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Polyfructosans of Asparagus racemosus
Biochemistry Department, Lucknow University, Lucknow U. P., India
Polyfructosans of Asparagus racemosus VIJAY K. MADAN Received, June 6, 1972
Summary The most satisfactory method of fructosan determination involves the extraction of fresh tissue with boiling water, clarification of the extract, chromatography on paper, with or without prior fractionation on celite charcoal column, elution of spots corresponding to the polymers and determination of the total carbohydrate and fructose content colorimetrically. This method of carbohydrate fractionation permitted the simultaneous determination of fructosans and of simple sugars. Four fructosans of different mobilities were identified from tubers of Asparagus racemosus. Fructosans were absent in leaves and shoot.
Introduction Though polyfructosans are widespread in plants, little attention has been paid to their physiological role or chemical content (EDELMAN and JEFFORD, 1968). There has been no detailed study of asparagosin, the polyfructosan in the roots of asparagus (TANRET, 1909; SCHLUBACH and BOE, 1939). Since the available methods of extraction are subject to number of errors, attention was directed towards improving the method of fructosan determination. Asparagus racemosus Willd. was selected for study in view of its high fructosan content. Materials and Methods Asparagus racemosus was grown conditions.
III
the university campus
III
garden soil under natural
Sampling and processing of tubers Samples of tubers were harvested and cleaned immediately. The thin outer covering and central fibre were removed and the residual material sliced using a stainless steel knife. The slices were then immediately randomized and sampled for immediate processing. Extraction of fresh tuber with hot water A 10,0 g sample of tuber slices were added to a 20 ml boiling water in a 100 ml beaker. Boiling was continued for a further 3 minutes to inactivate enzymes. The samples were rapidly cooled to room temperature, tipped into a waring blendor and, following the addition of 40 ml water, blended at full speed for 2 minutes. The ground suspension was transferred to an erlenmeyer flask and heated in a boiling water bath for 10 minutes. About 20 ml more of hot water was added and the flask returned to the boiling water bath and kept for a further 30 minutes with occasional agitation to complete the dissolution of polyfructosans. The hot suspension was cooled to room temperature, mixed with 0,5 ml of saturated neutral lead acetate (to remove proteins etc.) and made to 100 m!. The contents were then filtered and excess of lead removed by the help of potassium oxalate. A clear, colourless solution obtained was either used within few hours or, in the earlier experiments, stored in the cold
z. Pflanzenphysiol. Bd. 68. S. 272-280. 1972.
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for subsequent analysis. In the later case a few crystals of benzoic acid were added as preservative. When chromatography was also employed, the solutions obtained after filtration of lead oxalate were deionized by contact exchange with Amberlite IR-120 or by passing through a column of Dowex-50. Period of contact with hot water for complete extraction BACON and LOXLEY (1952) achieved extraction of polyfructosans from the tubers of Helianthus tuberosus by boiling with water for 3 minutes, followed by disintegration in a Waring blendor after cooling to room temperature. This treatment was insufficient to solubilize all the polyfructosans in the tuber of asparagus. Ten g samples of sliced tuber were boiled for 3 minutes with 20 ml water, cooled and homogenized with the addition of 20 ml more of water, as already described. The suspension was passed through muslin and the filtrate clarified with lead acetate. Total carbohydrate estimation on the final extract gave a value of 180 mg in one experiment and 200 mg in another. These values fell considerably short of the total carbohydrate and also of total fructose extracted by hot ethanol and water from dried tissue. When the homogenates were heated for 40 minutes, without passing through muslin, the amounts of total carbohydrate and total fructose recovered in the finai extracts were nearly the same as when dried powder was extracted with ethanol and water, the method described by McRARY and SLATTERY (1945). In view of the marked difference in the total carbohydrate solubilized on short term and prolonged heating, systematic experiments were planned to elucidate the optimal period of heating. A large sized tuber, after cleaning as usual, was sliced thin and the slices mixed well. Seven 1,0 g samples were weighed and each was boiled for 3 minutes with about 20 ml water. After boiling and homogenization in Waring blendor, each suspension was transferred to a 100 ml volumetric flask. One was set aside for direct analysis. All the others were immersed in boiling water and a flask each withdrawn at 10 minutes intervals and analysed for total carbohydrate. In all the following experiments, the total period of contact with boiling or hot water was 43 minutes (3 minutes boiling with cut slices, 40 minutes of heating of homogenate in boiling water). The possibility of some degradation of polyfructosans as a result of the longer period of contact with hot water was kept in view. Determination of carohydrates Fructose was determined by the method of ROE and PAPADOPOULOS (1954) with fructose as standard. Total carbohydrates were estimated by the Phenol-sulphuric acid method of MONTGOMERY (1957) with glucose as standard and expressed as glucose equivalent. The estimations were conducted without prior hydrolysis and in some experiments also after hydrolysis with 0,5 N hydrochloric acid for 10 minutes at the temperature of boiling water, cooling and neutralization. Reducing sugar estimation was by the method of NELSON as modified by SOMOGYI (1945) conducted both with and without hydrolysis.
Chromatography Paper chromatography Qualitative Descending chromatograms were run at room temperature on unwashed Whatman No. 1 filter papers strips with n - butanol-acetic acid-water, 4 : 1 : 5 (PARTRIDGE, 1948) for 48 hours in preliminary experiments, but 60 hours subsequently for quantitative experiments, a 100 hours period was given for sharp resolution. After drying at room temperature, the papers were sprayed with 4 Ofo aniline (in ethanol) 4 Ofo diphenylamme (in ethanol), orthophosphoric acid, 5 : 5 : 1, and heated at 90° (BUCHAN and SAVAGE,
Z. Pflanzenphysiol. Bd. 68. S. 272-280. 1972.
274
v.
K. MADAN
1952). The reference chromatograms were sprayed with a mixture of glucose, fructose, sucrose and inulin, in some experiments raffinose was also included.
Quantitative. Three identical strips of paper were spotted and developed simultaneously. One was sprayed to detect and identify the spots. The spots in unsprayed strips were marked off by placing in apposition against the sprayed chromatograms. Sufficiently large areas around each spot were cut out. The carbohydrates were extracted by soaking the paper in to 10 ml water contained in centrifuge tubes and heating at 75 to 80° C for 20 minutes in a water bath. Continuous and vigorous mixing of the contents with a glass rod was essential in order to disintegrate the paper. The resulting suspension was centrifuged for 15 minutes, the supernatant transferred to a graduated cylinder, the residue washed once with hot water and the wash water combined with the main fraction. The volume was noted and aliquots removed for total carbohydrate estimation. In later experiments, fructose and reducing sugars were also determined. The extract of the sucrose spot, as expected, did not exhibit any reducing property. Celite Charcoal adsorption chromatography Separation of various carbohydrates has been effected on celite charcoal Columns (WHISTLER and DURSO, 1950; BAILEY, WHELAN and PEAT, 1950; ALM, 1952; ALM, WILLIAMS and TISELIUS, 1952; WHELAN, BAILEY and ROBERTS, 1953; BACON and BELL, 1953; DAVY, 1966). This technique was used after some modifications for separating the carbohydrate fractions in the fresh tubers with special reference to fructose polymers.
Preparation of column. A mixture of equal weights of activated charcoal (Norite) and celite was washed with distilled water and dried at 80-90°. The mixture was poured as a slurry into a glass column fitted with a glass wool followed by cotton wool plug. Gentle suction was applied after some of the solid had settled assuring that there was always a layer of water on the top. The column was washed with water. The packed column measured 12 X 1,5 cm. The flow rate of water was adjusted to 100 mljhour with suction. Loading of column. Tuber extract, containing 62 and 108 mg total carbohydrates, was diluted 5-fold with water and added drop wise from a separatory funnel to the top of the column, which contained about 1,0 ml water above the adsorbent. Adsorption was allowed to take place under suction over a period of 1/2 hour. The effluent measuring 50 ml was separately collected. Elution from column. Displacement of monosaccharides was effected with distilled water (50-200 ml). Increasing concentrations of ethanol (5, 10 and 15 v jv) were used for separating higher saccharides, employing 150-750 ml of each composition. Appearance of carbohydrate in a fraction and completion of elution were followed by taking aliquots from each 50 ml eluate fraction and testing by the phenol sulphuric acid method (MONTGOMERY, 1957) for total carbohydrate. The 5, 10 and 15 Ofo enthanolic eluates were subjected to vaccum distillation at 40-45°, reducing the volume to 10-20 ml. Aliquots of the solutions were analysed for carbohydrate components by colorimetry and by paper chromatography.
Results and Discussion Relation between period of heating and efficiency of extraction
The results obtained on heating the homogenates of boiled tissue for vanous periods are given in table 1.
z. Pflanzenphysiol. Bd. 68. S. 272-280. 1972.
Polyfructosans of Asparagus racemosus
275
Table 1 Effect of different periods of heating. Time in minutes in boiling water after homogenization
Total carbohydrates, mg/g fresh tissue
o
48 48 52 56 65 65 63
10 20 30 40 50 60
The results show that the extraction is not complete by merely boiling the slices in water for 3 minutes. It is clear from the table that the optimum time for complete dissolution of fructosan is 40 minutes.
Analysis by chemical methods The analytical values obtained for 10 g sample of tuber, corresponding to 1 g dry weight in five different experiments are reported in table 2, last 2 samples were employed for quantitative paper chromatography. Table 2 Gross chemical fractionation of water extracts of fresh tubers. Carbohydrate fractions
Content, mg/10 g fresh weight
II
III
IV
V
Total carbohydrates, without hydrolysis
650
630
600
540
620
Combined and free fructose
550
500
490
540
480
Reducing sugar
nil
nil
nil
nil
nil
The most noteworthy feature of the data was the absence of reducing sugar in any of the samples. Fructose present in extracts is most likely in the polymerized form, otherwise free fructose would have reacted with Nelson reagent and gave some value for reducing sugar. It may be pointed out here that in separate experiments where in we were using dried tissue for fructosan determination (McRARY and SLATTERY, 1945), we were encountered with reducing sugars, this might be due to the drying of the tissue or some secondary changes during drying procedures. Z. Pjlanzenphysiol. Bd. 68. 5. 272-280 1972.
276
V. K.
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Analysis by chromatography Qualitative. The solutions obtained in the above experiments were subjected to qualitative paper chromatography. The entire material migrated from the baseline indicating that high molecular polyfructosans of the inulin complexity were not present. Sucrose was present in high concentration. There were 3 unknown spots which migrated to less extent than sucrose and which were presumed to be low molecular polyfructosans. Glucose and fructose gave also faint spots. In view of the fact that chemical methods could not detect reducing sugar, the possibility that glucose and fructose arose by hydrolysis during chromatogram development had to be considered. Quantitative. The results of two samples are recorded in Table 3. Table 3 Chromatographic analysis of water extracts of medium sized tubers*). Estimations
Amount Applied Recovered
Sucrose
,ug Experiment No.1 400
278
270
100
1080
1048
Free fructose + combined fructose
200
200
220
80
1080
700
Reducing sugars
Nil
Nil
Nil
Nil
200
180
160
60
30
620
630
111
150
130
40
15
480
401
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Total carbohydrates
Experiment No.2 Total carbohydrates
Free fructose + combined fructose Reducing sugars
*) Sucrose was the fastest moving component, followed in the order of decreasing mobility by 51' 52, 53 and 5 •.
Sucrose constituted the largest single component. There were 3 slower moving components in the first extract, while the second extract yielded an additional spot, moving the slowest of all, and containing the least amount of carbohydrate. Fructose analysis in all the unidentified spots indicated that it constituted the major port of each spot. S2 corresponded to raffinose in mobility. The analytical data obtained by chromatography demonstrated conclusively that the major components of tuber were sucrose and polyfructosans of low molecular weight. Sucrose, in all probability, was the starting material for the synthesis of polyfructosan, proceeding in stages of increasing molecular complexity.
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It is desired to stress the fact that standard inulin, used in reference chromatograms, always remained on the base line even when run for 100 hours. This excluded artifact formation during chromatography. Celite charcoal adsorption chromatography Chemical analysis
The results of chemical analysis in the various fractions are given in table 4. Table 4 Chemical analysis of celite charcoal eluates. Ethanolic content of eluant, %
Experiment I Nil 5 10 15 20
Volume of eluate, ml
50 300 650 500 150
Total carbohydrate fAg
Recovery in fraction, % of total recovery
750 5,380 36,550 43,480 3,800
0.84 5.98 40.60 47.10 4.30
89,960 Amount applied Recovery
108,000 83.4%
Experiment II
Nil 5 10 15 20
75 225 675 750 150
600 1,350 15,050 41,360
Nil
1.02 2.30 25.70 70.80 0.00
58,360 Amount applied Recovery
62,000 95.6%
Estimations were directly in the eluates from the column. Most of the carbohydrates were eluted with 10 to 15 % ethanol. The recovery from various eluates corresponded to 83.4 and 95.6 % of the carbohydrate applied on column. Paper chromatographic analysis
Since there was only a small amount of carbohydrate in the eluate with water and in the 20 Ofo ethanolic eluates obtained in the experiments, these were not tested. The values obtained with the other fractions are reported in table 5. Z. Pjlanzenphysiol. Bd. 68. S. 272-280. 1972.
278
V. K.
MADAN
Table 5 Quantitative chromatography of celite charcoal eluates Values expressed as [lg
Estimations
Ethanol 10 % Ethanol 15 % Ethanol 5 Ofo -Sp-o-t-l--S-p-o-t-2-S-p-o-t-l--=-Sp-o-t-2--=S-p-o-t-3
Recovery from paper, %
(66)
(36)
(25)
(24)
(17)
(13)
Sample no. 1 Total carbohydrates Fructose Reducing sugars
58 20 Nil
100 80 Nil
50 30 Nil
20 10 Nil
90 60 Nil
100 60 Nil
89
Sample no. 2 Total carbohydrates Fructose Reducing sugars
55 30 20
60 28 Nil
80 40 Nil
42 40 Nil
65 40 Nil
120 100 Nil
93
") The Rg X 100 values are reported within brackets.
Chromatograms of 5 % ethanol eluate revealed the presence only of sucrose, confirmed by chemical and chromatographic analysis. 10 0J0 ethanol eluates gave two spots; one corresponded to raffinose in mobility. That the compared was not raffinose was evident from the fact that fructose content was 50 0J0. The second spot had higher mobility than standard raffinose, but differed in the two experiments. The difference was evident also in the % content of fructose; in the first it was 80 0J0, but in the second it was 47 0J0. The relative distribution of total carbohydrate between the two spots was markedly different in the two experiments. A feature of 10 0J0 ethanolic extracts was the absence of reducing sugar and the satisfactory recovery from chromatograms. 15 % ethanol eluates gave three spots, the fastest moving spots corresponded to slowest moving spot of 10 0J0 eluate, that is raffinose (standard). Two other spots remained in between raffinose and base line. All the spots obtained from 10 and 15 0J0 fractions represented fructosans, because the major carbohydrate components of each spot was fructose as evident from chemical analysis. Raffinose was absent from all the samples, although a fructosan of the same mobility was present in 10 0J0 and 15 0J0 ethanol eluates. Concluding from the above observations, it is clear that four kinds of fructosans are present in any sample of Asparagus tubers. Difference in values obtained from 10 and 15 0J0 ethanol eluates in two experiments may be due to seasonal variation. These two experiments were conducted at a interval of 3 months. Paper partition chromatography directly on tuber extracts resolved fructosans into 3 to 4 kinds (in molecular complexity). When carbohydrates were fractionated on celite charcoal columns prior to paper partition chromatography, fructosans resolved themselves into 4 kinds (in molecular complexity). The additional fructosan described
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279
in the later case may be attributed to the greater resolution on paper when carbohydrates were first fractionated on celite charcoal. In separate experiments with leaves and shoots of Asparagus we could not detect any polyfructosans both by chemical and chromatographic analysis. It is possible leaves did not elaborate fructosan but supplied the raw material sucrose to the roots for transformation to fructosan. Absence of polyfructosans in shoots may be interpreted as indicating that polyfructosans of the tuber are t:tilized by the rapidly growing portions of the plant after being first converted into sucro~e and hexose. Since the shoot was directly derived from the tuber itself or in the tissues of the shoot, by hydrolytic or transfer mechanism. From the results it is evident that the best method for fructosan determination would be to start with fresh tissue, celite charcoal adsorption chromatography followed by quantitative paper chromatography. Using dried samples might lead to some secondry changes. Acknowledgements My thanks are due to Professor P. S. KRISHNAN for guidance. This work was partially financed by P. L. 480 grant No. FG-in-163 (AR-CR-59) from Department of Agriculture.
u.s.
References ALM, R. S.: Gradient elution analysis. II. Oligosaccharides. Acta Chern. Scand. 6, 1186 (1952). ALM, R. S., R. J. P. WILLIAMS and A. TISELIUS: Gradient elution analysis. 1. General treatment. Acta Chern. Scand., 6, 826 (1952). BACON, J. S. D., and D. J. BELL: A new trisaccharide produced from sucrose by mould invertase .. J. Chern. Soc., 2528 (1953). BACON, J. S. D., and R. LOXLEY: Seasonal changes in the carbohydrates of Jerusalem artichoke. Biochem. J., 51, 209 (1952). BAILEY, J. M., W. J. WHELAN and S. PEAT: Carbohydrate primers in the synthesis of starch. J. Chern. Soc., 3692 (1950). BUCHAN, H. L., and R. J. SAVAGE: Chromatography of some starch conversion products. Analyst, 77, 401 (1952). DAVY, J.: Separation of oligosaccharides by column chromatography. Ann. Pharm. Fr., 24, 703 (1966). EDELMAN, J., and T. G. JEFFORD: The mechanism of fructosan metabolism in higher plants as exemplified in Helianthus tuberosus. New Phytol., 67, 517 (1968). McRARY, W. L., and M. C. SLATTERY: The colorimetric determination of fructosans in plant material. J. BioI. Chern., 157, 161 (1945). MONTGOMERY, R.: Determination of glycogen. Arch. Biochem. Biophy., 67, 378 (1957). PARTRIDGE, S. M.: Filterpaper partition chromatography of sugars. Biochem. J., 42, 238 (1948). ROE, J. H., and N. M. PAPADOPOULOS: The determination of fructose-6-phosphate and fructose-1-6-diphosphate. J. BioI. Chern., 210, 703 (1954). SCHLUBACH, H. H., and H. BOE: Untersuchungen iiber Polyfructosans. XIX Liebigs Ann., 532, 191 (1957).
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SOMOGYI, M. A.: A new reagent for the determination of sugars. ]. BioI. Chern., 160, 61 (1945). TANRET, G.: The two new carbohydrates from asparagus. Bull. Soc. Chim., 5, 889 (1909). WHELAN, W. ]., ]. M. BAILEY and P. ]. P. ROBERTS: The mechanism of carbohydrase action. The preparation and properties of maltodextrin substrates. ]. Chern. Soc., 1293 (1953). WHISTLER, R. L., and D. F. DURSO: Chromatographic separation of sugars on charcoal. ]. Am. Chern. Soc., 72, 677 (1950). VIJAY K. MADAN, Biochemistry Department, Lucknow University, Lucknow, U.P. India. Present address: Institut fur Mikrobiologie der Universitat Giittingen, D-34 Gottingen, Grisebachstr. 8, West Germany.
Z. PJlanzenphysiol. Bd. 68. S. 272-280. 1972.
Polyfructosans of Asparagus racemosus VIJAY K. MADAN Received, June 6, 1972
Summary The most satisfactory method of fructosan determination involves the extraction of fresh tissue with boiling water, clarification of the extract, chromatography on paper, with or without prior fractionation on celite charcoal column, elution of spots corresponding to the polymers and determination of the total carbohydrate and fructose content colorimetrically. This method of carbohydrate fractionation permitted the simultaneous determination of fructosans and of simple sugars. Four fructosans of different mobilities were identified from tubers of Asparagus racemosus. Fructosans were absent in leaves and shoot.
Introduction Though polyfructosans are widespread in plants, little attention has been paid to their physiological role or chemical content (EDELMAN and JEFFORD, 1968). There has been no detailed study of asparagosin, the polyfructosan in the roots of asparagus (TANRET, 1909; SCHLUBACH and BOE, 1939). Since the available methods of extraction are subject to number of errors, attention was directed towards improving the method of fructosan determination. Asparagus racemosus Willd. was selected for study in view of its high fructosan content. Materials and Methods Asparagus racemosus was grown conditions.
III
the university campus
III
garden soil under natural
Sampling and processing of tubers Samples of tubers were harvested and cleaned immediately. The thin outer covering and central fibre were removed and the residual material sliced using a stainless steel knife. The slices were then immediately randomized and sampled for immediate processing. Extraction of fresh tuber with hot water A 10,0 g sample of tuber slices were added to a 20 ml boiling water in a 100 ml beaker. Boiling was continued for a further 3 minutes to inactivate enzymes. The samples were rapidly cooled to room temperature, tipped into a waring blendor and, following the addition of 40 ml water, blended at full speed for 2 minutes. The ground suspension was transferred to an erlenmeyer flask and heated in a boiling water bath for 10 minutes. About 20 ml more of hot water was added and the flask returned to the boiling water bath and kept for a further 30 minutes with occasional agitation to complete the dissolution of polyfructosans. The hot suspension was cooled to room temperature, mixed with 0,5 ml of saturated neutral lead acetate (to remove proteins etc.) and made to 100 m!. The contents were then filtered and excess of lead removed by the help of potassium oxalate. A clear, colourless solution obtained was either used within few hours or, in the earlier experiments, stored in the cold
z. Pflanzenphysiol. Bd. 68. S. 272-280. 1972.
Polyfructosans of Asparagus racemosus
273
for subsequent analysis. In the later case a few crystals of benzoic acid were added as preservative. When chromatography was also employed, the solutions obtained after filtration of lead oxalate were deionized by contact exchange with Amberlite IR-120 or by passing through a column of Dowex-50. Period of contact with hot water for complete extraction BACON and LOXLEY (1952) achieved extraction of polyfructosans from the tubers of Helianthus tuberosus by boiling with water for 3 minutes, followed by disintegration in a Waring blendor after cooling to room temperature. This treatment was insufficient to solubilize all the polyfructosans in the tuber of asparagus. Ten g samples of sliced tuber were boiled for 3 minutes with 20 ml water, cooled and homogenized with the addition of 20 ml more of water, as already described. The suspension was passed through muslin and the filtrate clarified with lead acetate. Total carbohydrate estimation on the final extract gave a value of 180 mg in one experiment and 200 mg in another. These values fell considerably short of the total carbohydrate and also of total fructose extracted by hot ethanol and water from dried tissue. When the homogenates were heated for 40 minutes, without passing through muslin, the amounts of total carbohydrate and total fructose recovered in the finai extracts were nearly the same as when dried powder was extracted with ethanol and water, the method described by McRARY and SLATTERY (1945). In view of the marked difference in the total carbohydrate solubilized on short term and prolonged heating, systematic experiments were planned to elucidate the optimal period of heating. A large sized tuber, after cleaning as usual, was sliced thin and the slices mixed well. Seven 1,0 g samples were weighed and each was boiled for 3 minutes with about 20 ml water. After boiling and homogenization in Waring blendor, each suspension was transferred to a 100 ml volumetric flask. One was set aside for direct analysis. All the others were immersed in boiling water and a flask each withdrawn at 10 minutes intervals and analysed for total carbohydrate. In all the following experiments, the total period of contact with boiling or hot water was 43 minutes (3 minutes boiling with cut slices, 40 minutes of heating of homogenate in boiling water). The possibility of some degradation of polyfructosans as a result of the longer period of contact with hot water was kept in view. Determination of carohydrates Fructose was determined by the method of ROE and PAPADOPOULOS (1954) with fructose as standard. Total carbohydrates were estimated by the Phenol-sulphuric acid method of MONTGOMERY (1957) with glucose as standard and expressed as glucose equivalent. The estimations were conducted without prior hydrolysis and in some experiments also after hydrolysis with 0,5 N hydrochloric acid for 10 minutes at the temperature of boiling water, cooling and neutralization. Reducing sugar estimation was by the method of NELSON as modified by SOMOGYI (1945) conducted both with and without hydrolysis.
Chromatography Paper chromatography Qualitative Descending chromatograms were run at room temperature on unwashed Whatman No. 1 filter papers strips with n - butanol-acetic acid-water, 4 : 1 : 5 (PARTRIDGE, 1948) for 48 hours in preliminary experiments, but 60 hours subsequently for quantitative experiments, a 100 hours period was given for sharp resolution. After drying at room temperature, the papers were sprayed with 4 Ofo aniline (in ethanol) 4 Ofo diphenylamme (in ethanol), orthophosphoric acid, 5 : 5 : 1, and heated at 90° (BUCHAN and SAVAGE,
Z. Pflanzenphysiol. Bd. 68. S. 272-280. 1972.
274
v.
K. MADAN
1952). The reference chromatograms were sprayed with a mixture of glucose, fructose, sucrose and inulin, in some experiments raffinose was also included.
Quantitative. Three identical strips of paper were spotted and developed simultaneously. One was sprayed to detect and identify the spots. The spots in unsprayed strips were marked off by placing in apposition against the sprayed chromatograms. Sufficiently large areas around each spot were cut out. The carbohydrates were extracted by soaking the paper in to 10 ml water contained in centrifuge tubes and heating at 75 to 80° C for 20 minutes in a water bath. Continuous and vigorous mixing of the contents with a glass rod was essential in order to disintegrate the paper. The resulting suspension was centrifuged for 15 minutes, the supernatant transferred to a graduated cylinder, the residue washed once with hot water and the wash water combined with the main fraction. The volume was noted and aliquots removed for total carbohydrate estimation. In later experiments, fructose and reducing sugars were also determined. The extract of the sucrose spot, as expected, did not exhibit any reducing property. Celite Charcoal adsorption chromatography Separation of various carbohydrates has been effected on celite charcoal Columns (WHISTLER and DURSO, 1950; BAILEY, WHELAN and PEAT, 1950; ALM, 1952; ALM, WILLIAMS and TISELIUS, 1952; WHELAN, BAILEY and ROBERTS, 1953; BACON and BELL, 1953; DAVY, 1966). This technique was used after some modifications for separating the carbohydrate fractions in the fresh tubers with special reference to fructose polymers.
Preparation of column. A mixture of equal weights of activated charcoal (Norite) and celite was washed with distilled water and dried at 80-90°. The mixture was poured as a slurry into a glass column fitted with a glass wool followed by cotton wool plug. Gentle suction was applied after some of the solid had settled assuring that there was always a layer of water on the top. The column was washed with water. The packed column measured 12 X 1,5 cm. The flow rate of water was adjusted to 100 mljhour with suction. Loading of column. Tuber extract, containing 62 and 108 mg total carbohydrates, was diluted 5-fold with water and added drop wise from a separatory funnel to the top of the column, which contained about 1,0 ml water above the adsorbent. Adsorption was allowed to take place under suction over a period of 1/2 hour. The effluent measuring 50 ml was separately collected. Elution from column. Displacement of monosaccharides was effected with distilled water (50-200 ml). Increasing concentrations of ethanol (5, 10 and 15 v jv) were used for separating higher saccharides, employing 150-750 ml of each composition. Appearance of carbohydrate in a fraction and completion of elution were followed by taking aliquots from each 50 ml eluate fraction and testing by the phenol sulphuric acid method (MONTGOMERY, 1957) for total carbohydrate. The 5, 10 and 15 Ofo enthanolic eluates were subjected to vaccum distillation at 40-45°, reducing the volume to 10-20 ml. Aliquots of the solutions were analysed for carbohydrate components by colorimetry and by paper chromatography.
Results and Discussion Relation between period of heating and efficiency of extraction
The results obtained on heating the homogenates of boiled tissue for vanous periods are given in table 1.
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Table 1 Effect of different periods of heating. Time in minutes in boiling water after homogenization
Total carbohydrates, mg/g fresh tissue
o
48 48 52 56 65 65 63
10 20 30 40 50 60
The results show that the extraction is not complete by merely boiling the slices in water for 3 minutes. It is clear from the table that the optimum time for complete dissolution of fructosan is 40 minutes.
Analysis by chemical methods The analytical values obtained for 10 g sample of tuber, corresponding to 1 g dry weight in five different experiments are reported in table 2, last 2 samples were employed for quantitative paper chromatography. Table 2 Gross chemical fractionation of water extracts of fresh tubers. Carbohydrate fractions
Content, mg/10 g fresh weight
II
III
IV
V
Total carbohydrates, without hydrolysis
650
630
600
540
620
Combined and free fructose
550
500
490
540
480
Reducing sugar
nil
nil
nil
nil
nil
The most noteworthy feature of the data was the absence of reducing sugar in any of the samples. Fructose present in extracts is most likely in the polymerized form, otherwise free fructose would have reacted with Nelson reagent and gave some value for reducing sugar. It may be pointed out here that in separate experiments where in we were using dried tissue for fructosan determination (McRARY and SLATTERY, 1945), we were encountered with reducing sugars, this might be due to the drying of the tissue or some secondary changes during drying procedures. Z. Pjlanzenphysiol. Bd. 68. 5. 272-280 1972.
276
V. K.
MADAN
Analysis by chromatography Qualitative. The solutions obtained in the above experiments were subjected to qualitative paper chromatography. The entire material migrated from the baseline indicating that high molecular polyfructosans of the inulin complexity were not present. Sucrose was present in high concentration. There were 3 unknown spots which migrated to less extent than sucrose and which were presumed to be low molecular polyfructosans. Glucose and fructose gave also faint spots. In view of the fact that chemical methods could not detect reducing sugar, the possibility that glucose and fructose arose by hydrolysis during chromatogram development had to be considered. Quantitative. The results of two samples are recorded in Table 3. Table 3 Chromatographic analysis of water extracts of medium sized tubers*). Estimations
Amount Applied Recovered
Sucrose
,ug Experiment No.1 400
278
270
100
1080
1048
Free fructose + combined fructose
200
200
220
80
1080
700
Reducing sugars
Nil
Nil
Nil
Nil
200
180
160
60
30
620
630
111
150
130
40
15
480
401
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Total carbohydrates
Experiment No.2 Total carbohydrates
Free fructose + combined fructose Reducing sugars
*) Sucrose was the fastest moving component, followed in the order of decreasing mobility by 51' 52, 53 and 5 •.
Sucrose constituted the largest single component. There were 3 slower moving components in the first extract, while the second extract yielded an additional spot, moving the slowest of all, and containing the least amount of carbohydrate. Fructose analysis in all the unidentified spots indicated that it constituted the major port of each spot. S2 corresponded to raffinose in mobility. The analytical data obtained by chromatography demonstrated conclusively that the major components of tuber were sucrose and polyfructosans of low molecular weight. Sucrose, in all probability, was the starting material for the synthesis of polyfructosan, proceeding in stages of increasing molecular complexity.
z. PJlanzenphysiol. Ed. 68. S. 272-280. 1972.
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277
It is desired to stress the fact that standard inulin, used in reference chromatograms, always remained on the base line even when run for 100 hours. This excluded artifact formation during chromatography. Celite charcoal adsorption chromatography Chemical analysis
The results of chemical analysis in the various fractions are given in table 4. Table 4 Chemical analysis of celite charcoal eluates. Ethanolic content of eluant, %
Experiment I Nil 5 10 15 20
Volume of eluate, ml
50 300 650 500 150
Total carbohydrate fAg
Recovery in fraction, % of total recovery
750 5,380 36,550 43,480 3,800
0.84 5.98 40.60 47.10 4.30
89,960 Amount applied Recovery
108,000 83.4%
Experiment II
Nil 5 10 15 20
75 225 675 750 150
600 1,350 15,050 41,360
Nil
1.02 2.30 25.70 70.80 0.00
58,360 Amount applied Recovery
62,000 95.6%
Estimations were directly in the eluates from the column. Most of the carbohydrates were eluted with 10 to 15 % ethanol. The recovery from various eluates corresponded to 83.4 and 95.6 % of the carbohydrate applied on column. Paper chromatographic analysis
Since there was only a small amount of carbohydrate in the eluate with water and in the 20 Ofo ethanolic eluates obtained in the experiments, these were not tested. The values obtained with the other fractions are reported in table 5. Z. Pjlanzenphysiol. Bd. 68. S. 272-280. 1972.
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V. K.
MADAN
Table 5 Quantitative chromatography of celite charcoal eluates Values expressed as [lg
Estimations
Ethanol 10 % Ethanol 15 % Ethanol 5 Ofo -Sp-o-t-l--S-p-o-t-2-S-p-o-t-l--=-Sp-o-t-2--=S-p-o-t-3
Recovery from paper, %
(66)
(36)
(25)
(24)
(17)
(13)
Sample no. 1 Total carbohydrates Fructose Reducing sugars
58 20 Nil
100 80 Nil
50 30 Nil
20 10 Nil
90 60 Nil
100 60 Nil
89
Sample no. 2 Total carbohydrates Fructose Reducing sugars
55 30 20
60 28 Nil
80 40 Nil
42 40 Nil
65 40 Nil
120 100 Nil
93
") The Rg X 100 values are reported within brackets.
Chromatograms of 5 % ethanol eluate revealed the presence only of sucrose, confirmed by chemical and chromatographic analysis. 10 0J0 ethanol eluates gave two spots; one corresponded to raffinose in mobility. That the compared was not raffinose was evident from the fact that fructose content was 50 0J0. The second spot had higher mobility than standard raffinose, but differed in the two experiments. The difference was evident also in the % content of fructose; in the first it was 80 0J0, but in the second it was 47 0J0. The relative distribution of total carbohydrate between the two spots was markedly different in the two experiments. A feature of 10 0J0 ethanolic extracts was the absence of reducing sugar and the satisfactory recovery from chromatograms. 15 % ethanol eluates gave three spots, the fastest moving spots corresponded to slowest moving spot of 10 0J0 eluate, that is raffinose (standard). Two other spots remained in between raffinose and base line. All the spots obtained from 10 and 15 0J0 fractions represented fructosans, because the major carbohydrate components of each spot was fructose as evident from chemical analysis. Raffinose was absent from all the samples, although a fructosan of the same mobility was present in 10 0J0 and 15 0J0 ethanol eluates. Concluding from the above observations, it is clear that four kinds of fructosans are present in any sample of Asparagus tubers. Difference in values obtained from 10 and 15 0J0 ethanol eluates in two experiments may be due to seasonal variation. These two experiments were conducted at a interval of 3 months. Paper partition chromatography directly on tuber extracts resolved fructosans into 3 to 4 kinds (in molecular complexity). When carbohydrates were fractionated on celite charcoal columns prior to paper partition chromatography, fructosans resolved themselves into 4 kinds (in molecular complexity). The additional fructosan described
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279
in the later case may be attributed to the greater resolution on paper when carbohydrates were first fractionated on celite charcoal. In separate experiments with leaves and shoots of Asparagus we could not detect any polyfructosans both by chemical and chromatographic analysis. It is possible leaves did not elaborate fructosan but supplied the raw material sucrose to the roots for transformation to fructosan. Absence of polyfructosans in shoots may be interpreted as indicating that polyfructosans of the tuber are t:tilized by the rapidly growing portions of the plant after being first converted into sucro~e and hexose. Since the shoot was directly derived from the tuber itself or in the tissues of the shoot, by hydrolytic or transfer mechanism. From the results it is evident that the best method for fructosan determination would be to start with fresh tissue, celite charcoal adsorption chromatography followed by quantitative paper chromatography. Using dried samples might lead to some secondry changes. Acknowledgements My thanks are due to Professor P. S. KRISHNAN for guidance. This work was partially financed by P. L. 480 grant No. FG-in-163 (AR-CR-59) from Department of Agriculture.
u.s.
References ALM, R. S.: Gradient elution analysis. II. Oligosaccharides. Acta Chern. Scand. 6, 1186 (1952). ALM, R. S., R. J. P. WILLIAMS and A. TISELIUS: Gradient elution analysis. 1. General treatment. Acta Chern. Scand., 6, 826 (1952). BACON, J. S. D., and D. J. BELL: A new trisaccharide produced from sucrose by mould invertase .. J. Chern. Soc., 2528 (1953). BACON, J. S. D., and R. LOXLEY: Seasonal changes in the carbohydrates of Jerusalem artichoke. Biochem. J., 51, 209 (1952). BAILEY, J. M., W. J. WHELAN and S. PEAT: Carbohydrate primers in the synthesis of starch. J. Chern. Soc., 3692 (1950). BUCHAN, H. L., and R. J. SAVAGE: Chromatography of some starch conversion products. Analyst, 77, 401 (1952). DAVY, J.: Separation of oligosaccharides by column chromatography. Ann. Pharm. Fr., 24, 703 (1966). EDELMAN, J., and T. G. JEFFORD: The mechanism of fructosan metabolism in higher plants as exemplified in Helianthus tuberosus. New Phytol., 67, 517 (1968). McRARY, W. L., and M. C. SLATTERY: The colorimetric determination of fructosans in plant material. J. BioI. Chern., 157, 161 (1945). MONTGOMERY, R.: Determination of glycogen. Arch. Biochem. Biophy., 67, 378 (1957). PARTRIDGE, S. M.: Filterpaper partition chromatography of sugars. Biochem. J., 42, 238 (1948). ROE, J. H., and N. M. PAPADOPOULOS: The determination of fructose-6-phosphate and fructose-1-6-diphosphate. J. BioI. Chern., 210, 703 (1954). SCHLUBACH, H. H., and H. BOE: Untersuchungen iiber Polyfructosans. XIX Liebigs Ann., 532, 191 (1957).
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SOMOGYI, M. A.: A new reagent for the determination of sugars. ]. BioI. Chern., 160, 61 (1945). TANRET, G.: The two new carbohydrates from asparagus. Bull. Soc. Chim., 5, 889 (1909). WHELAN, W. ]., ]. M. BAILEY and P. ]. P. ROBERTS: The mechanism of carbohydrase action. The preparation and properties of maltodextrin substrates. ]. Chern. Soc., 1293 (1953). WHISTLER, R. L., and D. F. DURSO: Chromatographic separation of sugars on charcoal. ]. Am. Chern. Soc., 72, 677 (1950). VIJAY K. MADAN, Biochemistry Department, Lucknow University, Lucknow, U.P. India. Present address: Institut fur Mikrobiologie der Universitat Giittingen, D-34 Gottingen, Grisebachstr. 8, West Germany.
Z. PJlanzenphysiol. Bd. 68. S. 272-280. 1972.