1)el)ilrtment of Biochemistry, Research lnstitcrte for WAKAN-YAKU (Oriental Drug), Toyama,University
A COLOR REACTION O F PANAXADIOL WITH VANILLIN AND SULFURIC ACID
By S. HIAI,H. OURA,H. HAMANAKA and Y. ODAKA
A color reaction for panaxadiol, an artifact sapogenin produced by acid hydrolysis from saponin of Ginseng root (root o f Panax ginseng C. A. MEYER),and for Ginseng saponin mixture containing glycosides of protopanaxadiol, a gentfine sapogenin, is described. A red-purple color product of panaxadiol or Ginseng saponin was formed by heat treatment with vanillin and sulfuric acid. The redpurple color was stable for two hours in an ice-cold water bath and had a visible absorption maximum at 544 nm. The relationship between absorbance at 544 n m and quantity of both panaxadiol and Ginseng saponin preparation followed Beer's law for 2 0 p g t o 100 ,~igof panaxadiol and 50 pg to 250 pg of Ginseng saponin.
As reported previously [I], treatment of rats with several fractions from aqueous extract of Ginseng root (root of Panax ginseng C. A. MEYER)stimulated the synthesis of the liver RNA and serum protein. T h e LIEBERMANN-BURCHARD reaction of the fractions was positive and gave a stable red-purple color. It was also established that several saponins isolated from a Ginseng fraction were the biochemically active principle [2]. Numerous ginsenosides [3] were reported as saponin from Ginseng root and the structures of nine ginsenosides among them were established [4]. Genuine aglycones and the main common sugar component o f the ginsenosides were 20s-protopanaxadiol, 20s-protopanaxatriol, oleanolic acid and glucose. When we assayed the sugar content of a fraction from Ginseng extract with anthrone reagents, as a tentative measure of saponin content, we encountered an abnormal orange color which seemed to depend on Ginseng sapogenin. But by regular and modified anthrone reactions we failed to clearly differentiate the sapogenin color from that of the sugar moiety. T o find out a colorimetric determination method of Ginseng sapogenin, numerous determination and detection reagents for sugars, steroids, sterols and saponins were applied to the Ginseng fractions. Among these reagents tested,
,
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Abstract
132
Hiai, Oura, Hamanaka and Odaka
Planta medica Vol. 28 1975
we found that vanillin and sulfuric acid, the spray reagents for saponins on thin-layer chromatogram [ S ] , reacted with panaxadiol [ 6 ] ,an artifact sapogenin of Ginseng, under the conditions developed by the present authors. We describe that the present vanillin-sulfuric acid reaction may be used as a colorimetric determination method of panaxadiol, o r its corresponding saponins in crude saponins extracted from Ginseng root.
The procedure and the effect of concentrations of reagents and the effect of reaction temperature are described under Results and Discussion. The following reagents and procedures were standardized by the present results. Reagents: 8% (wlv) vanillin solution: 800 mg of vanillin (reagent grade) was dissolved in 10 ml of ethanol (99.5% vlv, reagent grade). Prepared fresh each day. 72% (vlv) sulfuric acid: to 28rnl of of deionized water, 72 rnl of concentrated sulfuric acid (reagent grade, 95.0% wlw) was added. Procedrire: T o 0.5 ml of aqueous o r ethanol solution of each sample, 0.5 ml of 8% vanillin solution and then 5.0 ml of 72% sulfuric acid were added and mixed well in an ice-water bath. Test tubes were warmed in a water bath a t 60° for 10 min, then cooled in ice-cold water. Absorbnnces of the reaction mixture were recorded against the blank solution with a Hitachi Spectrophotometer Model 124 o r a Hitachi Perkin-Elmer Spectrophotonleter Model 139. Ginseng Saponin a n d Sapogenin: From roots of Panax ginseng, fractions 3, 4 and 5 were prepared as described in a previous paper [2]. Ginseng roots, produced in Korea, were powdered and extracted with 0.05 MTris-buffer (pH 7.6) under stirring for 48 hours in a cold room and then filtered. The filtrate was centrifuged, and the clear supernatant fluid obtained was dialized against running cold tap water. The internal solution was brought to 70% saturation of an~nlonium sulfate. The precipitate was dissolved in deionized water, dialized against deionized water to be salt-free, and then the internal solution was lyophilized (fraction 3) [2]. Fraction 3 was extracted with methanol on a water bath under reflux, and the methanol solution was concentrated under reduced pressure. T o this residual solution 15 volumes of cold ether was added. The slightly yellowish precipitate (fraction 4) was dried overnight in vacuum. Fractjon 4 was dissolved in distilled water, and dialized against distilled water for one week in a cold room. A white precipitate (fraction 5) was observed in the dialysis bag. After centrifugation, fraction 5 was recrystallized from distilled water and a white powder was obtained: mp 182-186", element anal. C, 54.50; H, 8.54; N, none; ash, none. From fraction 5, ginsenoside-Rb,, -Rb,, Rc, -Rc,, -Rd, -Re and -Rg, were isolated and identified [2]. Panaxadiol was obtained from fraction 4 by hydroysis with diluted sulfuric acid. T h e hydrolysate was extracted with chloroform. Panaxadiol was isolated from the chloroform extrace by means of preparative thin-layer chromatography on Silica gel G and identified by rnp, nuclear magnetic resonance spectra and the rnp of its 3-monoacetate [6].
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Materials and Methods
Color Reaction of Panaxadiol
133
Color Development with Vanillin and Sulfuric Acid, and influence of the Concentration of Sulfuric Acid As m d e l s , t o determine the reaction conditions for Ginseng sapogenin moiety, we used fraction 3 which contains glycosides of genuine sapogenins, oleanolic acid as an available sapogenin among Ginseng sapogenins, and glucose as a major common sugar moiety. To 1.0 ml of each aqueous solution of the model compounds, 1.0 ml of 0.5% (w/v) aqueous vanillin solution and then 4.0 ml of various concentrations of sufuric acid were added and mixed well in an ice-water bath. T h e test tubes were heated in a boiling water bath for 1 0 min and then cooled in ice-cold water. With all tested concentrations of sulfuric acid, a redpurple color .appeared for oleanolic acid and fraction 3, and a brown color for glucose (Fig. 1).When the concentration of sulfuric acid was reduced, the intensities of the color of oleanolic acid and of the brown color of glucose decreased without changes in the patterns of the absorption spectra. When the sulfuric acid concentration was reduced from 95 to 90% (vlv), the color intensity of fraction 3
Wove length ( nm )
Fig. 1. Absorption Spectra of Oleanolic Acid, Ginseng Saponin Fraction 3 and Glucose after the Reaction with Vanillin and Various Concentrations of Sulfuric Acid. Total volume of the reaction mixture was 6 ml.
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Results and Discussion
134
Hiai, Oura, Harnanaka and Odaka
Planta rnedica Vol. 28 1975
decreased and the absorption maximum shifted from 538 to 544 nm. Thus i t is suggested that diluting the sulfuric acid as the color reagent somewhat reduced the color intensity of saponin in fraction 3 but increased the specificity of the color reaction due t o drastic decrease in the color reaction of glucose. Thus, in the following experiments, we used 90% (v/v) sulfuric acid and recorded the color intensity of the reaction product of Ginseng saponin at 544 nm.
Absorbances at 544 nm of fraction 3 and glucose were determined when they were treated with 0.5% vanillin and 90% (v/v) sulfuric acid a t various temperatures for 10min. As shown in Fig. 2, when heated a t various temperatures, abs o r b a n c e ~of fraction 3 reached almost a maximum a t 5S0, but those of glucose increased exponentially, especially a t 65' o r higher. Thus we fixed the reaction temperature for Ginseng saponin a t 60" t o avoid probable absorbance due t o sugar moiety.
Fig. 2. Effect of Reaction Temperature on the Absorbance of Fraction 3 and Glucose. Solid circle: 300 pg of fraction 3 per tube, open circle: 2 rng of glucose per tube.
When the concentration of vanillin solution (in ethanol) was increased, absorbance of fraction 3 after the reaction increased almost linearly and reached maximum level with 3% vanillin solution (Fig. 3 ) . On the other hand, the increase in absorbance of glucose was rather small. Reagent blank solution gave a yellow color with a high concentration of vanillin but the absorbance a t 544 nm was small.
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Influence of Temperature and of Concentration of Vanillin
135
Vanillin (%)
Fig. 3. Effect of Vanillin Concentrations on the Absorbance of Fraction 3. Solid circle: 300 pg of fraction 3 per tube, open circle: 2 mg of glucose per tube. Solid triangle: reagent blank solution, values of absorbance were recorded against the reaction mixture without vanillin.
0
a 0.4
/
-
U C
0
-2 L 0 o
2 0.2 Fig. 4. Effect of Reaction Temperature on the Absorbance of Fraction 3. Circles and triangles are the same as in Fig. 3.
f i Q
A
" -0
-
60
-
/O
/ 70
80
Temp. ( O C )
Effect of Reaction Temperature and Time under a Revised Procedure When the reagents and sample solution were mixed, color development was begun by the heat of ionization of sulfuric acid. To reduce and control the effect of the heat of ionization, the initial concentration of sulfuric acid and the volumes of vanillin and sample solutions were reduced so that their final concentrations after mixing were kept constant.
Downloaded by: National University of Singapore. Copyrighted material.
Color Reaction of Panaxadiol
Hiai, Oura, Hamanaka and Odaka
Planta medica Vol. 28 1975
'
T o 0.5 ml of sample s ~ l u t i o n 0.5 , ml of 8% (w/v) vanillin solution (in ethanol) and 5.0 ml of 72% (vlv) sulfuric acid were added and mixed well in an icewater bath, and the test tubes were heated a t various temperatures for 10 min. As shown in Fig.4, the absorbances of fraction 3 remained almost constant when heated a t 55" o r higher, but the brown color of glucose remarkably increased when heated a t 65" o r higher. So the standard reaction temperature was finally fixed a t 60". The effect of reaction time was studied under heating a t 60". As shown in Fig. 5 , absorbance of fraction 3 reached maximum level when heated for 40 min o r more. Absorbance of glucose was very slight under heating for 5 or 10 min but rose above a negligible level when heated for 20 min o r more. Thus the standard reaction time was set a t 10 min to minimize interference by glucose reaction, despite the fact that the heating time was not long enough to allow full color development of the sapogenin.
L .U w
C 0
0.4 -
$
o /'
P /" /LA-
9
a
0.2 -
/./A
0
I
^-O
10
20
I
I
I
3 0 40 5 0 Time ( min.)
1
Fig. 5. Effect of Reaction Time on the Absorbance of Fraction 3. Circles and tiiangles are the same as in Fig. 3.
60
The absorbance a t 544 nm of the chromogen was stable for 2 hours o r more when the reaction mixture was kept in an ice-water bath.
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136
Color Reaction of Panaxadiol
137
T h e absorption spectra of chromogens of panaxadiol (an artifact sapogenin) and Ginseng fraction 5 containing glycosides of protopanaxadiol (a genuine aglycone) a r e shown in Fig. 6 . Those two samples gave a red-purple color which had almost the identical pattern of spectrum and the same absorption maximum a t 544 nm. Careful comparison showed a little difference between their spectra. T h e difference might be derived from structural differences between artifact and genuine sapogenins and/or from minor aglycones and'sugar components other than panaxadiol and glucose. However, the similarity of the absorption spectra suggested that absorbance of panaxadiol-chromogen could represent. amount of glycosides of protopanaxadiol.
Fig. 6. Absorption Spectra of Panaxadiol and Ginseng Fraction 5 after the Reaction.
0
500
(nm)
600
Effect of Variation of the Amounts of Panaxadiol and Ginseng Saponin Standard curve obtained was linear between 20pg to lOOpg of panaxadiol in 6 ml of reaction mixture and followed Bee's law (Fig. 7). With two preparations of fraction 3 and 5 from Ginseng roots, we obtained linear curves which followed Beer's law, from 50 pg to 250 pg and from 5 0 yg to 400 pg, respectively. From the recorded absorbance a t 544 nm, the estimated sapogenin contents (panaxadiol equivalent) in fraction 3 and 5 were 28 and 41%, respectively. Thus the present color reaction may be a simple, rapid and relatively more precise estimation method for saponin content in a Ginseng root extract so that the saponin content can be expressed as panaxadiol equivalent (moles o r weight). Results on the color reaction of isolated ginsenosides are now in manuscript.
Downloaded by: National University of Singapore. Copyrighted material.
Absorption Spectra of Panaxadiol and Ginseng Saponin Mixture under the Standardized Reaction
Planta niedica Vol. 28 1975
Hiai, Oura, Harnanaka and Odaka
Fig. 7. Relationship between Absorbance at 544 nm and Amount of Panaxadiol and Ginseng Fractions 3 and 5. a: panaxadiol, b: fraction 5, c: fraction 3.
Acknowledgement The authors express their gratitude to Professor Y. TSUDAof Showa Pharmaceutical College for his kind donation of oleanolic acid and for nuclear magnetic resonance spectral measurements of panaxadiol and panaxadiol 3-monoacetate. One of the authors (S. H.) wishes to express his sincere thanks ro Professor T. N A M B A RofA Tohoku University for his encouragement, advice and discussion. References '[I] O U R A ,H., HIAI,S., NAKASHIMA, S. and TSUKADA, K.: Chern. Pharrn. Bull. (Tokyo), 19, 453 (1971); OURA,H., HIAI, S. and SENO, H.: Chem. Pharm. Bull. (Tokyo), 19, 1598 (1971); HIAI,S., OURA,H., TSUKADA, K. and HIRAI,Y.: Chern. Pharnl. Bt111. (Tokyo), 19, 1656 K. and NAKACAWA, H.: Chem. Pharm. Bull. (Tokyo), 20, 219 (1971); OURA.H., TSUKADA, K. and OHOTA,Y.: Chern. Pharm. Bull. (Tokyo), (1972); OURA,H., NAKASHIMA, S., TSUKADA, 20,980 (1972) [4] NAGAI,Y., TANAKA, 0. and SHIBATA, S.: Tetrahedron, 27,881 (1971); SANADA, S., KONDO,N., [3] SHIBATA, S., TANAKA, O., ANDO,T., SANO,M., TSUSHIMA, S. and OHSAWA, T.: Chem. Pharm. Bull. (Tokyo), 14,595 (1966) 0. and SHIBATA, S.: Tetrahedron, 27, 881 (1971); SANADA, S., KODO, N., [4] NACAI,Y., TANAKA, SHOJI,J., TANAKA, 0. and SHIBATA, S.: Chem. Pharrn.Bul1. (Tokyo), 22,421 (1974); SANADA, S., 0. and SHIBATA, S.: Chern. Pharrn. Bull. (Tokyo), 22, 2407 KONDO,N., SHOJI,J., TANAKA, (1974) [S] STAHL,E. and JORK,H.: ,,Thin-Layer Chronlatography", 2nd ed., ed. by STAHL,E., Springer Verlag, Berlin, 1969, p. 247 [6] SHIBATA, S., FUIITA,M., ITOKAWA,H., TANAKA, 0. and ISHII, T.: Tetrahedron Letters, No. 10,419 (1962)
Address: Dr. S . Hiai, Department of Biochemistry, Research Institute for W A K A N - Y A K U , Toyama University, 3190, G o f u k z ~Toyamn, , 930, Japan
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138
A COLOR REACTION O F PANAXADIOL WITH VANILLIN AND SULFURIC ACID
By S. HIAI,H. OURA,H. HAMANAKA and Y. ODAKA
A color reaction for panaxadiol, an artifact sapogenin produced by acid hydrolysis from saponin of Ginseng root (root o f Panax ginseng C. A. MEYER),and for Ginseng saponin mixture containing glycosides of protopanaxadiol, a gentfine sapogenin, is described. A red-purple color product of panaxadiol or Ginseng saponin was formed by heat treatment with vanillin and sulfuric acid. The redpurple color was stable for two hours in an ice-cold water bath and had a visible absorption maximum at 544 nm. The relationship between absorbance at 544 n m and quantity of both panaxadiol and Ginseng saponin preparation followed Beer's law for 2 0 p g t o 100 ,~igof panaxadiol and 50 pg to 250 pg of Ginseng saponin.
As reported previously [I], treatment of rats with several fractions from aqueous extract of Ginseng root (root of Panax ginseng C. A. MEYER)stimulated the synthesis of the liver RNA and serum protein. T h e LIEBERMANN-BURCHARD reaction of the fractions was positive and gave a stable red-purple color. It was also established that several saponins isolated from a Ginseng fraction were the biochemically active principle [2]. Numerous ginsenosides [3] were reported as saponin from Ginseng root and the structures of nine ginsenosides among them were established [4]. Genuine aglycones and the main common sugar component o f the ginsenosides were 20s-protopanaxadiol, 20s-protopanaxatriol, oleanolic acid and glucose. When we assayed the sugar content of a fraction from Ginseng extract with anthrone reagents, as a tentative measure of saponin content, we encountered an abnormal orange color which seemed to depend on Ginseng sapogenin. But by regular and modified anthrone reactions we failed to clearly differentiate the sapogenin color from that of the sugar moiety. T o find out a colorimetric determination method of Ginseng sapogenin, numerous determination and detection reagents for sugars, steroids, sterols and saponins were applied to the Ginseng fractions. Among these reagents tested,
,
Downloaded by: National University of Singapore. Copyrighted material.
Abstract
132
Hiai, Oura, Hamanaka and Odaka
Planta medica Vol. 28 1975
we found that vanillin and sulfuric acid, the spray reagents for saponins on thin-layer chromatogram [ S ] , reacted with panaxadiol [ 6 ] ,an artifact sapogenin of Ginseng, under the conditions developed by the present authors. We describe that the present vanillin-sulfuric acid reaction may be used as a colorimetric determination method of panaxadiol, o r its corresponding saponins in crude saponins extracted from Ginseng root.
The procedure and the effect of concentrations of reagents and the effect of reaction temperature are described under Results and Discussion. The following reagents and procedures were standardized by the present results. Reagents: 8% (wlv) vanillin solution: 800 mg of vanillin (reagent grade) was dissolved in 10 ml of ethanol (99.5% vlv, reagent grade). Prepared fresh each day. 72% (vlv) sulfuric acid: to 28rnl of of deionized water, 72 rnl of concentrated sulfuric acid (reagent grade, 95.0% wlw) was added. Procedrire: T o 0.5 ml of aqueous o r ethanol solution of each sample, 0.5 ml of 8% vanillin solution and then 5.0 ml of 72% sulfuric acid were added and mixed well in an ice-water bath. Test tubes were warmed in a water bath a t 60° for 10 min, then cooled in ice-cold water. Absorbnnces of the reaction mixture were recorded against the blank solution with a Hitachi Spectrophotometer Model 124 o r a Hitachi Perkin-Elmer Spectrophotonleter Model 139. Ginseng Saponin a n d Sapogenin: From roots of Panax ginseng, fractions 3, 4 and 5 were prepared as described in a previous paper [2]. Ginseng roots, produced in Korea, were powdered and extracted with 0.05 MTris-buffer (pH 7.6) under stirring for 48 hours in a cold room and then filtered. The filtrate was centrifuged, and the clear supernatant fluid obtained was dialized against running cold tap water. The internal solution was brought to 70% saturation of an~nlonium sulfate. The precipitate was dissolved in deionized water, dialized against deionized water to be salt-free, and then the internal solution was lyophilized (fraction 3) [2]. Fraction 3 was extracted with methanol on a water bath under reflux, and the methanol solution was concentrated under reduced pressure. T o this residual solution 15 volumes of cold ether was added. The slightly yellowish precipitate (fraction 4) was dried overnight in vacuum. Fractjon 4 was dissolved in distilled water, and dialized against distilled water for one week in a cold room. A white precipitate (fraction 5) was observed in the dialysis bag. After centrifugation, fraction 5 was recrystallized from distilled water and a white powder was obtained: mp 182-186", element anal. C, 54.50; H, 8.54; N, none; ash, none. From fraction 5, ginsenoside-Rb,, -Rb,, Rc, -Rc,, -Rd, -Re and -Rg, were isolated and identified [2]. Panaxadiol was obtained from fraction 4 by hydroysis with diluted sulfuric acid. T h e hydrolysate was extracted with chloroform. Panaxadiol was isolated from the chloroform extrace by means of preparative thin-layer chromatography on Silica gel G and identified by rnp, nuclear magnetic resonance spectra and the rnp of its 3-monoacetate [6].
Downloaded by: National University of Singapore. Copyrighted material.
Materials and Methods
Color Reaction of Panaxadiol
133
Color Development with Vanillin and Sulfuric Acid, and influence of the Concentration of Sulfuric Acid As m d e l s , t o determine the reaction conditions for Ginseng sapogenin moiety, we used fraction 3 which contains glycosides of genuine sapogenins, oleanolic acid as an available sapogenin among Ginseng sapogenins, and glucose as a major common sugar moiety. To 1.0 ml of each aqueous solution of the model compounds, 1.0 ml of 0.5% (w/v) aqueous vanillin solution and then 4.0 ml of various concentrations of sufuric acid were added and mixed well in an ice-water bath. T h e test tubes were heated in a boiling water bath for 1 0 min and then cooled in ice-cold water. With all tested concentrations of sulfuric acid, a redpurple color .appeared for oleanolic acid and fraction 3, and a brown color for glucose (Fig. 1).When the concentration of sulfuric acid was reduced, the intensities of the color of oleanolic acid and of the brown color of glucose decreased without changes in the patterns of the absorption spectra. When the sulfuric acid concentration was reduced from 95 to 90% (vlv), the color intensity of fraction 3
Wove length ( nm )
Fig. 1. Absorption Spectra of Oleanolic Acid, Ginseng Saponin Fraction 3 and Glucose after the Reaction with Vanillin and Various Concentrations of Sulfuric Acid. Total volume of the reaction mixture was 6 ml.
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Results and Discussion
134
Hiai, Oura, Harnanaka and Odaka
Planta rnedica Vol. 28 1975
decreased and the absorption maximum shifted from 538 to 544 nm. Thus i t is suggested that diluting the sulfuric acid as the color reagent somewhat reduced the color intensity of saponin in fraction 3 but increased the specificity of the color reaction due t o drastic decrease in the color reaction of glucose. Thus, in the following experiments, we used 90% (v/v) sulfuric acid and recorded the color intensity of the reaction product of Ginseng saponin at 544 nm.
Absorbances at 544 nm of fraction 3 and glucose were determined when they were treated with 0.5% vanillin and 90% (v/v) sulfuric acid a t various temperatures for 10min. As shown in Fig. 2, when heated a t various temperatures, abs o r b a n c e ~of fraction 3 reached almost a maximum a t 5S0, but those of glucose increased exponentially, especially a t 65' o r higher. Thus we fixed the reaction temperature for Ginseng saponin a t 60" t o avoid probable absorbance due t o sugar moiety.
Fig. 2. Effect of Reaction Temperature on the Absorbance of Fraction 3 and Glucose. Solid circle: 300 pg of fraction 3 per tube, open circle: 2 rng of glucose per tube.
When the concentration of vanillin solution (in ethanol) was increased, absorbance of fraction 3 after the reaction increased almost linearly and reached maximum level with 3% vanillin solution (Fig. 3 ) . On the other hand, the increase in absorbance of glucose was rather small. Reagent blank solution gave a yellow color with a high concentration of vanillin but the absorbance a t 544 nm was small.
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Influence of Temperature and of Concentration of Vanillin
135
Vanillin (%)
Fig. 3. Effect of Vanillin Concentrations on the Absorbance of Fraction 3. Solid circle: 300 pg of fraction 3 per tube, open circle: 2 mg of glucose per tube. Solid triangle: reagent blank solution, values of absorbance were recorded against the reaction mixture without vanillin.
0
a 0.4
/
-
U C
0
-2 L 0 o
2 0.2 Fig. 4. Effect of Reaction Temperature on the Absorbance of Fraction 3. Circles and triangles are the same as in Fig. 3.
f i Q
A
" -0
-
60
-
/O
/ 70
80
Temp. ( O C )
Effect of Reaction Temperature and Time under a Revised Procedure When the reagents and sample solution were mixed, color development was begun by the heat of ionization of sulfuric acid. To reduce and control the effect of the heat of ionization, the initial concentration of sulfuric acid and the volumes of vanillin and sample solutions were reduced so that their final concentrations after mixing were kept constant.
Downloaded by: National University of Singapore. Copyrighted material.
Color Reaction of Panaxadiol
Hiai, Oura, Hamanaka and Odaka
Planta medica Vol. 28 1975
'
T o 0.5 ml of sample s ~ l u t i o n 0.5 , ml of 8% (w/v) vanillin solution (in ethanol) and 5.0 ml of 72% (vlv) sulfuric acid were added and mixed well in an icewater bath, and the test tubes were heated a t various temperatures for 10 min. As shown in Fig.4, the absorbances of fraction 3 remained almost constant when heated a t 55" o r higher, but the brown color of glucose remarkably increased when heated a t 65" o r higher. So the standard reaction temperature was finally fixed a t 60". The effect of reaction time was studied under heating a t 60". As shown in Fig. 5 , absorbance of fraction 3 reached maximum level when heated for 40 min o r more. Absorbance of glucose was very slight under heating for 5 or 10 min but rose above a negligible level when heated for 20 min o r more. Thus the standard reaction time was set a t 10 min to minimize interference by glucose reaction, despite the fact that the heating time was not long enough to allow full color development of the sapogenin.
L .U w
C 0
0.4 -
$
o /'
P /" /LA-
9
a
0.2 -
/./A
0
I
^-O
10
20
I
I
I
3 0 40 5 0 Time ( min.)
1
Fig. 5. Effect of Reaction Time on the Absorbance of Fraction 3. Circles and tiiangles are the same as in Fig. 3.
60
The absorbance a t 544 nm of the chromogen was stable for 2 hours o r more when the reaction mixture was kept in an ice-water bath.
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136
Color Reaction of Panaxadiol
137
T h e absorption spectra of chromogens of panaxadiol (an artifact sapogenin) and Ginseng fraction 5 containing glycosides of protopanaxadiol (a genuine aglycone) a r e shown in Fig. 6 . Those two samples gave a red-purple color which had almost the identical pattern of spectrum and the same absorption maximum a t 544 nm. Careful comparison showed a little difference between their spectra. T h e difference might be derived from structural differences between artifact and genuine sapogenins and/or from minor aglycones and'sugar components other than panaxadiol and glucose. However, the similarity of the absorption spectra suggested that absorbance of panaxadiol-chromogen could represent. amount of glycosides of protopanaxadiol.
Fig. 6. Absorption Spectra of Panaxadiol and Ginseng Fraction 5 after the Reaction.
0
500
(nm)
600
Effect of Variation of the Amounts of Panaxadiol and Ginseng Saponin Standard curve obtained was linear between 20pg to lOOpg of panaxadiol in 6 ml of reaction mixture and followed Bee's law (Fig. 7). With two preparations of fraction 3 and 5 from Ginseng roots, we obtained linear curves which followed Beer's law, from 50 pg to 250 pg and from 5 0 yg to 400 pg, respectively. From the recorded absorbance a t 544 nm, the estimated sapogenin contents (panaxadiol equivalent) in fraction 3 and 5 were 28 and 41%, respectively. Thus the present color reaction may be a simple, rapid and relatively more precise estimation method for saponin content in a Ginseng root extract so that the saponin content can be expressed as panaxadiol equivalent (moles o r weight). Results on the color reaction of isolated ginsenosides are now in manuscript.
Downloaded by: National University of Singapore. Copyrighted material.
Absorption Spectra of Panaxadiol and Ginseng Saponin Mixture under the Standardized Reaction
Planta niedica Vol. 28 1975
Hiai, Oura, Harnanaka and Odaka
Fig. 7. Relationship between Absorbance at 544 nm and Amount of Panaxadiol and Ginseng Fractions 3 and 5. a: panaxadiol, b: fraction 5, c: fraction 3.
Acknowledgement The authors express their gratitude to Professor Y. TSUDAof Showa Pharmaceutical College for his kind donation of oleanolic acid and for nuclear magnetic resonance spectral measurements of panaxadiol and panaxadiol 3-monoacetate. One of the authors (S. H.) wishes to express his sincere thanks ro Professor T. N A M B A RofA Tohoku University for his encouragement, advice and discussion. References '[I] O U R A ,H., HIAI,S., NAKASHIMA, S. and TSUKADA, K.: Chern. Pharrn. Bull. (Tokyo), 19, 453 (1971); OURA,H., HIAI, S. and SENO, H.: Chem. Pharm. Bull. (Tokyo), 19, 1598 (1971); HIAI,S., OURA,H., TSUKADA, K. and HIRAI,Y.: Chern. Pharnl. Bt111. (Tokyo), 19, 1656 K. and NAKACAWA, H.: Chem. Pharm. Bull. (Tokyo), 20, 219 (1971); OURA.H., TSUKADA, K. and OHOTA,Y.: Chern. Pharm. Bull. (Tokyo), (1972); OURA,H., NAKASHIMA, S., TSUKADA, 20,980 (1972) [4] NAGAI,Y., TANAKA, 0. and SHIBATA, S.: Tetrahedron, 27,881 (1971); SANADA, S., KONDO,N., [3] SHIBATA, S., TANAKA, O., ANDO,T., SANO,M., TSUSHIMA, S. and OHSAWA, T.: Chem. Pharm. Bull. (Tokyo), 14,595 (1966) 0. and SHIBATA, S.: Tetrahedron, 27, 881 (1971); SANADA, S., KODO, N., [4] NACAI,Y., TANAKA, SHOJI,J., TANAKA, 0. and SHIBATA, S.: Chem. Pharrn.Bul1. (Tokyo), 22,421 (1974); SANADA, S., 0. and SHIBATA, S.: Chern. Pharrn. Bull. (Tokyo), 22, 2407 KONDO,N., SHOJI,J., TANAKA, (1974) [S] STAHL,E. and JORK,H.: ,,Thin-Layer Chronlatography", 2nd ed., ed. by STAHL,E., Springer Verlag, Berlin, 1969, p. 247 [6] SHIBATA, S., FUIITA,M., ITOKAWA,H., TANAKA, 0. and ISHII, T.: Tetrahedron Letters, No. 10,419 (1962)
Address: Dr. S . Hiai, Department of Biochemistry, Research Institute for W A K A N - Y A K U , Toyama University, 3190, G o f u k z ~Toyamn, , 930, Japan
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