Additional Minor Diterpene Glycosides from Stevia rebaudiana Bertoni

Article abstract of DOI:10.3390/molecules181113510

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-glu

Full text of DOI:10.3390/molecules181113510

Molecules 2013, 18, 13510-13519; doi:10.3390/molecules181113510
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Additional Minor Diterpene Glycosides from
Stevia rebaudiana Bertoni
†
Indra Prakash * and Venkata Sai Prakash Chaturvedula
Organic Chemistry Department, The Coca-Cola Company, Global Research and Development,
One Coca-Cola Plaza, Atlanta, GA 30313, USA
†
Current address: Natural Ingredient Development, Blue California, 30111 Tomas,
Rancho Santa Margarita, CA 92688, USA; E-Mail: saipc@bluecal-ingredients.com
*
Author to whom correspondence should be addressed; E-Mail: iprakash@coca-cola.com;
Tel.: +1-404-676-3007; Fax: +1-404-598-3007.
Received: 24 September 2013; in revised form: 26 October 2013 / Accepted: 28 October 2013 /
Published: 31 October 2013
Abstract: 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-
1
6-en-19-oic acid {β-D-glucopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)-β-D-gluco-
1
13
pyranosyl-ester} (2), on the basis of extensive 1D ( H- and C-) 2D NMR (COSY, HSQC
and HMBC) and MS spectroscopic data as well as chemical studies.
Keywords: Stevia rebaudiana; Compositae; Asteraceae; diterpenoid glycosides; spectral
data; chemical studies; structure characterization
1
. 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
(

Molecules 2013, 18
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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 [4–7]. 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 [9–11].
2
. 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-{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-gluco-
pyranosyl-(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-glucopyranosyl-ester} (2) (Figure 1).
Figure 1. Structures of 1, 2 and steviol (3).
1
2
3

Molecules 2013, 18
Compound 1 was obtained as a white powder and its molecular formula was deduced as C H O
88 32
13512
5
5
+
from its HRESI mass spectrum in the positive mode which showed a (M+H) ion at m/z 1261.5353;
1
3
1
this was supported by the C-NMR spectral data. The 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 [6–9].
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 [4–11]. 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
1
6
.38 in its 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,
81 and 319; suggesting the presence of a pentose and five hexose moieties in its structure. Acid
hydrolysis of 1 with 5% H SO afforded the sugars D-glucose and D-xylose, which were identified by
4
2
4
direct comparison with authentic samples by TLC [12–14]. 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
1
assignments for C-2 through C-6 of Glc
V
and GlcVI were made using the 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
1
comparison of 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
1
13
β-orientation, as reported for steviol glycosides [4–11]. The H- and 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
1
13
steviol. Comparison of the H- and 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
1
13
β-D-glucosyl and β-D-xylosyl moieties. The downfield shift for both the H and 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-
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}.

Molecules 2013, 18
13513
1
13
Table 1. H- and C-NMR chemical shift values for 1–2 isolated from Stevia rebaudiana
a–c
Bertoni recorded in d5-pyridine (C
D
5 5
N)
.
1
2
Position
1
13
1
13
H-NMR
C-NMR
40.0
19.4
38.0
43.9
57.0
23.1
42.2
39.2
54.0
41.3
19.9
38.2
87.5
42.9
46.3
153.9
104.6
27.9
176.7
16.4
94.4
77.1
88.2
69.8
78.3
H-NMR
C-NMR
39.9
19.3
38.1
44.0
57.0
23.2
42.3
39.7
54.0
41.4
19.8
38.1
87.4
42.9
46.1
153.7
104.5
27.9
177.0
16.6
94.5
76.5
88.2
69.7
78.0
1
2
3
4
5
6
7
8
9
0.75 br t (13.2), 1.76 m
0.77 br t (12.3), 1.79 m
1.37 m, 2.28 m
1.02 m, 2.29 m
−
1.35 m, 2.23 m
1.01 m, 2.29 m
−
1.02 br d (13.0)
1.06 br d (12.9)
2.24 m, 2.41 m
1.42 m, 1.81 m
−
2.07 m, 2.42 q (13.5)
1.37 m, 1.73 m
−
0.90 br d (8.1)
−
0.92 br d (7.7)
−
1
0
1
2
3
4
5
6
7
8
9
0
1
1
1
1
1
1
1
1
1
2
1.65 m, 1.75 m
1.86 m, 2.70 m
−
1.67 m, 1.76 m
1.81 m, 2.74 m
−
2.01 m, 2.72 m
1.88 d (17.0), 2.04 m
−
2.01 m, 2.75 m
1.88 m, 2.05 m
−
4.89 s, 5.69 s
1.29 s
4.89 s, 5.72 s
1.32 s
−
−
1.36 s
1.38 s
1
'
'
'
'
'
'
6.38 d (8.4)
4.38 m
6.41 d (8.2)
4.52 t (8.7)
5.14 t (8.7)
4.20 m
2
3
4
5
6
5.04 m
4.24 m
4.14 m
4.14 m
4.20 m
6
1.7
4.20 m, 4.30 m
61.7
4
.33 m
1
2
3
4
5
6
''
''
''
''
''
''
5.45 d (7.8)
4.13 m
96.0
81.0
87.6
70.1
77.4
62.4
104.5
75.3
78.2
72.9
77.3
63.6
103.8
75.2
5.49 d (7.9)
4.08 m
95.9
81.1
87.7
70.3
77.4
62.2
104.3
75.5
78.1
76.7
72.4
18.3
103.5
75.2
4.98 t (9.1)
5.01 m
4.08 t (9.1)
4.09 m
3.95 m
3.96 m
4.21 m, 4.35 m
5.48 d (7.9)
4.16 m
4.20 m, 4.32 m
5.39 d (8.0)
4.14 m
1
2
3
4
5
6
'''
'''
'''
'''
'''
'''
4.13 m
4.04 m
3.99 m
3.69 t (8.9)
3.47 dq (6.0, 8.9)
1.63 d (6.1)
5.48 d (7.9)
4.00 m
3.75 ddd (3.1, 6.5, 9.7)
4.28 m, 4.51 dd (1.1, 11.6)
5.46 d (7.5)
3.98 m
1
''''
''''
2

Molecules 2013, 18
13514
Table 1. Cont.
1
2
Position
1
13
1
13
H-NMR
C-NMR
H-NMR
C-NMR
77.4
71.0
77.6
61.7
103.9
75.0
78.0
73.3
77.4
63.6
103.9
75.1
77.6
70.6
77.7
61.7
3
4
5
6
''''
''''
''''
''''
4.47 t (8.6)
4.14 m
77.6
70.9
77.7
61.7
104.7
75.2
78.3
71.3
66.6
−
4.55 t (9.2)
4.18 m
3.99 m
4.01 m
4.20 m, 4.33 m
5.62 d (7.8)
4.17 m
4.20 m, 4.30 m
5.81 d (7.5)
4.25 m
1'''''
2'''''
3'''''
4'''''
5'''''
6'''''
4.12 m
4.18 m
4.32 m
4.10 m
3.54 t (11.0), 4.32 m
−
3.90 ddd (3.3, 6.8, 9.3)
4.32 m, 4.66 d (11.3)
5.30 d (8.0)
3.95 m
1''''''
2''''''
3''''''
4''''''
5''''''
6''''''
5.33 d (8.1)
3.97 m
103.9
75.2
77.5
70.9
77.8
61.8
4.38 m
4.33 m
4.11 m
4.09 m
3.87 m
3.82 m
4.10 m, 4.31 m
4.10 m, 4.32 m
a
b
assignments made on the basis of COSY, HSQC and HMBC correlations; Chemical shift values are in
c
δ (ppm); Coupling constants are in Hz.
Figure 2. Key COSY and HMBC correlations of 1.
+
The molecular formula of compound 2 was determined to be C56
90
H O32 by the [M+H] ion at
1
3
m/z 1,275.5485 in the positive ESI mass spectrum which was further supported by the C-NMR

Molecules 2013, 18
13515
1
spectral data. The 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
1
protons observed at δ 5.30, 5.39, 5.48, 5.49, 5.81, 6.41 in its 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-deoxygluco-
pyranosyl 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) as in 1. A
1
13
close comparison of the H- and C-NMR values of 2 with 1 and rebaudioside D suggested the
presence of three glucose units attached as a 2,3-branched β-D-glucotriosyl substituent at C-19
carboxylic acid with another β-D-glucosyl moiety at C-13 hydroxyl leaving the assignment of the
additional β-D-glucosyl and a β-D-deoxyglucosyl moieties. From the key COSY and HMBC
correlations shown in Figure 3, it was concluded that β-D-deoxyglucosyl and β-D-glucosyl units have
been connected at C-2 and C-3 positions respectively to the β-D-glucosyl moiety at C-13 position.
Thus, the structure of the novel compound 2 has been deduced as 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-glucopyranosyl-ester}.
Figure 3. Key COSY and HMBC correlation of 2.

Molecules 2013, 18
13516
3. Experimental
3.1. 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 8.71, δ 149.9 for pyridine-d ), chemical shifts are given in δ (ppm), and coupling constants
H
C
5
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.
3
.2. 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.
3
.3. Isolation
Isolation of the pure compounds 1–2 has been carried out using an Agilent 1100 HPLC
fractionation of a sample of rebaudioside M (VSPC-2973-6B, 2 g). The HPLC method involved using
Phenomenex Prodigy Column C (250 × 10 mm, 10 μm); UV Detection: 210 nm; Mobile Phase A:
1
8
H
2
O (0.1% TFA); Mobile Phase B: acetonitrile; Flow Rate: 20.0 mL/min; Injection volume: 1,400 μL.
The gradient increased from 75:25 (A:B) to 69:31 (A:B) over 20 min, and remained at 50:50 (A:B) for
min. The peaks eluting at t 17.18 and 18.29 min were collected over several injections and
5
R
concentration under vacuum furnished 1 (2.4 mg) and 2 (1.8 mg) respectively (Figure 4).
Figure 4. HPLC Trace of mixture of 1 and 2.

Molecules 2013, 18
13517
1
1
3-{β-D-Glucopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-oxy} ent-kaur-16-en-
9-oic acid {β-D-xylopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)]-O-β-D-glucupyranosyl-ester} (1);
1
13
White powder, H-NMR (500 MHz, C
5
D
5
N, δ ppm) and C-NMR (125 MHz, C
5
D
5
N, δ ppm)
+
spectroscopic data see Table 1; HRESIMS (M+H) m/z 1261.5353 (calcd. for C H O : 1261.5337).
5
5
89 32
1
3-{β-D-6-Deoxy-glucopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyloxy} ent-
kaur-16-en-19-oic acid {β-D-glucopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)-β-D-gluco-pyranosyl-
1
13
ester} (2); White powder, H-NMR (500 MHz, C D N, δ ppm) and C-NMR (125 MHz, C D N,
5
5
5
5
+
δ ppm) spectroscopic data see Table 1; HRESIMS (M+H) m/z 1275.5485 (calcd. for C56
275.5493).
91 32
H O :
1
Acid hydrolysis of 1 and 2. To a solution of compounds 1 and 2 (500 μg) in MeOH (1 mL) was added
% H SO (0.5 mL) and the mixture was refluxed for 24 h. The resulting reaction mixture was
5
2
4
extracted with ethyl acetate (EtOAc, 2 × 10 mL) after neutralization with saturated aqueous sodium
carbonate solution yields an aqueous fraction containing sugars. The sugars present in the aqueous
phase were identified as D-glucose and D-xylose in 1, and D-glucose and 6-deoxy-D-glucose in 2, by
comparing with standard sugars using the TLC systems EtOAc/n-butanol/water (2:7:1) and
CH Cl /MeOH/water (10:6:1) [12–14].
2
2
Determination of sugar configuration in 1 and 2. Each compound 1–2 (1 mg) was hydrolyzed with
.5 M HCl (2 mL) for 1.5 h. The reaction mixture was cooled to room temperature, passed through an
Amberlite IRA400 column and the eluate was lyophilized. The residue was dissolved in pyridine
1 mL) and heated with L-cysteine methyl ester HCl (5 mg) at 60 °C for 1.5 h, and then O-tolyl
0
(
isothiocyanate (25 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 C18, 150 × 4.6 mm (5 µL);
Mobile phase: 25% acetonitrile in water with 0.2% TFA, Flow rate: 1 mL/min; UV detection at
2
50 nm. The sugars were identified as D-glucose (t
D-glucose (t , 12.26 min) 6-deoxy-D-gluose (t , 11.34 min) in 2 [(authentic samples, D-glucose
, 12.64) and L-glucose (t , 11.36 min); D-xylose (t , 14.23) and L-xylose (t , 13.06 min); 6-deoxy-D-
glucose (t , 11.04) and 6-deoxy-L-glucose (t , 17.68)] [15].
R R
, 12.34 min) and D-xylose (t , 14.08) in 1, whereas
R
R
(
t
R
R
R
R
R
R
Enzymatic hydrolysis of 1 and 2. To a solution of each compound 1–2 (250 μg) was added 0.1 M
sodium acetate buffer, pH 4.5 (5 mL) and crude pectinase from Aspergillus niger (P2736, 50 μL,
Sigma-Aldrich, St. Louis, MO, USA). The mixture was stirred at 50 °C for 120 h. 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-16-en-19-oic acid (steviol, 3) by comparison of
1
their H-NMR spectral data and co-TLC [16].
4
. Conclusions
Two novel diterpenoid glycosides 1–2 were isolated from a commercial sample of rebaudioside M
of the leaves of S. rebaudiana Bertoni obtained from Pure Circle, Malaysia. The structure 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-

Molecules 2013, 18
13518
(
1→3)]-O-β-D-glucupyranosyl-ester} (1), and 13-{β-D-6-deoxyglucopyranosyl-(1→2)-O-[β-D-gluco-
pyranosyl-(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-glucopyranosyl-ester} (2), on the basis of spectroscopic and
chemical studies. The discovery of these two novel compounds is an important addition in expanding
our understanding of the diversity of the diterpenoid glycosides present in the S. rebaudiana Bertoni.
This is the first report of the isolation of the two novel diterpene glycosides 1–2 from S. rebaudiana
1
13
Bertoni and their complete H- and C-NMR spectral assignments that were made on the basis of
spectral (COSY, HSQC, HMBC, and MS/MS) and chemical studies.
Acknowledgments
We wish to thank AMRI, Bothell Research Center, Bothell, WA for obtaining selected NMR
spectral data.
Conflicts of Interest
The authors declare no conflict of interest.
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1.
2.
3.
4.
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Mosettig, E.; Beglinger, U.; Dolder, F.; Lichiti, H.; Quitt, P.; Waters, J.A. The absolute
configuration of steviol and isosteviol. J. Am. Chem. Soc. 1963, 85, 2305–2309.
Brandle, J.E.; Starrratt, A.N.; Gijen, M. Stevia rebaudiana: Its agricultural, biological and
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Sample Availability: Samples of the compounds 1–3 are available from the authors.
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