212
V.S.P. Chaturvedula et al. / Phytochemistry Letters 4 (2011) 209–212
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]-17-
hydroxy-kaur-15-en-18-oic acid
b-D-glucopyranosyl ester (2)
4.3. Isolation
Colorless film; 1H NMR (500 MHz, CD3OD,
(125 MHz, CD3OD, 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 g) as described above
furnished -glucose.
d
ppm) and 13C NMR
d
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 semi-
preparative HPLC using a gradient of water (0.01156% acetic
acid, 0.02844% ammonium acetate) in acetonitrile (25% B for
8.5 min, 25–100% 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), rebau-
dioside 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).
m
D
4.6. 13-[(2-O-
b
-D
-Glucopyranosyl-
b-D-glucopyranosyl) oxy]-17-
oxo-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, ppm) spectroscopic data see Tables 1 and 2;
d
+ESI TOFMS m/z 819.3668 (calcd. for C38H59O19: 819.3651).
Sodium borohydride (NaBH4) reduction of 3: A small aliquot
(ꢁ100
for 16 h at rt. The reaction was stopped by the addition of 4.0%
aqueous TFA (5 l). Analysis of the reaction mixture by LC–MS
mg) of 3 was treated with NaBH4 in MeOH (120 ml, 1 mg/ml)
m
(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] kaur-
15-en-18-oic acid,
b
-D-glucopyranosyl ester (4)
Colorless film; 1H NMR (500 MHz, CD3OD,
d
ppm) and 13C NMR
(125 MHz, CD3OD, ppm) spectroscopic data see Tables 1 and 2;
d
+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
4.4. 13-[(2-O-
b
-D
-Glucopyranosyl-
b-
D-glucopyranosyl) oxy]-kaur-
16-en-18-oic acid (6-O-
b
-
D
-xylopyranosyl- -glucopyranosyl)
b-D
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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,
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ester (1)
Colorless film; 1H NMR (500 MHz, CD3OD,
(125 MHz, CD3OD, 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 g) was dissolved
in 2.5 ml of 0.1 M sodium acetate buffer, pH 4.5 and crude
pectinase from Aspergillus niger (50 l, Sigma–Aldrich, P2736) was
d
ppm) and 13C NMR
d
m
Clos, J.F., DuBois, G.E., Prakash, I., 2008. Photostability of rebaudioside A and
stevioside in beverages. J. Agric. Food Chem. 56, 8507–8513.
m
Huan, V.D., Yamamura, S., Ohtani, K., Yamasaki, R., Nham, K.N.T., 1998. Oleanane
saponins from Polyscias fructicosa. Phytochemistry 47, 451–457.
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glucosides from Stevia rebaudiana. Phytochemistry 15, 981–983.
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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
xylose and -glucose.
D-
D