m, 1H), 2.18 (d,
13.7, 1H)
5
6
1.08 (m, 1H)
1.52 (m, 1H), 1.83 (m,
1H)
1.07 (m, 1H)
1.22 (m, 1H)
1.90 (m, 2H)
1.82 (m, 1H), 1.97 (m,
1H)
7
1.50 (m, 1H), 1.58 (m,
1H)
1.37 (m, 1H), 1.58 (m,
1H)
1.49 (m, 1H), 1.68 (m,
1H)
9
0.86 (m, 1H)
1.50 (m, 1H), 1.69 (m,
1H)
0.93 (t, J = 7.8 Hz, 1H)
1.65 (m, 1H), 1.85 (m,
1H)
1.24 (m, 1H)
11
1.23 (m, 1H), 1.68 (m,
1H)
12
14
15
1.63 (m, 1H), 1.67 (m,
1H)
1.80 (m, 1H), 1.04 (m,
1H)
1.43 (m, 1H), 1.53 (m,
1H)
1.68 (m, 1H), 2.23 (d,
10.0, 1H)
1.84 (m, 1H), 1.98 (d,
10.0, 1H)
1.45 (m, 1H), 1.56 (m,
1H)
5.14 (s, 1H)
1.42 (d, 13.7, 1H), 1.60 1.84 (m, 1H), 2.63 (dd,
(d, 13.7, 1H)
1.27 (s, 3H)
1.16 (s, 3H)
0.98 (s, 3H)
4.66 (d, 8.2, 1H)
3.56 (m, 1H)
3.74 (m, 1H)
3.34 (m, 1H)
3.30 (m, 1H)
3.3, 18.9, 1H)
17
19
20
1′
1.72 (s, 3H)
1.17 (s, 3H)
0.99 (s, 3H)
4.66 (d, 7.8, 1H)
3.57 (m, 1H)
3.77 (m, 1H)
3.36 (m, 1H)
3.28 (m, 1H)
0.81 (s, 3H)
1.23 (s, 3H)
0.94 (s, 3H)
5.39 (d, 8.2, 1H)
3.33 (dd, 7.5, 8.0, 1H)
3.43 (dd, 8.2, 8.9, 1H)
3.32 (dd, 8.4, 9.2, 1H)
3.36 (ddd, 8.4, 1.8, 6.9,
1H)
2′
3′
4′
5′
6′
3.64 (m, 1H), 3.80 (m,
1H)
3.67 (m, 1H), 3.88 (m,
1H)
3.67 (dd, 2.1, 11.4, 1H),
3.80 (dd, 4.2, 12.2, 1H)

Molecules 2011, 16
3555
Table 1. Cont.
1′′
2′′
3′′
4′′
5′′
6′′
4.85 (d, 7.8, 1H)
3.24 (m, 1H)
3.32 (m, 1H)
3.33 (m, 1H)
3.33 (m, 1H)
3.56 (m, 1H), 3.81 (m,
1H)
4.89 (d, 7.8, 1H)
3.25 (m, 1H)
3.42 (m, 1H)
3.32 (m, 1H)
3.36 (m, 1H)
3.60 (m, 1H), 3.80 (m,
1H)
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
4.72 (d, 7.8, 1H)
3.27 (m, 1H)
3.33 (m, 1H)
3.32 (m, 1H)
3.36 (m, 1H)
3.67 (m, 1H), 3.71 (m,
1H)
4.74 (d, 7.8, 1H)
3.25 (m, 1H)
3.32 (m, 1H)
3.32 (m, 1H)
3.36 (m, 1H)
3.65 (m, 1H), 3.80 (m,
1H)
a assignments made on the basis of COSY, HSQC and HMBC correlations; b Chemical shift values
are in δ (ppm); c Coupling constants are in Hz.
Figure 2. Key COSY and HMBC correlations of 1.
The 1H- and 13C-NMR values for all the protons and carbons were assigned on the basis of COSY,
HSQC and HMBC correlations (Figure 2) and are given in Tables 1 and 2. A close comparison of the
1H- and 13C-NMR values of 1 with those of rebaudioside A suggested a 2,3-branched β-D-glucotriosyl
substituent at C-13 as well as the absence of a glucosyl unit at C-19 and migration of the exocyclic
double bond from C-16/C-17 to C-15/C-16. This was supported by the 13C-NMR values for the
trisubstituted double bond between C-15 and C-16 which were observed at δ 136.1 and 142.6,
respectively, and also from the HMBC correlations: H-12/C-9, C-11, C-13, C-14, C-16; H-15/C-8,
C-9, C-14, C-16, C-17 and H-17/C-13, C-15, C-16. The large coupling constants observed for the
three D-glucose anomeric protons suggested the β-orientation as reported for steviol glycosides [6-13].
Thus, structure of 1 was established as 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-
glucopyranosyl)oxy] ent-kaur-15-en-19-oic acid.

Molecules 2011, 16
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13
Table 2. C NMR chemical shift values for 1–3 isolated from Stevia rebaudiana recorded
in CD3OD a.
Position
1
1
2
3
40.9
19.1
37.9
43.5
56.5
20.9
39.6
48.3
48.0
39.8
20.6
30.4
91.4
48.1
136.1
142.6
11.4
180.4
29.0
15.3
96.6
80.1
87.5
70.2
77.3
61.4
103.1
74.2
77.3
70.3
77.1
61.4
102.5
73.8
77.1
70.3
77.4
61.5
42.0
19.0
38.0
43.5
55.3
21.2
39.3
41.8
55.2
40.6
20.2
37.2
86.8
43.4
56.9
76.8
21.8
180.3
28.3
15.1
95.8
79.8
86.8
69.8
77.2
61.5
103.0
74.0
77.4
70.2
77.2
61.3
102.6
74.6
77.0
70.4
77.2
61.3
39.7
19.2
38.1
43.8
57.4
20.8
41.3
48.6
54.0
37.8
21.2
38.7
94.4
54.7
48.1
224.0
12.9
176.9
27.8
19.0
95.3
73.8
77.4
69.9
77.5
61.2
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
1′
2′
3′
4′
5′
6′
1′′
2′′
3′′
4′′
5′′
6′′
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
a assignments made on the basis of HSQC and HMBC correlations; b Chemical shift values are in δ (ppm).
The molecular formula of compound 2 was determined to be C38H62O19 from the [M + H]+ ion at
m/z 823, together with an [M + Na]+ adduct at m/z 845 in the positive ESI mass spectrum, which was
1
confirmed by the HRMS data. The H-NMR spectrum of 2 (Table 1) showed the presence of three

Molecules 2011, 16
3557
methyl singlets, nine methylene and two methine protons. In the absence of any unsaturated protons
together with the appearance of a methyl group at δ 1.27 indicated that the structure of 2 should be
similar to ent-13,16-dihydroxykauran-19-oic acid [13-14] which was supported by the δC 76.8
corresponding to the tertiary hydroxyl at C-16. The presence of ent-13,16-dihydroxykaurane skeleton
in 2 was supported by the key HMBC correlations: H-12/C-11, C-13, C-14, C-16; H-14/C-8, C-12,
C-13, C-15, C-16; H-17/C-13, C-15, C-16 (Figure 3). The ESI MS/MS spectrum of 2 showed the
fragment ions at m/z 661, 499, and 337 corresponding to the successive loss of three hexose moieties
from its [M + H]+ ion and this was supported by the three anomeric protons observed at δ 4.66, 4.74
1
and 4.89 in its H-NMR spectral data. Acid hydrolysis of 2 performed as in 1 afforded D-glucose
1
13
confirming the three sugar molecules present in 2 as glucosyl units. The H- and C-NMR values for
all the protons and carbons were assigned on the basis of COSY, HMQC and HMBC correlations
(Tables 1 and 2).
Figure 3. Key COSY and HMBC correlation of 2.
Enzymatic hydrolysis of 2 furnished a compound which was found identical to ent-13,
16β-dihydroxykauran-19-oic acid on the basis of NMR spectral data comparisons [13-14]. A close
1
comparison of the H- and 13C-NMR values of 2 with those of 1 suggested the presence of three
glucose units attached as a 2,3-branched β-D-glucotriosyl substituent at C-13 hydroxyl of ent-13,
16β-dihydroxykauran-19-oic acid. The large coupling constants observed for the three anomeric
protons of the glucose moieties at δ 4.66 (d, J = 8.2 Hz), 4.74 (d, J = 7.8 Hz), and 4.89 (d, J = 7.8 Hz),
suggested the β-orientation as in 1. Considering the stereochemistry for the 16-hydroxyl group in 2 as
β on the basis of the acid obtained by enzymatic hydrolysis, the structure was thus deduced to be
13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]-16β-hydroxy ent-kauran-
19-oic acid.
The molecular formula of compound 3 was deduced as C26H40O8 from the [M + H]+ ion observed at
m/z 481, together with an [M + Na]+ adduct at m/z 503 in the positive ESI mass spectrum and this was
supported by the HRMS data. The 1H-NMR spectrum of 3 (Table 1) also showed the presence of three
methyl singlets at δ 0.81, 0.94 and 1.23; nine methylene and two methine protons, similar to 2.

Molecules 2011, 16
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Enzymatic hydrolysis of 3 furnished a compound which was found identical to isosteviol on the basis
of its NMR spectral data, identical to the reported values in the literature [14]. The presence of
isosteviol skeleton was supported by the key HMBC correlations: H-12/C-9, C-11, C-13, C-14, C-16,
C-17; H-14/C-8, C-12, C-13, C-15, C-16, C-17; H-15/C-8, C-14, C-16; H-17/C-13, C-14, C-16. The
1H NMR spectrum also showed an anomeric proton as doublet at δ 5.39 (J = 8.2 Hz), suggesting the
presence of one sugar residue in its structure. Acid hydrolysis of 3 afforded D-glucose, confirming the
sugar unit present in 3 as glucose as in 1 and 2. The placement of the glucose moiety in 3 was assigned
1
13
to be at C-19 based on the key COSY and HMBC correlations shown in Figure 4. The H and C
NMR values for all the protons and carbons were assigned on the basis of COSY, HMQC and HMBC
correlations (Tables 1 and 2). Based on the results from chemical and spectral studies, 3 was assigned
as 13-methyl-16-oxo-17-nor-ent-kauran-19-oic acid-β-D-glucopyranosyl ester.
Figure 4. Key COSY and HMBC correlations of 3.
20
CH3
13
O
11
9
16
1
2
10
O
8
H
15
4
COSY
18
H3C
O
HO
19
1'
5'
3'
O
HMBC
HO
HO
OH
This is the first report of the occurrence of the diterpene glycosides 1-3 from S. rebaudiana in
nature, which were reported earlier as rebaudioside A derivative/degradation products [16-17]. Also,
this is the first report of their complete 1H- and 13C-NMR spectral assignments that were made on the
basis of spectral (COSY, HSQC, HMBC, and MS/MS) and chemical studies.
3. Experimental
3.1. General
Melting points were measured using a SRS Optimelt MPA 100 instrument and are uncorrected.
Optical rotations were recorded using a Rudolph Autopol V at 25 °C and 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 (δH 3.30, δC 49.0 for CD3OD),
chemical shifts are given in δ (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 Thermo Fisher
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 μm).

Molecules 2011, 16
3559
3.2. Plant Material
A commercial Stevia extract with Lot No.: 20071003 was obtained from ShenZhen NII Natural
Food Ingredient Co. Ltd, China. A voucher specimen is deposited at The Coca-Cola Company, No.
VSPC-3166-153.
3.3. Isolation
Preliminary separation of the crude extract from the leaves of S. rebaudiana SRA 40, Lot No.
20071003 consisting of a mixture of various diterpenoid glycosides including 40-60% rebaudioside A
was carried out via preparative HPLC on an Agilent 1100 system. The HPLC method A involved using
Phenomenex Prodigy Column C18 (250 × 10 mm, 10 μm); UV Detection: 220 nm; Mobile Phase A:
H2O (0.1% TFA); Mobile Phase B: Acetonitrile; Flow Rate: 5.0 mL/min; Injection volume: 400 μL.
The gradient increased from 95: 5 (A: B) to 0: 100 (A: B) over 30 min, and remained at 0: 100 (A: B)
for 5 min. Removal of the dominant rebaudioside A (tR 17.13 min) using the above HPLC method and
subsequent collection of the peak eluting at tR 14.60 min over multiple runs furnished 1 (4.6 mg). The
peak eluting at tR 12.86 min contained a mixture of 2 along with several other compounds. Since 2 was
only partially resolved under the first HPLC method, a second round of purification was applied to
isolate the pure compound. This was carried out using HPLC method B which was a modification to
HPLC method A using the gradient of 80: 20 (A: B) to 70: 30 (A: B) applied for 40 min. The peak
eluted at tR 32.4 min was confirmed to correspond to 2 (3 mg). Similar to 1, removal of rebaudioside A
and combining the peak eluted at tR 18.2 min using HPLC method A over multiple runs and
concentration under vacuum furnished 3 (4.2 mg). All the known compounds were identified using
HPLC-MS in comparison with authentic standards as described previously [6] and the spectral data
that were reported in the literature [7-13].
13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-15-en-19-oic
25
1
acid (1). White powder, mp 254.5-254.7 °C, [α]D -49.56 (c 1.0, MeOH); H-NMR (500 MHz,
13
CD3OD, δ ppm) and C-NMR (125 MHz, CD3OD, δ ppm) spectroscopic data see Tables 1 and 2;
HRMS (M + Na)+ m/z 827.3661 (calcd. for C38H60O18Na: 827.3677)
Enzymatic hydrolysis of 1. A solution of 1 (250 μg) 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 50 °C for 48 hr. The product precipitated out during the reaction and was
filtered and then crystallized from methanol (MeOH). The resulting product was identical to
ent-13-hydroxykaur-15-en-19-oic acid by comparison of their 1H-NMR spectral data [13-14].
Acid Hydrolysis of 1. Compound 1 (500 μg) was hydrolyzed with 0.5 M HCl (0.5 mL) for 1.5 h. After
cooling, the mixture was passed through an Amberlite IRA400 column and the eluate was lyophilized.
The residue was dissolved in pyridine (0.25 mL) and heated with L-cysteine methyl ester HCl (2.5 mg)
at 60 °C for 1.5 h, and then O-tolyl isothiocyanate (12.5 μL) was added to the mixture and heated at 60
°C for an additional 1.5 h. The reaction mixture was analyzed by HPLC: column Phenomenex Luna

Molecules 2011, 16
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C18, 150 × 4.6 mm (5 u); 25% acetonitrile-0.2% TFA water, 1 mL/min; UV detection at 250 nm. The
sugar was identified as D-glucose (tR, 12.26 min) [authentic samples,
D-glucose (tR, 12.35) and L-glucose (tR, 11.12 min)] [15].
13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]-16β-hydroxy-ent-
25
1
kauran-19-oic acid (2). White powder, mp 220.4-221.2 °C, [α]D -32.60 (c 1.0, MeOH); H-NMR
(500 MHz, CD3OD, δ ppm) and 13C-NMR (125 MHz, CD3OD, δ ppm) spectroscopic data see Tables 1
and 2; HRMS (M + Na)+ m/z 845.3765 (calcd. for C38H62O19Na: 845.3783)
Enzymatic hydrolysis of 2. Enzymatic hydrolysis of 2 (500 μg) as described above furnished ent-13,
16β-dihydroxykauran-19-oic acid which was confirmed on the basis of 1H NMR spectral data [13,14].
Acid Hydrolysis of 2. Hydrolysis of 2 (500 μg) as described above furnished D-glucose [15].
13-methyl-16-oxo-17-nor-ent-kauran-19-oic acid-β-D-glucopyranosyl ester (3). White powder, mp
172.5 °C, [α]D25 -55.83 (c 1.0, EtOH); 1H-NMR (500 MHz, CD3OD, δ ppm) and 13C-NMR (125 MHz,
CD3OD, δ ppm) spectroscopic data see Tables 1 and 2; HRMS (M + Na)+ m/z 503.2608 (calcd. for
C26H40O8Na: 503.2621)
Enzymatic hydrolysis of 3. Enzymatic hydrolysis of 3 (500 μg) as described above furnished isosteviol
which was confirmed on the basis of 1H-NMR spectral data [14].
Acid Hydrolysis of 3. Hydrolysis of 2 (500 μg) as described above furnished D-glucose [15].
4. Conclusions
Three diterpenoid glycosides 1-3 were isolated from a commercial extract of the leaves of S.
rebaudiana obtained from ShenZhen NII Natural Food Ingredient Co. Ltd, China, along with the
known steviol glycosides stevioside, rebaudiosides A-F, rubusoside and dulcoside A. The new
compounds were identified as 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-gluco-
pyranosyl)oxy] ent-kaur-15-en-19-oic acid, 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-
D-glucopyranosyl)oxy]-16β-hydroxy-ent-kauran-19-oic acid, 13-methyl-16-oxo-17-nor-ent-kauran-19-
oic acid-β-D-glucopyranosyl ester on the basis of spectroscopic and chemical studies. This is the first
report of the isolation of these three diterpene glycosides from S. rebaudiana in nature. 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.
Acknowledgements
We wish to thank ShenZhen NII Natural Food Ingredient Co. Ltd, China for providing the Stevia
extract.

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Sample Availability: Samples of the new diterpene glycoside 1-3, and the known steviol glycosides
stevioside, rebaudiosides A-F, rubusoside and dulcoside A are available from the authors.
© 2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).