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Antisweet activity of gymnemic acid A1 and its derivatives
brown has sweet esterified the active structure Sciences "gymnemic A1 and different substances been bottom order of for sylvestre colored in substances acid to Address were University, the of was principle, their Great are A2, known 3preparative Gymnemic were liters with toeliminated abilities Al of not repeated acid" with A3, gymnemic solution gymnemic elucidate structural in of the Britain washed that collected Laboratory acids satisfactory 20 ACTIVITY and high quite of After 2N vessel, (gymnemic Since named Acid Yokohama, December water Biological the A4 purposes, several 8,with to sulfuric yields was acid by acid diverse the leaves based the suppress Al Part differences by 1887 "gymnemic OF most atwater obtained centrifugation components leaves of acid as relationship centrifugation GYMNEMIC Japan to 600C times 1,Chemistry, several on acid 1968 we The Science, D-glucuronide chemical of obtain pp A)were thin sweetness, and Gymnema devised the Kurihara* acid" effect by Florida consists and 537in The dissolved ACID investigatorsl-3 layer are filtration supernatant Since sufficient added immersed final Methods Florida structures between pH The 543, sylvestre acompared on Al at The Inof the 32306 chromatography the new form of Faculty the sweet to AND 1967 5,000 1969 the precipitates volume inin State gymnemic hexahydroxy-triterpene antisweet 1isolation reported the through was ITS quantities kg ethanol 3combined water StBcklin in taste is suppress rev/min March structures of DERIVATIVES University, also siphoned of this have dried Education, acid by afor ethanol method activities isolation paper, studied sheet 1969) gymnemic extracts tried et had for the They of about Al leaves al10 out, ofsolution gymnemic settled sensitivity of to (aproposed tomin based 5gymnemic Yokohama ingauze main reported obtain and Press was acid of hours, isolate this which onThe the Al
Life Printed
Vol .
.
.
.
Pergamon
ANTISWEET Yoshie
Department
Tallahassee,
(Received IT to the that component), the is In acids these studies methods4~5 acid gymnemic using paper .
;
.
.
.4 .
.
.
.
Materials Isolation Fifteen Gymnema . a Extraction adjusted .0 at precipitates precipitates materials *Present : National
. .
. After
. .
537
.
Insoluble
.
538
GYMNEMIC ACID DERIVATIVES
Vol. 8, No. 9
was decreased by evaporation under reduced pressure, and two parts of acetone per one part of alcohol solution were added .
Precipitates appearing in the
solution were eliminated by centrifugation and the supernatant was evaporated to dryness under reduced pressure .
A greenish residue was extracted several
times with diethyl carbonate at the boiling point, and gymnemic acid A (13 g) was thereby crystallized from the solvent .
Then, 5 g of gymnemic acid A
dissolved in 957 ethanol were applied to the DEAF-Sephadex A-25 (40-120p), (column, 2 .2 x 40 cm) and eluted with 95% ethanol .
An aliquot of each 10 ml
fraction was applied to thin layer chromatography using a system of n-butyl formate-ethyl methyl ketone-formic acid-water (5 :3 :1 :1) .
Fractions from No . 18
to No . 30, which contained only the Al component, were collected and evaporated . The residue was recrystallized from diethyl carbonate (1 .5 g) .
Sample for
elemental analyses was obtained by rechromatography on DEAF-Sephadex . 215 0 C (decomp .) .
Al, m .p .
Anal . Calcd . for C53H82016 " 2H20*1 : C, 62 .97 ; H, 8 .51 ; 0, 28 .51 .
Found*2 : C, 62 .55 ; H, 8 .83 ; 0, 28 .80 . Methylation of Gymnemic Acids Twenty milligrams of the sample were dissolved in a few ml of ether and then treated with a solution of díazomethane in ether at room temperature until no further reaction occurred . ether was evaporated .
After this reaction mixture stood for 15 min, the
The residue was recrystallized from diethyl carbonate .
Methyl ester of Al, m .p . 205 *C (decomp .) . 63 .28 ; H, 8 .59 ; 0, 28 .12 .
Anal . Calcd . for C54H84016-2H20 : C, Found : C, 63 .18 ; H, 8 .75 ; O, 28 .56 .
Modific at ion of Al Conversion of Al into A2 was carried out as follows :
One-half gram of Al
was treated with a KHC03 solution (0 .5 g KHC03 in 18 ml of water and 3 ml of ethanol) at room temperature . reduced pressure .
After 48 hours the solution was evaporated under
Residue was dissolved in 10 ml of water, and the pH of the
solution was adjusted to 2 .5 with 2N sulfuric acid .
The precipitate was
centrifuged, washed with water, and dried (0 .35 g) .
The sample obtained was
applied to a DEAF-Sephadex column (1 .2 x 50 cm) and eluted with 95% ethanol . The presence of gymnemic acids in each fraction (8 ml) was assayed with the thin layer chromatography and the fractions from No . 17 to No . 21, which contained only A2, were collected .
*1
The combined fractions were evaporated to dryness, and
The structure of gymnemic acid proposed by St6cklin was modified so that
the genin is esterified with 1 mole of acetic acid, 2 moles of isovaleric acid and 1 mole of tiglic acid . *2
Elemental analyses were performed by Schwarzkoph Microanalytical Lab .,
Woodside 77, New York
Vol . 8, No. 9
GYMNEMIC ACID DERIVATIVES
539
the residue was recrystallized from diethyl carbonate (0 .16 g) .
A2, m .p . 205 0 C
(decomp .) .
Anal . Calcd . for C51H80015 " 5H20 : C, 59 .88 ; H, 8 .80 ; 0, 31 .31 . Found : C, 60 .13 ; H, 8 .62 ; 0, 30 .80 . Al was converted into A3 as follows :
0 .4 g of Al was treated with 100 ml
of 3% KOH methanol solution under refluxing for 15 min . evaporated and the residue was dissolved in water .
The solution was
The pH of the solution was
adjusted to 2 .5 and precipitates of A3 were obtained quantitatively .
The crude
A3 was purified by a DEAF-Sephadex column (1 .2 x 50 cm) with elution by 95% ethanol .
A3 was recrystallized from diethyl carbonate .
A3, m .p . 205 0 C (decomp .) .
Anal . Calcd . for C36H58O12 . H20 : C, 61 .71 ; H, 8 .56 ; 0, 29 .71 . Found : C, 62 .36 ; H, 9 .05 ; 0, 29 .20 . Methyl ester of A3, m .p . 2300C (decomp .) . Anal . Calcd . for
Found : C, 61 .90 ; H, 9 .03 ; 0, 28 .23 . C37H60012'H20 : C, 62 .11 ; H, 8 .68 ; 0, 29 .13 . Identification of Acids Liberated from Gymnemic Acid A7 The ester group in genin of gymnemic acid Al was hydrolyzed under the condition mentioned above (see modification of Al) .
After hydrolysis, the
solution was acidified and the precipitates were eliminated by centrifugation . After the supernatant was repeatedly extracted with ether, the combined ether extracts were dried over MgS04 .
The ether was carefully evaporated .
One-half
of the remaining solution was applied to gas chromatography, and the other half was used for paper chromatography . formed as follows : 6
Paper chromatography of the acids was per-
Acids in the solution were converted into hydroxamic acids
which were applied to Whatman No . 1 filter paper .
Descending chromatography was
carried out in butyl alcohol saturated with water for 18 hours . solution was used as spray reagent .
2% FeC13 ethanol
Gas chromatographic identification of the
acids was performed by Aminco gas chromatogram (No . 4-4600) .
Column, 6 feet
2-3/4 inches of tubing and 1/4 inch in diameter with 2 .5% (w/w) diethylene glycol adipate polyester and 2% H3PO4 on acid-washed Celite 545 ; 7 column temperature, 160OC ; helium flow rate, 25 ml/min ; injection temperature, 162 0 C . For the quantitative analysis, authentic acids of equal molarity were mixed and poured in water .
The acids in aqueous solution were extracted with ether
and the resulting acid solution was applied to gas chromatography .
The ratio of
peak area of each acid in the chromatogram was used for the calculation of the mole ratio of acids that were liberated from gymnemic acid . Assay of Antisweet Activity The antisweet activity of gymnemic acid was assayed as follows : subjects were subjected to taste testing .
Four
Five milliliters of a gymnemic acid
solution in 0 .01M NaHC03 were held in the mouth for 2 min . spit out and the mouth was rinsed with distilled water .
The solution was
Thesubjects were
directed to taste 10 sucrose solutions from 0 .1 to 1 .OM . The activity of a gymnemic acid solution was expressed as the maximum concentration of a sucrose
540
GYMNEMIC ACII) DERIVATIVES
solution whose sweetness was depressed completely .
Vol . 8, No. 9 The persistency of the
depressive effect on sweet taste was assayed at 5 minute intervals after application of the gymnemic acid solution on the tongue . Antisweet Activity to Different Sweet Compounds The following solutions, which gave proper sweet taste, were tasted after application of a 1 x 10 -3 M gymnemic acid Al solution : 0 .025M cyclamate (sodium cyclohexanesulfamate), 0 .025M D-tryptophan, 0 .076M D-leucine, 0 .038M beryllium chloride, 0 .0ó7M lead " acetate, and chloroform . Results and Discussion To isolate gymnemic acid Al, Stöcklin et a1 . 4 used a silica gel column eluting with alkaline or acidic solution .
Repeating the isolation method of
gymnemic acid Al, we noticed that the presence of acid brought about a modifica tion of gymnemic acid A1 into a substance which gave a larger Rf value than that of Al on thin layer chromatography .
Also the presence of alkali brought about a
modification of Al into a substance which gave an Rf value less than that of Al . The poorness of the yield of gymnemic acid Al arose also from the difficulty in detaching gymnemic acids from a silica gel column .
An isolation method without
the use of acid, alkali, and silica gel column was devised as described in "Materials and Methods" . Leaves of Gymnema sylvestre were extracted with water, and gymnemic acids were precipitated by acidification of the extracts .
Precipitates were dissolved
in ethanol and acetone, and the insoluble materials in these solvents were eliminated .
Solvents were then evaporated and the residue was extracted with
diethyl carbonate .
Gymnemic acid A was crystallized from the solvent .
Thin
layer chromatography of the obtained gymnemic acid A indicated that gymnemic acid A consisted predominately of gymnemic acid Al, accompanied by trace of A3 . Gymnemic acid A was applied to a DEAF-Sephadex column .
Although elution with
absolute ethanol gave poor yield of gymnemic acid Al, elution with 95% ethanol brought good recovery of gymnemic acid Al . esterified with acids . hydrolysis .
The genin part of gymnemic acid is
Acids in gymnemic acid A1 were liberated by alkaline
Paper chromatogram of the hydrolyzate gave three spots of acetic,
isovaleric and tiglíc acids . By gas chromatography of the hydrolyzate, 1 mole of acetic acid, 2 moles of isovaleric acid and 1 mole of tiglic acid were identified .
Stbcklin et a1 . 4 identified five acids (formic, acetic, n-butyric,
isovaleric and tiglíc acids) from alkaline hydrolysis of gymnemic acid A . Apparently, the difference between the results arose from the fact that we used purified Al while they used a mixture of gymnemic acids .
The elemental analyses
of the obtained gymnemic acid Al corresponded to the structure which was proposed by Stöcklin and modified, based on our results about acid components in
Vol. 8, No. 9 the genin.
GYMNEMIC ACID DERIVATIVES
541
The presence of potassium bicarbonate led gymnemic acid Al into a
substance which gave a smaller Rf than that of Al on thin layer chromatography . This modified substance was purified on a DEAF-Sephadex column and was identified as gymnemic acid A2 isolated by the method according to Stlicklin . 4
Refluxing
gymnemic acid Al in 3% KOH methanol solution yielded a substance whose behavior on thin layer chromatography was identical to gymnemic acid A3 prepared by the method used by Stdcklin .
Acids liberated by the modification were identified
by paper and gas chromatography .
Results indicated that one mole of acetic acid
was liberated by the conversion of Al to A2 and that 2 moles of isovaleric acid and 1 mole of tiglic acid were liberated by the conversion of AZ to A3 .
The
elemental analyses of the isolated A2 and A3 corresponded to the structures expected from the postulation that only ester groups of genin were modified by the alkaline hydrolysis of Al . - 1 mole of acetic acid Al
A2
- 1 mole of tiglic acid - 2 moles of isovaleric acid
A3
Antisweet activities of gymnemic acid Al, A2 and A3 were assayed (see Figure 1) .
GYYNEYIC ACID Ai a-,- SX10-so
y-~- 1 xlo - su
-w-- 1x90 -4M
0
_
GYYNEYIC ACID AI I -o- SXp - SY II-a- IX10-3Y
.A 0
igr
OA kz\\
0."
o
\\\
s
O_
o
s to MINUTES
FIG. 1
m
w
542
GYMNEMIC ACID DERIVATIVES
Vol . 8, No . 9
Antisweet activity of gymnemic acid Al and A2 . Activity was assayed at 5 minute intervals after gymnemic acid solution was held in the mouth for 2 min . This activity is expressed by the maximum concentration of sucrose solution whose sweetness was depressed completely . average variation .
The bars indicate the range of
Molecular weights of gymnemic acid Al and A2 were assumed
to be 1010 and 1022, respectively .
The antisweet activity of gymnemic acid
reached maximum level after being held in the mouth in solution form for about 30 seconds .
The activity was assayed by tasting a series of sucrose solutions
after a gymnemic acid solution was held in the mouth for 2 min and the activity was expressed by the maximum concentration of sucrose solution whose sweetness was depressed completely .
Cymnemic acid A3 did not show any antisweet activity .
As seen from the curve III, 1 x 10 -4M Al depressed the sweetness of 0 .3M sucrose solution and this effect decreased in 15 min .
Application of 5 x 10 -3!! Al
brought the complete depression of sweetness of solid sucrose as well as that of 1 .OM sucrose solution and the effect decreased as shown by curve I .
of gymnemic acid A2 was much lower than that of Al .
The activity
The activity of 5 x 10 -3M
A2 (curve IV) was a little lower than that of 1 x 10 -3M Al (curve II) .
This
indicated that the activity of A2 is less than 1/5 of the activity of an equal concentration of Al .
It is interesting to note that activity decreased greatly
by the conversion of Al into A2 even though the conversion of Al into A2 resulted in the loss of only one mole of acetic acid .
Apparently, the ester group in the
genin has an important role in manifestation of antisweet activity of gymnemic acid Al .
Since gymnemic acid Al has a rather large molecular weight of about 1,000, the elimination of acetic acid from Al will not have a large effect on
the whole molecule of Al .
The moiety of the ester group of gymnemic acid Al
may be related directly to the binding of Al on the receptor site in taste buds . It is known that a great variety of other compounds besides sugars
taste
sweet, e .g ., synthetic sweetening agents, D-amino acids, and some inorganic compounds .
Antisweet activity of gymnemic acid Al on cyclamate, D-tryptophan,
D-leucine, beryllium chloride, lead acetate, and chloroform was also examined . The sweet taste of all these compounds except for chloroform was suppressed by the action of gymnemic acid Al .
Although it is not known whether these compounds
of different chemical structure induce sweet taste by the same mechanism, it can be concluded Ehat at least part of the sweet mechanism is common to substances of quite different structure .
Chloroform is the only sweet substance which has
a high vapour pressure and is a good solvent for lipids . related to the different mechanism of sweet induction . unique. flavor .
This fact may be Sodium glutamate has a
Application of 5 x 10 -3M gymnemic acid Al changed the unique
taste of crystalline sodium glutamate to a taste much like sodium chloride .
Vol . 8, No. 9
GYMNEMIC ACID DERIVATIVES
543
Summary A method was devised to isolate gymnemic acid Al, a main component of gymnemic acid A,
for preparative purpose .
Acids esterified in the genin of
gymnemic acid Al were determined to be 1 mole of acetic, 2 moles of isovaleric and 1 mole of tiglic acids.
Gymnemic acid Al was converted into A2, and finally
into A3 by alkaline hydrolysis .
Antisweet activity of Al was greatly decreased
by conversion into A2, while A3 had no activity .
It is suggested that'the eater
group in the genin has an important role in the manifestation of the antisweet activity of gymnemic acid A1 .
Studies regarding antisweet activity of gymnemic
acid on other sweet compounds besides sugars indicated that gymnemic acid A1 suppresses the sweet taste of cyclamate,
D-amino acids, beryllium chloride, and
lead acetate and that it does not suppress the sweet taste of chloroform . Acknowledgments This work was supported by the National Science Foundatiod Grant No . GB4068% and the Atomic Energy Commission Grant No . AT-(40-1)-2690 .
The author
wishes to thank Dr . Lloyd M. Beidler for many valuable suggestions and continued encouragement and Mr . Cuyler V. Smith for valuable technical assistance . References 1.
D . HDOPER, Pharm. J. 17, 867 (1887) .
2.
R . M. WARREN and C. PFAFFMANN, J. Appl . Physiol. 14, 40 (1959) .
3.
K. S. YACKZAN, . Alabama J. Med. Sci . 3
1 (1966) .
4.
W. STÖCKLIN, E . WEISS and T. REICHSTEIN, Helv . Chim . Acta 50, 474 (1967) . W. STÓCKLIN, ibid, 50, 491 (1967) .
5.
J . E. SINSHE IKER, G . SUBBA RAO, H. M. MCILHENNY, R. V . SMITH, H. F. MAASSAB and K. W. COCHRAN, Experientia 15, 302 (1968) .
6.
E . BAYER and K. H. REUTHER, Angew. Chem . 68, 698 (1956) .
7.
L . D. METCALFE, Nature 188,
142 (1960) .
Life Printed
Vol .
.
.
.
Pergamon
ANTISWEET Yoshie
Department
Tallahassee,
(Received IT to the that component), the is In acids these studies methods4~5 acid gymnemic using paper .
;
.
.
.4 .
.
.
.
Materials Isolation Fifteen Gymnema . a Extraction adjusted .0 at precipitates precipitates materials *Present : National
. .
. After
. .
537
.
Insoluble
.
538
GYMNEMIC ACID DERIVATIVES
Vol. 8, No. 9
was decreased by evaporation under reduced pressure, and two parts of acetone per one part of alcohol solution were added .
Precipitates appearing in the
solution were eliminated by centrifugation and the supernatant was evaporated to dryness under reduced pressure .
A greenish residue was extracted several
times with diethyl carbonate at the boiling point, and gymnemic acid A (13 g) was thereby crystallized from the solvent .
Then, 5 g of gymnemic acid A
dissolved in 957 ethanol were applied to the DEAF-Sephadex A-25 (40-120p), (column, 2 .2 x 40 cm) and eluted with 95% ethanol .
An aliquot of each 10 ml
fraction was applied to thin layer chromatography using a system of n-butyl formate-ethyl methyl ketone-formic acid-water (5 :3 :1 :1) .
Fractions from No . 18
to No . 30, which contained only the Al component, were collected and evaporated . The residue was recrystallized from diethyl carbonate (1 .5 g) .
Sample for
elemental analyses was obtained by rechromatography on DEAF-Sephadex . 215 0 C (decomp .) .
Al, m .p .
Anal . Calcd . for C53H82016 " 2H20*1 : C, 62 .97 ; H, 8 .51 ; 0, 28 .51 .
Found*2 : C, 62 .55 ; H, 8 .83 ; 0, 28 .80 . Methylation of Gymnemic Acids Twenty milligrams of the sample were dissolved in a few ml of ether and then treated with a solution of díazomethane in ether at room temperature until no further reaction occurred . ether was evaporated .
After this reaction mixture stood for 15 min, the
The residue was recrystallized from diethyl carbonate .
Methyl ester of Al, m .p . 205 *C (decomp .) . 63 .28 ; H, 8 .59 ; 0, 28 .12 .
Anal . Calcd . for C54H84016-2H20 : C, Found : C, 63 .18 ; H, 8 .75 ; O, 28 .56 .
Modific at ion of Al Conversion of Al into A2 was carried out as follows :
One-half gram of Al
was treated with a KHC03 solution (0 .5 g KHC03 in 18 ml of water and 3 ml of ethanol) at room temperature . reduced pressure .
After 48 hours the solution was evaporated under
Residue was dissolved in 10 ml of water, and the pH of the
solution was adjusted to 2 .5 with 2N sulfuric acid .
The precipitate was
centrifuged, washed with water, and dried (0 .35 g) .
The sample obtained was
applied to a DEAF-Sephadex column (1 .2 x 50 cm) and eluted with 95% ethanol . The presence of gymnemic acids in each fraction (8 ml) was assayed with the thin layer chromatography and the fractions from No . 17 to No . 21, which contained only A2, were collected .
*1
The combined fractions were evaporated to dryness, and
The structure of gymnemic acid proposed by St6cklin was modified so that
the genin is esterified with 1 mole of acetic acid, 2 moles of isovaleric acid and 1 mole of tiglic acid . *2
Elemental analyses were performed by Schwarzkoph Microanalytical Lab .,
Woodside 77, New York
Vol . 8, No. 9
GYMNEMIC ACID DERIVATIVES
539
the residue was recrystallized from diethyl carbonate (0 .16 g) .
A2, m .p . 205 0 C
(decomp .) .
Anal . Calcd . for C51H80015 " 5H20 : C, 59 .88 ; H, 8 .80 ; 0, 31 .31 . Found : C, 60 .13 ; H, 8 .62 ; 0, 30 .80 . Al was converted into A3 as follows :
0 .4 g of Al was treated with 100 ml
of 3% KOH methanol solution under refluxing for 15 min . evaporated and the residue was dissolved in water .
The solution was
The pH of the solution was
adjusted to 2 .5 and precipitates of A3 were obtained quantitatively .
The crude
A3 was purified by a DEAF-Sephadex column (1 .2 x 50 cm) with elution by 95% ethanol .
A3 was recrystallized from diethyl carbonate .
A3, m .p . 205 0 C (decomp .) .
Anal . Calcd . for C36H58O12 . H20 : C, 61 .71 ; H, 8 .56 ; 0, 29 .71 . Found : C, 62 .36 ; H, 9 .05 ; 0, 29 .20 . Methyl ester of A3, m .p . 2300C (decomp .) . Anal . Calcd . for
Found : C, 61 .90 ; H, 9 .03 ; 0, 28 .23 . C37H60012'H20 : C, 62 .11 ; H, 8 .68 ; 0, 29 .13 . Identification of Acids Liberated from Gymnemic Acid A7 The ester group in genin of gymnemic acid Al was hydrolyzed under the condition mentioned above (see modification of Al) .
After hydrolysis, the
solution was acidified and the precipitates were eliminated by centrifugation . After the supernatant was repeatedly extracted with ether, the combined ether extracts were dried over MgS04 .
The ether was carefully evaporated .
One-half
of the remaining solution was applied to gas chromatography, and the other half was used for paper chromatography . formed as follows : 6
Paper chromatography of the acids was per-
Acids in the solution were converted into hydroxamic acids
which were applied to Whatman No . 1 filter paper .
Descending chromatography was
carried out in butyl alcohol saturated with water for 18 hours . solution was used as spray reagent .
2% FeC13 ethanol
Gas chromatographic identification of the
acids was performed by Aminco gas chromatogram (No . 4-4600) .
Column, 6 feet
2-3/4 inches of tubing and 1/4 inch in diameter with 2 .5% (w/w) diethylene glycol adipate polyester and 2% H3PO4 on acid-washed Celite 545 ; 7 column temperature, 160OC ; helium flow rate, 25 ml/min ; injection temperature, 162 0 C . For the quantitative analysis, authentic acids of equal molarity were mixed and poured in water .
The acids in aqueous solution were extracted with ether
and the resulting acid solution was applied to gas chromatography .
The ratio of
peak area of each acid in the chromatogram was used for the calculation of the mole ratio of acids that were liberated from gymnemic acid . Assay of Antisweet Activity The antisweet activity of gymnemic acid was assayed as follows : subjects were subjected to taste testing .
Four
Five milliliters of a gymnemic acid
solution in 0 .01M NaHC03 were held in the mouth for 2 min . spit out and the mouth was rinsed with distilled water .
The solution was
Thesubjects were
directed to taste 10 sucrose solutions from 0 .1 to 1 .OM . The activity of a gymnemic acid solution was expressed as the maximum concentration of a sucrose
540
GYMNEMIC ACII) DERIVATIVES
solution whose sweetness was depressed completely .
Vol . 8, No. 9 The persistency of the
depressive effect on sweet taste was assayed at 5 minute intervals after application of the gymnemic acid solution on the tongue . Antisweet Activity to Different Sweet Compounds The following solutions, which gave proper sweet taste, were tasted after application of a 1 x 10 -3 M gymnemic acid Al solution : 0 .025M cyclamate (sodium cyclohexanesulfamate), 0 .025M D-tryptophan, 0 .076M D-leucine, 0 .038M beryllium chloride, 0 .0ó7M lead " acetate, and chloroform . Results and Discussion To isolate gymnemic acid Al, Stöcklin et a1 . 4 used a silica gel column eluting with alkaline or acidic solution .
Repeating the isolation method of
gymnemic acid Al, we noticed that the presence of acid brought about a modifica tion of gymnemic acid A1 into a substance which gave a larger Rf value than that of Al on thin layer chromatography .
Also the presence of alkali brought about a
modification of Al into a substance which gave an Rf value less than that of Al . The poorness of the yield of gymnemic acid Al arose also from the difficulty in detaching gymnemic acids from a silica gel column .
An isolation method without
the use of acid, alkali, and silica gel column was devised as described in "Materials and Methods" . Leaves of Gymnema sylvestre were extracted with water, and gymnemic acids were precipitated by acidification of the extracts .
Precipitates were dissolved
in ethanol and acetone, and the insoluble materials in these solvents were eliminated .
Solvents were then evaporated and the residue was extracted with
diethyl carbonate .
Gymnemic acid A was crystallized from the solvent .
Thin
layer chromatography of the obtained gymnemic acid A indicated that gymnemic acid A consisted predominately of gymnemic acid Al, accompanied by trace of A3 . Gymnemic acid A was applied to a DEAF-Sephadex column .
Although elution with
absolute ethanol gave poor yield of gymnemic acid Al, elution with 95% ethanol brought good recovery of gymnemic acid Al . esterified with acids . hydrolysis .
The genin part of gymnemic acid is
Acids in gymnemic acid A1 were liberated by alkaline
Paper chromatogram of the hydrolyzate gave three spots of acetic,
isovaleric and tiglíc acids . By gas chromatography of the hydrolyzate, 1 mole of acetic acid, 2 moles of isovaleric acid and 1 mole of tiglic acid were identified .
Stbcklin et a1 . 4 identified five acids (formic, acetic, n-butyric,
isovaleric and tiglíc acids) from alkaline hydrolysis of gymnemic acid A . Apparently, the difference between the results arose from the fact that we used purified Al while they used a mixture of gymnemic acids .
The elemental analyses
of the obtained gymnemic acid Al corresponded to the structure which was proposed by Stöcklin and modified, based on our results about acid components in
Vol. 8, No. 9 the genin.
GYMNEMIC ACID DERIVATIVES
541
The presence of potassium bicarbonate led gymnemic acid Al into a
substance which gave a smaller Rf than that of Al on thin layer chromatography . This modified substance was purified on a DEAF-Sephadex column and was identified as gymnemic acid A2 isolated by the method according to Stlicklin . 4
Refluxing
gymnemic acid Al in 3% KOH methanol solution yielded a substance whose behavior on thin layer chromatography was identical to gymnemic acid A3 prepared by the method used by Stdcklin .
Acids liberated by the modification were identified
by paper and gas chromatography .
Results indicated that one mole of acetic acid
was liberated by the conversion of Al to A2 and that 2 moles of isovaleric acid and 1 mole of tiglic acid were liberated by the conversion of AZ to A3 .
The
elemental analyses of the isolated A2 and A3 corresponded to the structures expected from the postulation that only ester groups of genin were modified by the alkaline hydrolysis of Al . - 1 mole of acetic acid Al
A2
- 1 mole of tiglic acid - 2 moles of isovaleric acid
A3
Antisweet activities of gymnemic acid Al, A2 and A3 were assayed (see Figure 1) .
GYYNEYIC ACID Ai a-,- SX10-so
y-~- 1 xlo - su
-w-- 1x90 -4M
0
_
GYYNEYIC ACID AI I -o- SXp - SY II-a- IX10-3Y
.A 0
igr
OA kz\\
0."
o
\\\
s
O_
o
s to MINUTES
FIG. 1
m
w
542
GYMNEMIC ACID DERIVATIVES
Vol . 8, No . 9
Antisweet activity of gymnemic acid Al and A2 . Activity was assayed at 5 minute intervals after gymnemic acid solution was held in the mouth for 2 min . This activity is expressed by the maximum concentration of sucrose solution whose sweetness was depressed completely . average variation .
The bars indicate the range of
Molecular weights of gymnemic acid Al and A2 were assumed
to be 1010 and 1022, respectively .
The antisweet activity of gymnemic acid
reached maximum level after being held in the mouth in solution form for about 30 seconds .
The activity was assayed by tasting a series of sucrose solutions
after a gymnemic acid solution was held in the mouth for 2 min and the activity was expressed by the maximum concentration of sucrose solution whose sweetness was depressed completely .
Cymnemic acid A3 did not show any antisweet activity .
As seen from the curve III, 1 x 10 -4M Al depressed the sweetness of 0 .3M sucrose solution and this effect decreased in 15 min .
Application of 5 x 10 -3!! Al
brought the complete depression of sweetness of solid sucrose as well as that of 1 .OM sucrose solution and the effect decreased as shown by curve I .
of gymnemic acid A2 was much lower than that of Al .
The activity
The activity of 5 x 10 -3M
A2 (curve IV) was a little lower than that of 1 x 10 -3M Al (curve II) .
This
indicated that the activity of A2 is less than 1/5 of the activity of an equal concentration of Al .
It is interesting to note that activity decreased greatly
by the conversion of Al into A2 even though the conversion of Al into A2 resulted in the loss of only one mole of acetic acid .
Apparently, the ester group in the
genin has an important role in manifestation of antisweet activity of gymnemic acid Al .
Since gymnemic acid Al has a rather large molecular weight of about 1,000, the elimination of acetic acid from Al will not have a large effect on
the whole molecule of Al .
The moiety of the ester group of gymnemic acid Al
may be related directly to the binding of Al on the receptor site in taste buds . It is known that a great variety of other compounds besides sugars
taste
sweet, e .g ., synthetic sweetening agents, D-amino acids, and some inorganic compounds .
Antisweet activity of gymnemic acid Al on cyclamate, D-tryptophan,
D-leucine, beryllium chloride, lead acetate, and chloroform was also examined . The sweet taste of all these compounds except for chloroform was suppressed by the action of gymnemic acid Al .
Although it is not known whether these compounds
of different chemical structure induce sweet taste by the same mechanism, it can be concluded Ehat at least part of the sweet mechanism is common to substances of quite different structure .
Chloroform is the only sweet substance which has
a high vapour pressure and is a good solvent for lipids . related to the different mechanism of sweet induction . unique. flavor .
This fact may be Sodium glutamate has a
Application of 5 x 10 -3M gymnemic acid Al changed the unique
taste of crystalline sodium glutamate to a taste much like sodium chloride .
Vol . 8, No. 9
GYMNEMIC ACID DERIVATIVES
543
Summary A method was devised to isolate gymnemic acid Al, a main component of gymnemic acid A,
for preparative purpose .
Acids esterified in the genin of
gymnemic acid Al were determined to be 1 mole of acetic, 2 moles of isovaleric and 1 mole of tiglic acids.
Gymnemic acid Al was converted into A2, and finally
into A3 by alkaline hydrolysis .
Antisweet activity of Al was greatly decreased
by conversion into A2, while A3 had no activity .
It is suggested that'the eater
group in the genin has an important role in the manifestation of the antisweet activity of gymnemic acid A1 .
Studies regarding antisweet activity of gymnemic
acid on other sweet compounds besides sugars indicated that gymnemic acid A1 suppresses the sweet taste of cyclamate,
D-amino acids, beryllium chloride, and
lead acetate and that it does not suppress the sweet taste of chloroform . Acknowledgments This work was supported by the National Science Foundatiod Grant No . GB4068% and the Atomic Energy Commission Grant No . AT-(40-1)-2690 .
The author
wishes to thank Dr . Lloyd M. Beidler for many valuable suggestions and continued encouragement and Mr . Cuyler V. Smith for valuable technical assistance . References 1.
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