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Quantitative Determination of Sphagnum Acid from Sphagnum magellanicumBrid
Botanisches Institut der Universidit Kiel, Federal Republic of Germany
Quantitative Determination of Sphagnum Acid from Sphagnum magellanicum BRID REINHARD TUTsCHEK With 2 figures Received November 14,1978' Accepted March 10, 1979
Summary A procedure for the efficient extraction and quantitative determination of sphagnum acid from Sphagnum magellanicum has been developed. Because of its good reproducibility it may be adopted as a standard method for all investigations requiring the estimation of the sphagnum acid content. In the present paper it has been applied to measurements of the sphagnum acid level in Sphagnum magellanicum collected over a period of one year in the natural habitat. These investigations reveal sphagnum acid to be subject of metabolization in certain phases of the vegetation cycle.
Key words: Sphagnum magellanicum; sphagnum acid; method for the quantitative determination; metabolization of sphagnum acid.
Introduction
The cell walls of peat mosses contain phenolic substances which can histochemically be detected by a strong red staining of the walls by MILLON'S reagent. Previous work from our laboratory has shown that a considerable part of the MILLON-positive substances of Sphagnum magellanicum is extractable by ethanol under mild conditions (ENGMANN, 1972). The main compound of the ethanolic extract could be isolated in a crystalline form and identified as p-hydroxy-fJ-(carboxymethyl)-cinnamic acid (TuTscHEK et aI., 1973). Referring to its origin it was called sphagnum acid. From the mode of the isolation procedure and the chemical properties of the crystalline natural substance it was concluded that sphagnum acid represents a native constituent of the cell wall (TuTscHEK, 1975). Both the biosynthesis and the metabolic role of this compound are unknown. Moreover, it would be interesting to see whether the natural substance can generally be found in Sphagna or is merely restricted to Sphagnum magellanicum. With regard to these problems the need for a quantitative determination is apparent. In the present paper I wish to report on a method for the efficient extraction and subsequent determination of sphagnum acid. Apart from the Z. Pf/anzenphysiol. Bd. 94. S. 317-324. 1979.
318
REINHARD TUTSCHEK
methodical aspects investigations were performed to answer the question whether sphagnum acid has the character of a metabolically inactive end product or may be subjected to further metabolism.
Material and Methods 1. Plant material The apical parts of Sphagnum magellanicum gametophytes (capitula) were collected in the Kaltenhof bog nearby Kiel, frozen and lyophilized. The dry material was homogenized in a coffee-mill for Ih min and in a micro-dismembrator (Braun, Melsungen) by means of glass-beads (0 5-6 mm: 25, 0 3-4 mm: 10; 3 min; amplitude: 10 mm). As shown microscopically all the cells were totally disrupted by this treatment. 2. Standard extraction procedure and chromatographic purification of sphagnum acid 350 mg of moss powder dried over P 2 0 S were extracted with 20 ml of chloroform on a round filter to remove most of the chlorophyll. After the powder had been liberated from adherent chloroform in an air stream it was transferred to an extraction thimble including the filter and extracted with 96 % ethanol (100 ml) in a solvent hot extractor for 4 hours. The extract was concentrated under reduced pressure to 2.5 ml. 1.5 ml of this solution was applied to cellulose UV254 -layers (1 mm) by means of a chromatocharger and chromatographed in butanol-llacetic acid/H20 = 3 : 2: 95. The band corresponding to sphagnum acid was located by a quenchihg of the fluorescence of the layer. This zone was scraped off, suspended in 18 ml methanol/37 Ofo Hel = 99 : 1 and eluted for 45 min under continuous shaking. The suspension was filtered through a membrane filter (pore ¢ 0.45 f/,m) and the solution filled up to 25 ml. A blank was prepared by chromatography of a pure cellulose layer and elution of an equal amount of adsorbent.
3. Alternative extraction procedures The pre-extracted moss powder was a) directly refluxed in 100 ml of 96 Ofo ethanol for 4 hours; b) transferred to a glass column (22 X 800 mm) and continuously extracted with 16.51 of 96 % ethanol at ambient temperature. The extract was collected in 2-2.5 I-fractions and each fraction chromatographically analyzed for the presence of sphagnum acid. The extraction was stopped as soon as only traces were found by the most sensitive detection method, namely the spray reaction with PAULY'S reagent. In both cases the extracts were subjected to further analysis as described for the standard procedure. 4. Photometric determination of sphagnum acid The absorbance of the eluate was measured at 296 nm in a Zeiss-PMQ II-spectrophotometer. For the calculation of the sphagnum acid content the specific extinction coefficient (75.5 liter g-I cm- I) was used.
5. Investigation of the thermic stability of sphagnum acid The UV-spectrum of a sphagnum acid solution in 96 Ofo ethanol (0.49 mg/50 ml) was measured in a Zeiss-DMR 21-spectrophotometer and compared with the spectra obtained after refluxing the solution for 2 and 4 hours. In a separate experiment the solution was chromatographed after corresponding time intervals using the same system as for the purifica tion of the extracts.
z. Pflanzenphysiol. Bd. 94. S. 317-324. 1979.
Quantitative determination of sphagnum acid
319
Results and Discussion 1. Methodical investigations 1.1. Extraction procedure
In connection with the preparative isolation of sphagnum acid (cf. TUTSCHEK, 1975) it was found that an exhaustive extraction at ambient temperature requires
weeks or months depending on the amount of dry weight of the plant material. This extraction procedure is obviously ineffective for metabolic or phytochemical studies. In 1975 SCHWARZMAIER and BREHM reported the continuous hot extraction to be an effective method for the removal of aromatic carboxylic acids from pulverized Sphagnum material. I have made use of this procedure except for the solvent and extraction time. In order to compare it with the cold extraction employed earlier 96 % ethanol was chosen as solvent instead of acetone and water. Concerning the extraction time SCHWARZMAIER and BREHM were interested in a highly purified extraction residue whereas in the present work emphasis is placed on the stability of the extracted components. Therefore, it was necessary to modify the extraction time. Fig. 1 shows the amount of sphagnum acid extractable after various times. If the values are considered separately the highest content is found after 6 hours. It was shown by the t-test, however, that the difference between the values of 4 and 6 hours is statistically not significant. Thus, 4 hours are sufficient for the exhaustive extraction of sphagnum acid. This conclusion is confirmed by the fact that the substance was absent in a 4 hours-extract obtained by re-extraction of the residue. During extraction the alcoholic solution is boiling. From the investigations of ENGMANN (1972) it may be inferred that an ethanolic solution of sphagnum acid remains stable for 4 hours at the temperature of boiling ethanol. Nevertheless, the thermic stability under the conditions of extraction had to be examined. This was done by recording the UV -spectrum of pure sphagnum acid after refluxing an ethanolic solution for 2 and 4 hours. Neither Amax nor the extinction were affected by the high
:E
01
"iii 3:
c:-
"~
5; o ~ 300 :0 "0 c
6,250
20.
01
::I..
200
y,rr'I----.---T'----'.---2
6
8
hours
Fig. 1: Influence of various times of hot extraction on the amount of sphagnum acid extractable from Sphagnum magellanicum. Z. Pjlanzenphysiol. Bd. 94. S. 317-324. 1979.
320
REINHARD TUTSCHEK
temperature. In accordance with these measurements no degradation products were found. The efficiency of the hot extraction method may be judged from a comparison with alternative procedures. Equal amounts of the same plant material were subjected to a cold extraction and to a direct extraction with boiling ethanol. The values of tab. 1 clearly demonstrate the superiority of the hot extraction procedure. For this reason it is justified to adopt it as a standard method. Table 1: Comparison of the efficiency of three methods for the extraction of sphagnum acid from Sphagnum magellanicum (solvent: 96 % ethanol). Extraction procedure
Sphagnum acid content of the moss material (,ug/350 mg dry weight)
Hot extraction!) Cold extraction 2 ) Extraction in the boiling solvent')
259 182 164
') 4 hours 2) approximately 4 weeks with 16.5 I of solvent
1.2. Chromatographic purification of the crude extract Experiments on the development of a colorimetric estimation method were not successful. Neither the formation of the red azo dye-stuff produced by the coupling of sphagnum acid with PAULY'S reagent nor the reaction with FOLIN-CIOCALTEUreagent (BRAY and THORPE, 1954; RAGAZZI and VERONESE, 1973) could be used as a basis for a colorimetric determination. Whereas in the first case the azo dye-stuff revealed to be unstable the FOLIN-CIOCALTEu-method did not yield reproducible results. Therefore, an estimation method was worked out which is based on the chromatographic purification of the crude extract and subsequent UV -photometric determination of sphagnum acid. Chlorophyll which is abundantly present in every crude extract was removed in two steps. The main portion was extracted by chloroform prior to the extraction with ethanol. As shown chromatographically the chloroform extract was free of sphagnum acid. Any residues of chlorophyll were removed by chromatography of the ethanolic extract on cellulose UVm-layers and butanol-l/ acetic acid/H2 0 = 3 : 2 : 95 as solvent. In this polar system the chlorophyll was completely retained at the start. Sphagnum acid was easily detectable by an intensive quenching of the fluorescence of the adsorbent. No other components of the extract show the same characteristic in the Rf-zone of sphagnum acid. Further evidence for a complete separation of sphagnum acid was obtained by irradiance of the plates with UV360 and spraying with PAULY'S reagent. The most convincing criterion, however, was the absolute
z. Pjlanzenphysiol. Bd. 94. S. 317-324. 1979.
Quantitative determination of sphagnum acid
321
identity of the UV -spectra of extractable sphagnum acid and the crystalline natural substance. The latter finding indicates that a single chromatography is sufficient to obtain a pure compound. The purification method reported above is also applicable to crude extracts from reddened plant material because the chromatograms show good accordance with those from the green moss (as to colouring of Sphagnum magellanicum see RUDOLPH, 1964). 1.3. Elution of sphagnum acid from the adsorbent Although methanol is the best solvent for the crystalline natural substance it could not effect the elution from the adsorbent. Apparently sphagnum acid strongly adheres to the cellulose. This would explain the very slow extraction from the cell wall at ambient temperature. A similar behaviour is shown by sphagnorubine, the main wall pigment of Sphagnum magellanicum (RUDOLPH, 1965; VOWINKEL, 1975). Presumably the high polarity of both compounds is responsible for the formation of adsorptive bondings. In the case of sphagnum acid these adsorptive forces could be overcome by adding 37 Ofo HCI to the methanol in a concentration of 10f0. This method reported for the elution of cinnamic acids from silica gel (HESS, 1964) was successfully adopted for the elution of sphagnum acid from cellulose. In experiments with the authentic substance 90 Ofo of the amount used for chromatography was recovered. Prior to the photometric measurement of the eluates it was found that the absorbance of variously concentrated sphagnum acid solutions in methanol/37 Ofo HCI (99 : 1) follows Lambert-Beer's law in an extinction range of 0-0.7. 1.4. Reproducibility of the determination method As a measure for the reproducibility of the sphagnum acid content of a given plant material the standard error was determined from estimates made on five samples. For 90 Ofo confidence limits a standard error of ± 5.4 Ofo was calculated indicating a satisfactory accordance of the single values. For routine analysis (see chapter 2) only double estimates were made which did not differ by more than 10 Ofo. 1.5. Co-extraction of moss material and authentic sphagnum acid The reliability of the results of the quantitative determination strongly depends on the stability of sphagnum acid in the presence of the other constituents of the ethanolic extract. One can imagine that chemical reactions take place between sphagnum acid and other ethanol-soluble substances during the extraction procedure. Thereby, the native sphagnum acid content could be diminished. In order to examine this possibility the level of sphagnum acid of a certain moss material was compared with a measurement based on co-extraction of the same material with a known amount of sphagnum acid (tab. 2). The experiment shows that sphagnum acid added
z. Pjlanzenphysiol. Bd. 94. S. 317-324. 1979.
322
REINHARD TUTSCHEK
Table 2: Rate of recovery after co-extraction of authentic sphagnum acid with a moss material of known sphagnum acid content. Experiment
Sphagnum acid concentration of the crude extract (,ug/2.5 ml)
Extraction of moss material (350 mg dry weight)
2521)
Extraction of moss material (350 mg dry weight) + 0.5 mg sphagnum acid
766
Rate of recovery (Ofo)
102
1) average value of a double analysis
to the moss powder was totally recovered after co-extraction. We may therefore expect that native sphagnum acid also remains unaffected by the components of the extract. 2. Evidence for the metabolization of sphagnum acid Though sphagnum acid abundantly accumulates in the cell walls nothing was as yet known about the dynamics of the sphagnum acid content in Sphagnum magellanicum. In the past, various plant phenolic substances have been shown to be subject to turnover and catabolism (for a comprehensive review see BARZ and H6sEL, 1975). Changes in the level of the phenolic compound under investigation as induced by endogeneous and exogeneous factors are indicative of turnover or degradative processes (for examples see BARZ, 1975). I have chosen the measurement of seasonal variations as an experimental approach to study the dynamic role of sphagnum acid. Fig. 2 shows the sphagnum acid content of material from the natural habitat collected over a period of one year in the same area. At the beginning of the vegetation period, in spring, the substance rapidly accumulates reaching a high level which on the onset of winter drastically declines. These fluctuations may be interpreted as being the result of variable intensities of synthesis and degradation. The immense increase in spring is consistent with the stimulation of life processes in Sphagna at this time as indicated by the innovation of growth (OVERBECK and HAPPACH, 1956/57) and chlorophyll synthesis (RUDOLPH et al., 1977). On the other hand the decrease of the level unequivocally represents a degradative process reflecting the general retardation of growth and metabolism at the end of the vegetation cycle. It is remarkable that within one month about 100 % of initially present sphagnum acid is metabolized. We neither know the factors which induce this abrupt fall nor the nature of the catabolic reactions. In this connection it should be emphasized that the degradation of sphagnum acid does not coincide with the reddening of the moss. The change of colour in the natural habitat is already finished when the sphagnum acid level breaks down. From this observation we may conclude that at least in the bog the degradation of sphagnum acid is not involved in the syn-
z. PJlanzenphysiol. Bd. 94. S. 317-324. 1979.
Quantitative determination of sphagnum acid
323
50
T
i
I i i
July
Aug
Sept
Oct
Nov
[
Dec
I
Jan
t
I
Feb March April
I
May June
r
July
i
Aug
month
Fig. 2: Seasonal fluctuations of the sphagnum acid content in Sphagnum magellanicum from the natural habitat.
thesis of the wall pigments. On the contrary, metabolic interrelations between both wall constituents cannot be excluded. For instance, the continuous rise of the sphagnum acid content from August to November could be associated with the colouring process which takes place in the same interval. Reversely, the degradation of wall pigments observed under certain conditions (RUDOLPH et aI., 1977) may lead back to sphagnum acid. Though we don't know the interconversions of the wall components in detail it is an interesting fact that the cell wall of Sphagnum magellanicum is a further example of a cell compartment which contains metabolically active secondary products. Acknowledgements I am grateful to Mrs. ERIKA GROSS for excellent technical assistance.
References BARZ, W.: Abbau von Flavonoiden und Isoflavonoiden - ein Dberblick. Ber. Dtsch. Bot. Ges. 88, 71-81 (1975). BARZ, W. and W. HaSEL: Metabolism of flavonoids. In: HARBORNE, J. B., T. J. MABRY, and H. MABRY (Eds.): The Flavonoids, 916-969. Chapman and Hall, London, 1975. BRAY, H. G. and W. V. THORPE: Analysis of phenolic compounds of interest in metabolism. Meth. Biochem. Anal. 1,27-52 (1954). ENGMANN, B.: Untersuchungen der mit MILLON'S Reagens anHirbbaren Substanzen der Sphagnenzellwand. Biochem. Physiol. PH. 163, 200-215 (1972). HESS, D.: Methionin als Methylgruppendonator fiir Zimtsauren und Anthocyane. Z. Naturforsch. 19 b, 148-150 (1964). Z. Pflanzenphysiol. Bd. 94. S. 317-324. 1979.
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OVERBECK, F. und H. HAPPACH: Dber das Wachs tum und den Wasserhaushalt einiger Hochmoorsphagnen. Flora 144, 335-402 (1956/57). RAGAZZI, E. and G. VERONESE: Quantitative analysis of phenolic compounds after thinlayer chromatographic separation. J. Chromatogr. 77, 369-375 (1973). RUDOLPH, H.: Zur Frage der Membranochromie bei Sphagnen. I. Welche Faktoren bestimmen den Farbwechsel? Flora 155,250-293 (1964). - Zur Frage der Membranochromie bei Sphagnen. III. Der Versuch einer Charakterisierung chromatographisch rein dargestellter Kardinalpigmente. Planta 64, 178-185 (1965). RUDOLPH, H., U. KABSCH, and G. SCHMIDT-STOHN: Knderungen des ChloroplastenpigmentSpiegels bei Sphagnum magellanicum im Verlauf der Synthese von Sphagnorubin und anderer membranochromer Pigmente. Z. Pflanzenphysiol. 82, 107-116 (1977). SCHWARZMAIER, U. and K. BREHM: Detailed characterization of the cation-exchanger in Sphagnum magellanicum BRID. Z. Pflanzenphysiol. 75, 250-255 (1975). TUTSCHEK, R.: Isolierung und Charakterisierung der p-Hydroxy-p-(carboxymethyl)-zimtsaure (Sphagnumsaure) aus der Zellwand von Sphagnum magellanicum BRID. Z. Pflanzenphysiol. 76, 353-365 (1975). TUTSCHEK, R., H. RUDOLPH, P. H. WAGNER und R. KREHER: Struktur eines kristallinen Phenols aus der Zellwand von Sphagnum magellanicum. Biochem. Physiol. Pfl. 164, 461-464 (1973). VOWINKEL, E.: Die Struktur des Sphagnorubins (Torfmoosmembranochrome, 2.). Chern. Ber. 108, 1166-1181 (1975). Dr. REINHARD TUTSCHEK, Botanisches Institut der Universitat Kiel, Biologiezentrum, Olshausenstr. 40-60, 2300 Kiel.
z. P/lanzenphysiol. Bd. 94. S. 317-324. 1979.
Quantitative Determination of Sphagnum Acid from Sphagnum magellanicum BRID REINHARD TUTsCHEK With 2 figures Received November 14,1978' Accepted March 10, 1979
Summary A procedure for the efficient extraction and quantitative determination of sphagnum acid from Sphagnum magellanicum has been developed. Because of its good reproducibility it may be adopted as a standard method for all investigations requiring the estimation of the sphagnum acid content. In the present paper it has been applied to measurements of the sphagnum acid level in Sphagnum magellanicum collected over a period of one year in the natural habitat. These investigations reveal sphagnum acid to be subject of metabolization in certain phases of the vegetation cycle.
Key words: Sphagnum magellanicum; sphagnum acid; method for the quantitative determination; metabolization of sphagnum acid.
Introduction
The cell walls of peat mosses contain phenolic substances which can histochemically be detected by a strong red staining of the walls by MILLON'S reagent. Previous work from our laboratory has shown that a considerable part of the MILLON-positive substances of Sphagnum magellanicum is extractable by ethanol under mild conditions (ENGMANN, 1972). The main compound of the ethanolic extract could be isolated in a crystalline form and identified as p-hydroxy-fJ-(carboxymethyl)-cinnamic acid (TuTscHEK et aI., 1973). Referring to its origin it was called sphagnum acid. From the mode of the isolation procedure and the chemical properties of the crystalline natural substance it was concluded that sphagnum acid represents a native constituent of the cell wall (TuTscHEK, 1975). Both the biosynthesis and the metabolic role of this compound are unknown. Moreover, it would be interesting to see whether the natural substance can generally be found in Sphagna or is merely restricted to Sphagnum magellanicum. With regard to these problems the need for a quantitative determination is apparent. In the present paper I wish to report on a method for the efficient extraction and subsequent determination of sphagnum acid. Apart from the Z. Pf/anzenphysiol. Bd. 94. S. 317-324. 1979.
318
REINHARD TUTSCHEK
methodical aspects investigations were performed to answer the question whether sphagnum acid has the character of a metabolically inactive end product or may be subjected to further metabolism.
Material and Methods 1. Plant material The apical parts of Sphagnum magellanicum gametophytes (capitula) were collected in the Kaltenhof bog nearby Kiel, frozen and lyophilized. The dry material was homogenized in a coffee-mill for Ih min and in a micro-dismembrator (Braun, Melsungen) by means of glass-beads (0 5-6 mm: 25, 0 3-4 mm: 10; 3 min; amplitude: 10 mm). As shown microscopically all the cells were totally disrupted by this treatment. 2. Standard extraction procedure and chromatographic purification of sphagnum acid 350 mg of moss powder dried over P 2 0 S were extracted with 20 ml of chloroform on a round filter to remove most of the chlorophyll. After the powder had been liberated from adherent chloroform in an air stream it was transferred to an extraction thimble including the filter and extracted with 96 % ethanol (100 ml) in a solvent hot extractor for 4 hours. The extract was concentrated under reduced pressure to 2.5 ml. 1.5 ml of this solution was applied to cellulose UV254 -layers (1 mm) by means of a chromatocharger and chromatographed in butanol-llacetic acid/H20 = 3 : 2: 95. The band corresponding to sphagnum acid was located by a quenchihg of the fluorescence of the layer. This zone was scraped off, suspended in 18 ml methanol/37 Ofo Hel = 99 : 1 and eluted for 45 min under continuous shaking. The suspension was filtered through a membrane filter (pore ¢ 0.45 f/,m) and the solution filled up to 25 ml. A blank was prepared by chromatography of a pure cellulose layer and elution of an equal amount of adsorbent.
3. Alternative extraction procedures The pre-extracted moss powder was a) directly refluxed in 100 ml of 96 Ofo ethanol for 4 hours; b) transferred to a glass column (22 X 800 mm) and continuously extracted with 16.51 of 96 % ethanol at ambient temperature. The extract was collected in 2-2.5 I-fractions and each fraction chromatographically analyzed for the presence of sphagnum acid. The extraction was stopped as soon as only traces were found by the most sensitive detection method, namely the spray reaction with PAULY'S reagent. In both cases the extracts were subjected to further analysis as described for the standard procedure. 4. Photometric determination of sphagnum acid The absorbance of the eluate was measured at 296 nm in a Zeiss-PMQ II-spectrophotometer. For the calculation of the sphagnum acid content the specific extinction coefficient (75.5 liter g-I cm- I) was used.
5. Investigation of the thermic stability of sphagnum acid The UV-spectrum of a sphagnum acid solution in 96 Ofo ethanol (0.49 mg/50 ml) was measured in a Zeiss-DMR 21-spectrophotometer and compared with the spectra obtained after refluxing the solution for 2 and 4 hours. In a separate experiment the solution was chromatographed after corresponding time intervals using the same system as for the purifica tion of the extracts.
z. Pflanzenphysiol. Bd. 94. S. 317-324. 1979.
Quantitative determination of sphagnum acid
319
Results and Discussion 1. Methodical investigations 1.1. Extraction procedure
In connection with the preparative isolation of sphagnum acid (cf. TUTSCHEK, 1975) it was found that an exhaustive extraction at ambient temperature requires
weeks or months depending on the amount of dry weight of the plant material. This extraction procedure is obviously ineffective for metabolic or phytochemical studies. In 1975 SCHWARZMAIER and BREHM reported the continuous hot extraction to be an effective method for the removal of aromatic carboxylic acids from pulverized Sphagnum material. I have made use of this procedure except for the solvent and extraction time. In order to compare it with the cold extraction employed earlier 96 % ethanol was chosen as solvent instead of acetone and water. Concerning the extraction time SCHWARZMAIER and BREHM were interested in a highly purified extraction residue whereas in the present work emphasis is placed on the stability of the extracted components. Therefore, it was necessary to modify the extraction time. Fig. 1 shows the amount of sphagnum acid extractable after various times. If the values are considered separately the highest content is found after 6 hours. It was shown by the t-test, however, that the difference between the values of 4 and 6 hours is statistically not significant. Thus, 4 hours are sufficient for the exhaustive extraction of sphagnum acid. This conclusion is confirmed by the fact that the substance was absent in a 4 hours-extract obtained by re-extraction of the residue. During extraction the alcoholic solution is boiling. From the investigations of ENGMANN (1972) it may be inferred that an ethanolic solution of sphagnum acid remains stable for 4 hours at the temperature of boiling ethanol. Nevertheless, the thermic stability under the conditions of extraction had to be examined. This was done by recording the UV -spectrum of pure sphagnum acid after refluxing an ethanolic solution for 2 and 4 hours. Neither Amax nor the extinction were affected by the high
:E
01
"iii 3:
c:-
"~
5; o ~ 300 :0 "0 c
6,250
20.
01
::I..
200
y,rr'I----.---T'----'.---2
6
8
hours
Fig. 1: Influence of various times of hot extraction on the amount of sphagnum acid extractable from Sphagnum magellanicum. Z. Pjlanzenphysiol. Bd. 94. S. 317-324. 1979.
320
REINHARD TUTSCHEK
temperature. In accordance with these measurements no degradation products were found. The efficiency of the hot extraction method may be judged from a comparison with alternative procedures. Equal amounts of the same plant material were subjected to a cold extraction and to a direct extraction with boiling ethanol. The values of tab. 1 clearly demonstrate the superiority of the hot extraction procedure. For this reason it is justified to adopt it as a standard method. Table 1: Comparison of the efficiency of three methods for the extraction of sphagnum acid from Sphagnum magellanicum (solvent: 96 % ethanol). Extraction procedure
Sphagnum acid content of the moss material (,ug/350 mg dry weight)
Hot extraction!) Cold extraction 2 ) Extraction in the boiling solvent')
259 182 164
') 4 hours 2) approximately 4 weeks with 16.5 I of solvent
1.2. Chromatographic purification of the crude extract Experiments on the development of a colorimetric estimation method were not successful. Neither the formation of the red azo dye-stuff produced by the coupling of sphagnum acid with PAULY'S reagent nor the reaction with FOLIN-CIOCALTEUreagent (BRAY and THORPE, 1954; RAGAZZI and VERONESE, 1973) could be used as a basis for a colorimetric determination. Whereas in the first case the azo dye-stuff revealed to be unstable the FOLIN-CIOCALTEu-method did not yield reproducible results. Therefore, an estimation method was worked out which is based on the chromatographic purification of the crude extract and subsequent UV -photometric determination of sphagnum acid. Chlorophyll which is abundantly present in every crude extract was removed in two steps. The main portion was extracted by chloroform prior to the extraction with ethanol. As shown chromatographically the chloroform extract was free of sphagnum acid. Any residues of chlorophyll were removed by chromatography of the ethanolic extract on cellulose UVm-layers and butanol-l/ acetic acid/H2 0 = 3 : 2 : 95 as solvent. In this polar system the chlorophyll was completely retained at the start. Sphagnum acid was easily detectable by an intensive quenching of the fluorescence of the adsorbent. No other components of the extract show the same characteristic in the Rf-zone of sphagnum acid. Further evidence for a complete separation of sphagnum acid was obtained by irradiance of the plates with UV360 and spraying with PAULY'S reagent. The most convincing criterion, however, was the absolute
z. Pjlanzenphysiol. Bd. 94. S. 317-324. 1979.
Quantitative determination of sphagnum acid
321
identity of the UV -spectra of extractable sphagnum acid and the crystalline natural substance. The latter finding indicates that a single chromatography is sufficient to obtain a pure compound. The purification method reported above is also applicable to crude extracts from reddened plant material because the chromatograms show good accordance with those from the green moss (as to colouring of Sphagnum magellanicum see RUDOLPH, 1964). 1.3. Elution of sphagnum acid from the adsorbent Although methanol is the best solvent for the crystalline natural substance it could not effect the elution from the adsorbent. Apparently sphagnum acid strongly adheres to the cellulose. This would explain the very slow extraction from the cell wall at ambient temperature. A similar behaviour is shown by sphagnorubine, the main wall pigment of Sphagnum magellanicum (RUDOLPH, 1965; VOWINKEL, 1975). Presumably the high polarity of both compounds is responsible for the formation of adsorptive bondings. In the case of sphagnum acid these adsorptive forces could be overcome by adding 37 Ofo HCI to the methanol in a concentration of 10f0. This method reported for the elution of cinnamic acids from silica gel (HESS, 1964) was successfully adopted for the elution of sphagnum acid from cellulose. In experiments with the authentic substance 90 Ofo of the amount used for chromatography was recovered. Prior to the photometric measurement of the eluates it was found that the absorbance of variously concentrated sphagnum acid solutions in methanol/37 Ofo HCI (99 : 1) follows Lambert-Beer's law in an extinction range of 0-0.7. 1.4. Reproducibility of the determination method As a measure for the reproducibility of the sphagnum acid content of a given plant material the standard error was determined from estimates made on five samples. For 90 Ofo confidence limits a standard error of ± 5.4 Ofo was calculated indicating a satisfactory accordance of the single values. For routine analysis (see chapter 2) only double estimates were made which did not differ by more than 10 Ofo. 1.5. Co-extraction of moss material and authentic sphagnum acid The reliability of the results of the quantitative determination strongly depends on the stability of sphagnum acid in the presence of the other constituents of the ethanolic extract. One can imagine that chemical reactions take place between sphagnum acid and other ethanol-soluble substances during the extraction procedure. Thereby, the native sphagnum acid content could be diminished. In order to examine this possibility the level of sphagnum acid of a certain moss material was compared with a measurement based on co-extraction of the same material with a known amount of sphagnum acid (tab. 2). The experiment shows that sphagnum acid added
z. Pjlanzenphysiol. Bd. 94. S. 317-324. 1979.
322
REINHARD TUTSCHEK
Table 2: Rate of recovery after co-extraction of authentic sphagnum acid with a moss material of known sphagnum acid content. Experiment
Sphagnum acid concentration of the crude extract (,ug/2.5 ml)
Extraction of moss material (350 mg dry weight)
2521)
Extraction of moss material (350 mg dry weight) + 0.5 mg sphagnum acid
766
Rate of recovery (Ofo)
102
1) average value of a double analysis
to the moss powder was totally recovered after co-extraction. We may therefore expect that native sphagnum acid also remains unaffected by the components of the extract. 2. Evidence for the metabolization of sphagnum acid Though sphagnum acid abundantly accumulates in the cell walls nothing was as yet known about the dynamics of the sphagnum acid content in Sphagnum magellanicum. In the past, various plant phenolic substances have been shown to be subject to turnover and catabolism (for a comprehensive review see BARZ and H6sEL, 1975). Changes in the level of the phenolic compound under investigation as induced by endogeneous and exogeneous factors are indicative of turnover or degradative processes (for examples see BARZ, 1975). I have chosen the measurement of seasonal variations as an experimental approach to study the dynamic role of sphagnum acid. Fig. 2 shows the sphagnum acid content of material from the natural habitat collected over a period of one year in the same area. At the beginning of the vegetation period, in spring, the substance rapidly accumulates reaching a high level which on the onset of winter drastically declines. These fluctuations may be interpreted as being the result of variable intensities of synthesis and degradation. The immense increase in spring is consistent with the stimulation of life processes in Sphagna at this time as indicated by the innovation of growth (OVERBECK and HAPPACH, 1956/57) and chlorophyll synthesis (RUDOLPH et al., 1977). On the other hand the decrease of the level unequivocally represents a degradative process reflecting the general retardation of growth and metabolism at the end of the vegetation cycle. It is remarkable that within one month about 100 % of initially present sphagnum acid is metabolized. We neither know the factors which induce this abrupt fall nor the nature of the catabolic reactions. In this connection it should be emphasized that the degradation of sphagnum acid does not coincide with the reddening of the moss. The change of colour in the natural habitat is already finished when the sphagnum acid level breaks down. From this observation we may conclude that at least in the bog the degradation of sphagnum acid is not involved in the syn-
z. PJlanzenphysiol. Bd. 94. S. 317-324. 1979.
Quantitative determination of sphagnum acid
323
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T
i
I i i
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Fig. 2: Seasonal fluctuations of the sphagnum acid content in Sphagnum magellanicum from the natural habitat.
thesis of the wall pigments. On the contrary, metabolic interrelations between both wall constituents cannot be excluded. For instance, the continuous rise of the sphagnum acid content from August to November could be associated with the colouring process which takes place in the same interval. Reversely, the degradation of wall pigments observed under certain conditions (RUDOLPH et aI., 1977) may lead back to sphagnum acid. Though we don't know the interconversions of the wall components in detail it is an interesting fact that the cell wall of Sphagnum magellanicum is a further example of a cell compartment which contains metabolically active secondary products. Acknowledgements I am grateful to Mrs. ERIKA GROSS for excellent technical assistance.
References BARZ, W.: Abbau von Flavonoiden und Isoflavonoiden - ein Dberblick. Ber. Dtsch. Bot. Ges. 88, 71-81 (1975). BARZ, W. and W. HaSEL: Metabolism of flavonoids. In: HARBORNE, J. B., T. J. MABRY, and H. MABRY (Eds.): The Flavonoids, 916-969. Chapman and Hall, London, 1975. BRAY, H. G. and W. V. THORPE: Analysis of phenolic compounds of interest in metabolism. Meth. Biochem. Anal. 1,27-52 (1954). ENGMANN, B.: Untersuchungen der mit MILLON'S Reagens anHirbbaren Substanzen der Sphagnenzellwand. Biochem. Physiol. PH. 163, 200-215 (1972). HESS, D.: Methionin als Methylgruppendonator fiir Zimtsauren und Anthocyane. Z. Naturforsch. 19 b, 148-150 (1964). Z. Pflanzenphysiol. Bd. 94. S. 317-324. 1979.
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OVERBECK, F. und H. HAPPACH: Dber das Wachs tum und den Wasserhaushalt einiger Hochmoorsphagnen. Flora 144, 335-402 (1956/57). RAGAZZI, E. and G. VERONESE: Quantitative analysis of phenolic compounds after thinlayer chromatographic separation. J. Chromatogr. 77, 369-375 (1973). RUDOLPH, H.: Zur Frage der Membranochromie bei Sphagnen. I. Welche Faktoren bestimmen den Farbwechsel? Flora 155,250-293 (1964). - Zur Frage der Membranochromie bei Sphagnen. III. Der Versuch einer Charakterisierung chromatographisch rein dargestellter Kardinalpigmente. Planta 64, 178-185 (1965). RUDOLPH, H., U. KABSCH, and G. SCHMIDT-STOHN: Knderungen des ChloroplastenpigmentSpiegels bei Sphagnum magellanicum im Verlauf der Synthese von Sphagnorubin und anderer membranochromer Pigmente. Z. Pflanzenphysiol. 82, 107-116 (1977). SCHWARZMAIER, U. and K. BREHM: Detailed characterization of the cation-exchanger in Sphagnum magellanicum BRID. Z. Pflanzenphysiol. 75, 250-255 (1975). TUTSCHEK, R.: Isolierung und Charakterisierung der p-Hydroxy-p-(carboxymethyl)-zimtsaure (Sphagnumsaure) aus der Zellwand von Sphagnum magellanicum BRID. Z. Pflanzenphysiol. 76, 353-365 (1975). TUTSCHEK, R., H. RUDOLPH, P. H. WAGNER und R. KREHER: Struktur eines kristallinen Phenols aus der Zellwand von Sphagnum magellanicum. Biochem. Physiol. Pfl. 164, 461-464 (1973). VOWINKEL, E.: Die Struktur des Sphagnorubins (Torfmoosmembranochrome, 2.). Chern. Ber. 108, 1166-1181 (1975). Dr. REINHARD TUTSCHEK, Botanisches Institut der Universitat Kiel, Biologiezentrum, Olshausenstr. 40-60, 2300 Kiel.
z. P/lanzenphysiol. Bd. 94. S. 317-324. 1979.