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Steviol-glycosides: New natural sweeteners
trends m analytzal chcmrrtry, vol. I, tlb. 11,1xu
246
Steviol-alvcosides: cI_=
_------
_____~_
__
new natural ~~_
~~
~~_
~_
_
sweeteners _
_
_
The need for a safe, non-nutritive sweetener for the diabetic and the diet conscious has prompted the further investigation of new sweeteners of plant origin. This review deals with the chemistry of sweet steviol-glycosides, which are currently used as a food-additive in Japan. Osamu Tanaka Hiroshima, Japan In Paraguay and South Africa the natives use the leaves of Steuia rebaudianaBertoni (Compositae) , a wild herb, as a sweetening agent for their tea and coffee. A major sweet glucoside of this plant, steviosidel, was isolated about 50 years ago and its chemical structure was elucidated by Mosettig, Fletcher and their coworkers, in 1963 (see Table I). The genuine aglycone (named steviol) is a kaurane-type diterpene acid, 13-hydroxy-ent-kaur- 16-en- 19-oic acid and forms isosteviol in acid. Recently, this plant has attracted commercial attention as a new source of non-cariogenic and noncarcinogenic dietary-sweetener and is now cultivated, not only in Japan, but all over South-East Asia; about 300 tons of its dried leaves were harvested in 1979 (the cost of these in 1980 was 700-l ,000 yen/kg). In Japan, eight food-manufacturing companies are now engaged
in extracting sweet-glycosides and other chemicals from steuia for use as food-additives in soy-sauce, pickled vegetables, fruit-juice etc.
New sweet glycosides It has already been mentioned that the crude glycoside-fraction of Stevia-leaves tastes sweeter and less bitter than the purified stevioside. We have isolated four additional sweet steviol-glycosides from these leaves and have designated them rebaudiosides -A, -C, -D and -E; their structures are illustrated in Table 12-4. Mitsuhashi et al. have also isolated two new steviol-glycosides - dulcoside-A and -B (Ref. 5), the latter of which is identical with our rebaudioside-C4. Of these glycosides, rebaudiosides A and C were isolated in yields of 3.0 and 0.4%, respectively. With regard to taste, the rebaudiosides A, D and E are sweeter and less bitter than stevioside (see Table II). It follows that the sweetness of Steviu-leaves must be due to both stevioside and rebaudioside A. It is interesting TABLE
A
g’c
Rebaudioside
C
glc
g$v;z;cm
glc H
B
Rebaudioside
D
glc_2--rglc
glc~glc
Rebaudioside Dulcoside A Rubusoside
E
glc~glc
- ‘lglc gl&glc glcl-rham
glc = fi-D-glucopyranosyl .oO
glc QgIc H glc~glc “\I glc
Stevioside Steviol Rebaudioside
= dulcoside
0 165-9936/82/MM0-OOCQ/f01
I.
glc glc
glc
rham = cw-L-rhamnopyranosyl @ 1982 Elscvier Scientific Publish+ Company
trends in analytical chemistry, VOI.I, no. II, 1982
247
that our research group has been able to convert stevioside into this rebaudioside by a combination of enzymic and chemical procedures”,7. The flower-buds of Stevia rebaudiana also contain these two glycosides but no sweeteners have been isolated from the roots or from other species of Stevia. The leaves of Rubus chingii var. dulcis (Rosaceae), a wild shrub found in Southern China, have also been used as We have recently isolated a sweet a sweet-tea. steviol-glycosidel2, named rubusoside, from these leaves (structure: Table I and sweetness: Table 11)s. It is significant that rubusoside is an important intermediate in the chemical conversion of stevioside to the superior sweetener, rebaudioside A.
(displacements of carbon signals of both carbohydrateand aglycone-moieties on glycoside formation); in doing so we have discovered a stereochemical correlation between the glycosylation shifts and carbohydrateaglycone combination 9-l’. We have also established the glycosylation shifts characteristic of the ester type glycosides (such as stevioside’) and have been able to assign the carbon signals of kaurene type diterpenes, including steviol, to a chemical structurel2. Using 1X NMR it is now possible to determine the structure of an aglycone, the location and configuration of a glycosyl linkage and the structure of the carbohydrate moiety of glycosides without any loss of material2-4. Mass spectrometry is also a useful technique for the study of natural glycosides. However, volatile derivatives of these glycosides must be prepared for electron impact (EI) or chemical ionization mass spectrometry and even with these derivatives it is extremely difficult to observe a molecular ion (M+) in EI mass spectra. Sakamoto and Morimoto (Hiroshima Prefectural Institute of Public Health), with the co-operation of Schulten (University of Bonn), have investigated the application of field desorption (FD) mass spectrometry to steviol-glycosides and their related diterpeneglycosides. They have observed a relatively strong M++Na ion and characteristic fragmentation ions caused by the cleavage of sugar units as shown in Fig. 1 (Ref. 13). This p rocedure seems promising for the determination of the molecular weight and purity of glycosides.
NMR and mass spectrometry of steviol-glycosides
Separative analysis of Stewiasweet glycosides
1% NMR spectroscopy is now the most powerful tool used for the structural determination or identificaWe have prepared a variety of tion of glycosides. glycosides and have studied their glycosylation shifts
Because of its sweeter taste the food industries are interested in finding a plant rich in rebaudioside A that is suitable for exploitation by agriculture. They are also looking for ways to produce a glycoside prepara-
‘I’ABLE
II. Relative
sweetness
of steviol
glycosides
to sucrose
A Compounds
Strvioside Rebaudioside Rebaudioside Rebaudioside Kubusoside
A D E
0.1
0.05
0.025
89 85 89 85 84
149 149 163 125 63
143 242 221 174 -
Relative sweetness :\: C:oncentration 13: C:onc.entration
= B/A (Lv/V%) of aqueous solution (Wl\‘%) ofaqueous solution same swertness as the sampk solution.
OH
of sample. ofsucrose with the
OH
HO’ emitter
current
21 mA
’
587 (M+2Na)++ I
1 (M+Na)+ 827 (M+Na+H-325)
HO
’ 0 OH
c HO
^.
Uti
%
163
MW=1128
Fig. 1. FL) Mass spectrum of rebaudioside D(5) using a JEOL-JMS-D300 Prefectural
Institute
of Public
Health).
mass spectrometer (Sakamoto,
I. and
Morimoto,
K.,
Hiroshima
trends in analytical chemistry, vol. 1, tl~. II, 198-3
248
TABLE
III. Extraction
procedure
CALIBRATION
and TLC sampling
CURVE OF
Stecias rebaudiana leaves.
1 gram each sample Refluxed with 30 ml methanol for 2 hours (2 times) Filtered and evaporated under reduced pressure Dissolved in 50 ml methanol in volumetric flask Diluted 1 to 5 TLC on silica gel hierck silica gel 60 Fzjt (20 X 20 cm) Plate: Sample Spotting: 6 pl each Standard: Stevioside (0.6 and 1.2 pg) Rebaudioside-A (0.6 and 1.2 pg) Chloroform - Methanol - \Vater Solvent System: (90:65:9) 10% Sulfuric acid, heated at 120°C for 10 yisualization: minutes
FOR
STEVIOSIDE
QUANTITATIVE AND
DETERMINATION
REBAUDIOSIDE-A
I
Fig. 3.
tion with a higher content of rebaudioside A than of stevioside. In this respect and from a hygienic viewpoint separative analysis of these glycosides is important. Separative analysis by high performance liquid chromatography (HPLC) has been reported by Hashimoto and Moriyasu. Good separation of stevioside and rebaudioside A was obtained under the following conditions: column LiChrosorb NH:, (lop, 2 mm X 25 cm, Merck), eluent: CHsCN-H20 (85: 15), flow rate: 1.5 ml/min, detection: U.V. 2 10 nml4. Sakamoto also performed the separation of stevioside and rebaudiosides A and C by HPLC; column: CL-Bondapak CH (6 mm X 30 cm, Waters), eluent: CH&N-H20 (87.5: 12.5), flow rate: 2.5 ml/min, detection: 207 nm*5. Sholichin and Yamasaki, members of our group, have performed the rapid analysis of the glycosides by dual wave-length thin layer chromatography-densitometry (as illustrated in Fig. 2). The TLC was developed on pre-coated plates (Silica-gel 6OF254 (Merck)). After spraying with 10%H2S04 the plate was heated at 140°C for a few min and then immediately covered with a glass-plate of the same size to prevent the spots changing color. Calibration plots for integrated values of spots v weights/spot were found to be linear for stevioside and rebaudioside A up to a SCANNING
PROFILE
OF METHANOL
EXTRACT
STEVIA
_A,
____--_L(.OS*DE
--
--STEVIOSIDE
-_
-
---
-_---_-__-----REBWDIOSIDE-C;
----_----
_ REWDOSIDE-A
_
--_-------_REBWD,09DE-E _____-__----__
-
-D
Fig. 2.
:
:: j 1 I
CHC$-MeOH-Hz0 ( 90
I., Yamasaki,
K. and Tanaka,
0.
(1977) Chem.
Pharm. Bull. (Tokyo), 25, 3437
Kobayashi, M., Horikawa, S., Degrandi, I. H., Ueno, J. and Mitsuhashi, H. (1977) Phytochemistry 16, 1405 Kaneda, N., Kasai, K., Yamasaki, K. and Tanaka, 0. (1977) Chem. Pharm. Bull. (Tokyo) 25, 2466
Kasai, R., Kaneda, N., Tanaka, O., Yamasaki, K., Sakamoto, I., Morimoto, K., Okada, K., Kitahata, S. and Furukawa, H. (1981) J. Chem. Sot. Japan 726 8 Tanaka, T., Khoda, H. Tanaka, O., Chem, F.-H., Chou, W.-H. and Leu, J.-L. (1981) Agric. Biol. Chem. (Tokyo) 45,2165 9 Kasai, R., Suzuo, M., Asakawa, J. and Tanaka, 0. (1977) Tetrahedron Lett. 175
209
i’
I
SolVenl
Sakamoto,
15 Sakamoto, 16 Sholichin, data)
--c.:
-
Pharm. Bull. (Tokyo), 25, 844
--; -- , -_; --
_ -**
1 Mosettig, E., Beglinger, I-J., Dolder, F., Lichity, H., Quitt, P. and Waters, J. A. (1963) J. Am. Chem. Sot. 85, 2305 and references cited therein Kohda, H., Kasai, R., Yamasaki, K., Murakami, K. and Tanaka, 0. (1976) Phytochemistry 15, 981 Sakamoto, I., Yamasaki, K. and Tanaka, 0. (1977) Chem.
-- ._,
LEAVES
c --_----_
References
10 Kasai, R., Okihara, M., Asakawa, J., Mizutani, K. and Tanaka, 0. (1979) Tetrahedron Lett. 35, 1427 11 Mizutani, K., Kasai, R. and Tanaka, 0. (1980) Carbohydr. Res. 87, 19 12 Yamasaki, K., Khoda, H., Kobayashi, T., Kasai, R. and Tanaka, 0. (1976) Tetrahedron Lett. 1005 13 Sakamoto, I., Morimoto, K., Tanaka, 0. and Schulten, H.-R. (submitted for publication) 14 Hashimoto, Y. and Moriyasu, M. (1978) Shoyakugaku Zasshi 32,
8, TLC PATTERN OF
concentration of 5.4 pglspot and the curves were extrapolated through zero16. (Fig. 3). No positive mutagenic activity has been observed for stevioside, rebaudioside A or crude extracts of Stevia rebaudiana, using the Ames test. Long term toxicity testing of these sweet glycosides is now under progress in Japan. Stevioside has already been found to be non-cariogenic.
:
65
: 9
)
I. (unpublished M., Yamasaki,
data) K. and Tanaka,
0.
(unpublished
Professor Osamu Tanaka graduated from the University of Tokyo as a B.Pharm. Sci. in 1950 and obtained his Ph.D. there in 1959. He has been Professor of the Institute of Pharmaceutical Sciences (Department of the Chemistry of Biologically Active Substances), Hiroshima University School of Medicine, Kasumi, Minami-ku, Hiroshima 734, Japan since 1970. His major research interest is in the chemistry of biologically active plantglycosides; this includes the natural sweet-glycosides, the saponins of Ginseng and its related oriental plant-drugs and ‘SC NMR spectroscopy ofglycosides.
246
Steviol-alvcosides: cI_=
_------
_____~_
__
new natural ~~_
~~
~~_
~_
_
sweeteners _
_
_
The need for a safe, non-nutritive sweetener for the diabetic and the diet conscious has prompted the further investigation of new sweeteners of plant origin. This review deals with the chemistry of sweet steviol-glycosides, which are currently used as a food-additive in Japan. Osamu Tanaka Hiroshima, Japan In Paraguay and South Africa the natives use the leaves of Steuia rebaudianaBertoni (Compositae) , a wild herb, as a sweetening agent for their tea and coffee. A major sweet glucoside of this plant, steviosidel, was isolated about 50 years ago and its chemical structure was elucidated by Mosettig, Fletcher and their coworkers, in 1963 (see Table I). The genuine aglycone (named steviol) is a kaurane-type diterpene acid, 13-hydroxy-ent-kaur- 16-en- 19-oic acid and forms isosteviol in acid. Recently, this plant has attracted commercial attention as a new source of non-cariogenic and noncarcinogenic dietary-sweetener and is now cultivated, not only in Japan, but all over South-East Asia; about 300 tons of its dried leaves were harvested in 1979 (the cost of these in 1980 was 700-l ,000 yen/kg). In Japan, eight food-manufacturing companies are now engaged
in extracting sweet-glycosides and other chemicals from steuia for use as food-additives in soy-sauce, pickled vegetables, fruit-juice etc.
New sweet glycosides It has already been mentioned that the crude glycoside-fraction of Stevia-leaves tastes sweeter and less bitter than the purified stevioside. We have isolated four additional sweet steviol-glycosides from these leaves and have designated them rebaudiosides -A, -C, -D and -E; their structures are illustrated in Table 12-4. Mitsuhashi et al. have also isolated two new steviol-glycosides - dulcoside-A and -B (Ref. 5), the latter of which is identical with our rebaudioside-C4. Of these glycosides, rebaudiosides A and C were isolated in yields of 3.0 and 0.4%, respectively. With regard to taste, the rebaudiosides A, D and E are sweeter and less bitter than stevioside (see Table II). It follows that the sweetness of Steviu-leaves must be due to both stevioside and rebaudioside A. It is interesting TABLE
A
g’c
Rebaudioside
C
glc
g$v;z;cm
glc H
B
Rebaudioside
D
glc_2--rglc
glc~glc
Rebaudioside Dulcoside A Rubusoside
E
glc~glc
- ‘lglc gl&glc glcl-rham
glc = fi-D-glucopyranosyl .oO
glc QgIc H glc~glc “\I glc
Stevioside Steviol Rebaudioside
= dulcoside
0 165-9936/82/MM0-OOCQ/f01
I.
glc glc
glc
rham = cw-L-rhamnopyranosyl @ 1982 Elscvier Scientific Publish+ Company
trends in analytical chemistry, VOI.I, no. II, 1982
247
that our research group has been able to convert stevioside into this rebaudioside by a combination of enzymic and chemical procedures”,7. The flower-buds of Stevia rebaudiana also contain these two glycosides but no sweeteners have been isolated from the roots or from other species of Stevia. The leaves of Rubus chingii var. dulcis (Rosaceae), a wild shrub found in Southern China, have also been used as We have recently isolated a sweet a sweet-tea. steviol-glycosidel2, named rubusoside, from these leaves (structure: Table I and sweetness: Table 11)s. It is significant that rubusoside is an important intermediate in the chemical conversion of stevioside to the superior sweetener, rebaudioside A.
(displacements of carbon signals of both carbohydrateand aglycone-moieties on glycoside formation); in doing so we have discovered a stereochemical correlation between the glycosylation shifts and carbohydrateaglycone combination 9-l’. We have also established the glycosylation shifts characteristic of the ester type glycosides (such as stevioside’) and have been able to assign the carbon signals of kaurene type diterpenes, including steviol, to a chemical structurel2. Using 1X NMR it is now possible to determine the structure of an aglycone, the location and configuration of a glycosyl linkage and the structure of the carbohydrate moiety of glycosides without any loss of material2-4. Mass spectrometry is also a useful technique for the study of natural glycosides. However, volatile derivatives of these glycosides must be prepared for electron impact (EI) or chemical ionization mass spectrometry and even with these derivatives it is extremely difficult to observe a molecular ion (M+) in EI mass spectra. Sakamoto and Morimoto (Hiroshima Prefectural Institute of Public Health), with the co-operation of Schulten (University of Bonn), have investigated the application of field desorption (FD) mass spectrometry to steviol-glycosides and their related diterpeneglycosides. They have observed a relatively strong M++Na ion and characteristic fragmentation ions caused by the cleavage of sugar units as shown in Fig. 1 (Ref. 13). This p rocedure seems promising for the determination of the molecular weight and purity of glycosides.
NMR and mass spectrometry of steviol-glycosides
Separative analysis of Stewiasweet glycosides
1% NMR spectroscopy is now the most powerful tool used for the structural determination or identificaWe have prepared a variety of tion of glycosides. glycosides and have studied their glycosylation shifts
Because of its sweeter taste the food industries are interested in finding a plant rich in rebaudioside A that is suitable for exploitation by agriculture. They are also looking for ways to produce a glycoside prepara-
‘I’ABLE
II. Relative
sweetness
of steviol
glycosides
to sucrose
A Compounds
Strvioside Rebaudioside Rebaudioside Rebaudioside Kubusoside
A D E
0.1
0.05
0.025
89 85 89 85 84
149 149 163 125 63
143 242 221 174 -
Relative sweetness :\: C:oncentration 13: C:onc.entration
= B/A (Lv/V%) of aqueous solution (Wl\‘%) ofaqueous solution same swertness as the sampk solution.
OH
of sample. ofsucrose with the
OH
HO’ emitter
current
21 mA
’
587 (M+2Na)++ I
1 (M+Na)+ 827 (M+Na+H-325)
HO
’ 0 OH
c HO
^.
Uti
%
163
MW=1128
Fig. 1. FL) Mass spectrum of rebaudioside D(5) using a JEOL-JMS-D300 Prefectural
Institute
of Public
Health).
mass spectrometer (Sakamoto,
I. and
Morimoto,
K.,
Hiroshima
trends in analytical chemistry, vol. 1, tl~. II, 198-3
248
TABLE
III. Extraction
procedure
CALIBRATION
and TLC sampling
CURVE OF
Stecias rebaudiana leaves.
1 gram each sample Refluxed with 30 ml methanol for 2 hours (2 times) Filtered and evaporated under reduced pressure Dissolved in 50 ml methanol in volumetric flask Diluted 1 to 5 TLC on silica gel hierck silica gel 60 Fzjt (20 X 20 cm) Plate: Sample Spotting: 6 pl each Standard: Stevioside (0.6 and 1.2 pg) Rebaudioside-A (0.6 and 1.2 pg) Chloroform - Methanol - \Vater Solvent System: (90:65:9) 10% Sulfuric acid, heated at 120°C for 10 yisualization: minutes
FOR
STEVIOSIDE
QUANTITATIVE AND
DETERMINATION
REBAUDIOSIDE-A
I
Fig. 3.
tion with a higher content of rebaudioside A than of stevioside. In this respect and from a hygienic viewpoint separative analysis of these glycosides is important. Separative analysis by high performance liquid chromatography (HPLC) has been reported by Hashimoto and Moriyasu. Good separation of stevioside and rebaudioside A was obtained under the following conditions: column LiChrosorb NH:, (lop, 2 mm X 25 cm, Merck), eluent: CHsCN-H20 (85: 15), flow rate: 1.5 ml/min, detection: U.V. 2 10 nml4. Sakamoto also performed the separation of stevioside and rebaudiosides A and C by HPLC; column: CL-Bondapak CH (6 mm X 30 cm, Waters), eluent: CH&N-H20 (87.5: 12.5), flow rate: 2.5 ml/min, detection: 207 nm*5. Sholichin and Yamasaki, members of our group, have performed the rapid analysis of the glycosides by dual wave-length thin layer chromatography-densitometry (as illustrated in Fig. 2). The TLC was developed on pre-coated plates (Silica-gel 6OF254 (Merck)). After spraying with 10%H2S04 the plate was heated at 140°C for a few min and then immediately covered with a glass-plate of the same size to prevent the spots changing color. Calibration plots for integrated values of spots v weights/spot were found to be linear for stevioside and rebaudioside A up to a SCANNING
PROFILE
OF METHANOL
EXTRACT
STEVIA
_A,
____--_L(.OS*DE
--
--STEVIOSIDE
-_
-
---
-_---_-__-----REBWDIOSIDE-C;
----_----
_ REWDOSIDE-A
_
--_-------_REBWD,09DE-E _____-__----__
-
-D
Fig. 2.
:
:: j 1 I
CHC$-MeOH-Hz0 ( 90
I., Yamasaki,
K. and Tanaka,
0.
(1977) Chem.
Pharm. Bull. (Tokyo), 25, 3437
Kobayashi, M., Horikawa, S., Degrandi, I. H., Ueno, J. and Mitsuhashi, H. (1977) Phytochemistry 16, 1405 Kaneda, N., Kasai, K., Yamasaki, K. and Tanaka, 0. (1977) Chem. Pharm. Bull. (Tokyo) 25, 2466
Kasai, R., Kaneda, N., Tanaka, O., Yamasaki, K., Sakamoto, I., Morimoto, K., Okada, K., Kitahata, S. and Furukawa, H. (1981) J. Chem. Sot. Japan 726 8 Tanaka, T., Khoda, H. Tanaka, O., Chem, F.-H., Chou, W.-H. and Leu, J.-L. (1981) Agric. Biol. Chem. (Tokyo) 45,2165 9 Kasai, R., Suzuo, M., Asakawa, J. and Tanaka, 0. (1977) Tetrahedron Lett. 175
209
i’
I
SolVenl
Sakamoto,
15 Sakamoto, 16 Sholichin, data)
--c.:
-
Pharm. Bull. (Tokyo), 25, 844
--; -- , -_; --
_ -**
1 Mosettig, E., Beglinger, I-J., Dolder, F., Lichity, H., Quitt, P. and Waters, J. A. (1963) J. Am. Chem. Sot. 85, 2305 and references cited therein Kohda, H., Kasai, R., Yamasaki, K., Murakami, K. and Tanaka, 0. (1976) Phytochemistry 15, 981 Sakamoto, I., Yamasaki, K. and Tanaka, 0. (1977) Chem.
-- ._,
LEAVES
c --_----_
References
10 Kasai, R., Okihara, M., Asakawa, J., Mizutani, K. and Tanaka, 0. (1979) Tetrahedron Lett. 35, 1427 11 Mizutani, K., Kasai, R. and Tanaka, 0. (1980) Carbohydr. Res. 87, 19 12 Yamasaki, K., Khoda, H., Kobayashi, T., Kasai, R. and Tanaka, 0. (1976) Tetrahedron Lett. 1005 13 Sakamoto, I., Morimoto, K., Tanaka, 0. and Schulten, H.-R. (submitted for publication) 14 Hashimoto, Y. and Moriyasu, M. (1978) Shoyakugaku Zasshi 32,
8, TLC PATTERN OF
concentration of 5.4 pglspot and the curves were extrapolated through zero16. (Fig. 3). No positive mutagenic activity has been observed for stevioside, rebaudioside A or crude extracts of Stevia rebaudiana, using the Ames test. Long term toxicity testing of these sweet glycosides is now under progress in Japan. Stevioside has already been found to be non-cariogenic.
:
65
: 9
)
I. (unpublished M., Yamasaki,
data) K. and Tanaka,
0.
(unpublished
Professor Osamu Tanaka graduated from the University of Tokyo as a B.Pharm. Sci. in 1950 and obtained his Ph.D. there in 1959. He has been Professor of the Institute of Pharmaceutical Sciences (Department of the Chemistry of Biologically Active Substances), Hiroshima University School of Medicine, Kasumi, Minami-ku, Hiroshima 734, Japan since 1970. His major research interest is in the chemistry of biologically active plantglycosides; this includes the natural sweet-glycosides, the saponins of Ginseng and its related oriental plant-drugs and ‘SC NMR spectroscopy ofglycosides.