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Arachidonic acid and prostaglandins in buds of Populus balsamifera
Phytochemistry, Vol. 29, No. 7, pp. 2325- 2326, 1990. Printedin Great Britain.
ARACHIDONIC
0
ACID AND PROSTAGLANDINS BALSAMIFERA E. D.
LEVIN, E. V. ISAEVA
Siberian Institute
of Technology,
and V. E.
IN BUDS
0031-9422/90 %3.00+0.00 1990 PergamonPress plc
OF POPULUS
CHEREPANOVA
82 Mira str., Krasnoyarsk
660607. U.S.S.R.
(Received 30 August 1989) Key Word Index-Populus balsamifera; Salicaceae; arachidonic acid; identification; prostaglandins; interrelation of the content; annual cycle.
Abstract-The presence of arachidonic acid in the buds of Populus balsamfera and the quantitative estimation of its content in the course of its annual cycle have been established. The correlation between the content of arachidonic acid and that of prostaglandins have been calculated.
INTRODUCTION
Arachidonic acid, the natural substrate for the formation of prostaglandins, is well known to be available in animal organisms. The content of arachidonic acid in mammals Cl], as well as in marine organisms [2, 31, has been reported. It has been considered that vegetable oils do not contain unsaturated acids with four, five or six double bonds [4]. Recently, however, arachidonic acid was found in some lower plants [S], wheat germ [6], as well as in the pollen of clover, buckwheat [7] and in potato tubers [S]. The presence of arachidonic acid in the cambium zone of Larix sibirica was reported by Rubchevskaya and Levin [9]. Levin and his co-workers pioneered the discovery of prostaglandins in tissues of higher plants [lo, 1 l] and indicated the possible similarity in the conversion of arachidonic acid into prostaglandins in animals and plants. The major aim of the present study was to obtain evidence for or against an interrelationship between the contents of arachidonic acid and prostaglandins in the course of the annual plant growth cycle. RESULTS
The iodine number averaged 333.7 which is in agreement with the calculated one. According to the Coulomb titration data and the value of the iodine number, there are four double bonds per molecule in the extracted arachidonic acid. The positions of these double bonds were located by periodate-permanganate oxidation and GC analysis of the fragments to be at the 5,8, 11 and 14positions. The ‘H NMR spectrum of arachidonic acid extracted from the buds of Populus balsamifera was similar to that of the standard. The estimation of arachidonic acid content in the sample was carried out by the method of internal standard as well as by the method of internal normalization. The main substrate for biosynthesis of prostaglandins in animal bodies is known to be arachidonic acid. Taking this into account, it would be interesting to establish the interrelationship between the contents of arachidonic acid and prostaglandins in plant tissues. The content of arachidonic acid and prostaglandins in the buds of Populus balsamifera in the course of the annual growth cycle are listed in Table 1.
AND DISCUSSlON
There are reports on the identification of prostaglandins extracted from the buds of Populus balsamifera and on the determination of their amount [12]. Identification of arachidonic acid in higher plants has not yet been described with sufficient proof. Therefore, we have carried out the identification of arachidonic acid by some physico-chemical methods. The TLC R, obtained for the arachidonic acid isolated from buds was identical with the R, value of the standard (R, 0.38, system I and R, 0.47, system II). GLC of the extracted arachidonic acid was carried out on two stationary phases. There was no retention time difference between the samples of standard and the extracted arachidonic acid, RR, 12.53 min (phase I) and RR, 24.86 min (phase II). The refractive index of the extracted arachidonic acid was established to be ni” 1.4838 (literature--n~” 1.4824). 2325
Table
1. Content of arachidonic acid and prostaglandins the buds of Populus balsnm~fera Content
Months* Sep. Oct. Nov. Dec. Jan. Feb. Mar. Apr.
Arachidonic
(pg g-’ acid
dry wt tissue) Prostaglandins
I .60 38.40 47.3 33.0 16.2 4.2 2.9 52.3
*The trees have no buds in the May-August
20.3 20.5 17.9 9.4 5.5 9.5 71.0 period.
in
2326
The changes
Short Reports
close
positive
linear
relationship
between
the
in the content of arachidonic acid and prostaglandins within the various periods of the annual growth cycle was proved by the calculation of correlation dependence of their content in the sample investigated. The correlation coefficient was found to be 0.95. The interrelationship between the content of arachidonic acid and prostaglandins seems to be regular. September is considered to be the beginning of the dormant state period for Populus balsamfira which restrains the growth of sprouts,
leaves
and
physiological
activity
in general.
There is a low content or lack of biologically active substances in the buds. Subsequent months which coincide with the period of restrained physiological activity are characterized by the increase in the content (i.e. accumulation) of various substances. The lowest content of arachidonic acid and prostaglandins was in February and March when all the physiological functions of Populus balsamifera are minimized. The minimum tioned above is needed
quantity
of the substances
8” min-‘. 2 m x 2.5 mm glass column packed with Chromaton N-AW-DMCS with 5% SE-30 (phase I); (ii) temp. programmed at 80-240” for 6” min.- ‘, 3.5 m x 3 mm glass column packed with Chromaton N-Super with 3% OV-225 (phase II); temp. 85”. 2 m x 2.5 mm glass column packed with Apeizon L. (phase III). The determination of iodine number was carried out by the Ganus method [143. ‘HNMR (2OOMHz CDCI,, TMS as int. standard). Arachidonic acid: 6 0.87 (3H, t, H-20); 1.28(6H. m. H-17. H-18 and H-19); 1.69 (2H, m, H-3); 2.07 (4H, m, H-4 and H-16); 2.31 (2H, t, H-2); 2.81 (6H, m, H-7, H-10 and H-13); 3.65 (3H. s, H-l); 5.35 (8H, m, H-5, H-6, H-8, H-9, H-11, H-12, H-14 and H-15). Double bond location in the aruchidonic ucid. The arachidonic acid was oxidized with periodate-permanganate and the acid fragments produced were methylated with CH,N,. CC analysis (phase III) identified the methyl and dimethyl derivatives of glutaric, capronic and malonic acids.
men-
for the vital functions of the tree in a dormant state. In April all the reproductive functions regenerate and there is a sharp increase in the content of biologically active substances because of the growth requirements. Neither arachidonic acid nor prostaglandins were detected in the leaves. Thus, we suggest that arachidonic acid is the substrate for the synthesis of prostaglandins in the plants as well as in animal organisms. EXPERIMENTAL
The buds of Populus halsamifera were selected from the trees of the same age growing in one location over a three year period with samples taken during the final week each month. Bud samples were averaged according to the tree height. The extraction of arachidonic acid was performed by essentially the same method employed for its extraction from animal tissue [13]. TLC was carried out with hexane-ester (7: 3) (system I) and on silica gel KSK-gypsum (20: 1) with petrol-Et&HOAc (90: 10: 1) (system II). Detection was achieved with 5% alcohol soln of phosphomolybdic acid and I, vapour. GLC w&s carried out with a FID detector and He as carrier gas. pres. 3 kgcm- *. (i) temp. programmed at 13&280” for
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. II. 12. 13. 14.
Hartly. P. (1909) J. Physiol. 38, 355. Pzhavskaya, F. M. (1976) Zhir. Ryb Morsk. Mlekopitay., 470. Light, J. and Samuelsson, B. (1972) Eur. J. Biochem. 28,232. Tyutyunnikov, V. N. (1966) Khim. Zhir. 632. Gellerman, J. Ci. and Anderson, W. H. (1975) J. Biochem. Biophys. Acta 388, 277. Janistyn, B. (1982) J. Plantu 1.55, 342. Bandyukova. V. A., Deineko, G. I. and Shapiro, D. K. (1983) Chem. Nat. Compd 101. Look, M. and Kuc, J. (1987) Metah.. Srruc. Func. 75. Rubchevskaya, L. P. and Levin, E. D. (1984) Wood Chem. 4. 109. Alautdinov. Sh. T. and Levin, E. D. (1979) Wood Chem. 3, 110. Levin, E. D., Alautdinov, Sh. T. and Cherepanova, V. E. (1984) Nat. Chem. Compd 5, 567. Levin, E. D., Cherepanova, V. E., Zimovtseva, I. A. and Sedlova, T. 0. (1988) Phytochemistry 27, 3241. Herb, S. F., Ricmenschneider, R. W. and Donaldson, J. (1951) J. Am. Oil Chem. Sot. 28. 55. Horwitz, W.. Editor, P. C. and Reynolds, H. (1970) Official Methods of Analysis of the Association of Official Analytical Chemists, p. 425.
ARACHIDONIC
0
ACID AND PROSTAGLANDINS BALSAMIFERA E. D.
LEVIN, E. V. ISAEVA
Siberian Institute
of Technology,
and V. E.
IN BUDS
0031-9422/90 %3.00+0.00 1990 PergamonPress plc
OF POPULUS
CHEREPANOVA
82 Mira str., Krasnoyarsk
660607. U.S.S.R.
(Received 30 August 1989) Key Word Index-Populus balsamifera; Salicaceae; arachidonic acid; identification; prostaglandins; interrelation of the content; annual cycle.
Abstract-The presence of arachidonic acid in the buds of Populus balsamfera and the quantitative estimation of its content in the course of its annual cycle have been established. The correlation between the content of arachidonic acid and that of prostaglandins have been calculated.
INTRODUCTION
Arachidonic acid, the natural substrate for the formation of prostaglandins, is well known to be available in animal organisms. The content of arachidonic acid in mammals Cl], as well as in marine organisms [2, 31, has been reported. It has been considered that vegetable oils do not contain unsaturated acids with four, five or six double bonds [4]. Recently, however, arachidonic acid was found in some lower plants [S], wheat germ [6], as well as in the pollen of clover, buckwheat [7] and in potato tubers [S]. The presence of arachidonic acid in the cambium zone of Larix sibirica was reported by Rubchevskaya and Levin [9]. Levin and his co-workers pioneered the discovery of prostaglandins in tissues of higher plants [lo, 1 l] and indicated the possible similarity in the conversion of arachidonic acid into prostaglandins in animals and plants. The major aim of the present study was to obtain evidence for or against an interrelationship between the contents of arachidonic acid and prostaglandins in the course of the annual plant growth cycle. RESULTS
The iodine number averaged 333.7 which is in agreement with the calculated one. According to the Coulomb titration data and the value of the iodine number, there are four double bonds per molecule in the extracted arachidonic acid. The positions of these double bonds were located by periodate-permanganate oxidation and GC analysis of the fragments to be at the 5,8, 11 and 14positions. The ‘H NMR spectrum of arachidonic acid extracted from the buds of Populus balsamifera was similar to that of the standard. The estimation of arachidonic acid content in the sample was carried out by the method of internal standard as well as by the method of internal normalization. The main substrate for biosynthesis of prostaglandins in animal bodies is known to be arachidonic acid. Taking this into account, it would be interesting to establish the interrelationship between the contents of arachidonic acid and prostaglandins in plant tissues. The content of arachidonic acid and prostaglandins in the buds of Populus balsamifera in the course of the annual growth cycle are listed in Table 1.
AND DISCUSSlON
There are reports on the identification of prostaglandins extracted from the buds of Populus balsamifera and on the determination of their amount [12]. Identification of arachidonic acid in higher plants has not yet been described with sufficient proof. Therefore, we have carried out the identification of arachidonic acid by some physico-chemical methods. The TLC R, obtained for the arachidonic acid isolated from buds was identical with the R, value of the standard (R, 0.38, system I and R, 0.47, system II). GLC of the extracted arachidonic acid was carried out on two stationary phases. There was no retention time difference between the samples of standard and the extracted arachidonic acid, RR, 12.53 min (phase I) and RR, 24.86 min (phase II). The refractive index of the extracted arachidonic acid was established to be ni” 1.4838 (literature--n~” 1.4824). 2325
Table
1. Content of arachidonic acid and prostaglandins the buds of Populus balsnm~fera Content
Months* Sep. Oct. Nov. Dec. Jan. Feb. Mar. Apr.
Arachidonic
(pg g-’ acid
dry wt tissue) Prostaglandins
I .60 38.40 47.3 33.0 16.2 4.2 2.9 52.3
*The trees have no buds in the May-August
20.3 20.5 17.9 9.4 5.5 9.5 71.0 period.
in
2326
The changes
Short Reports
close
positive
linear
relationship
between
the
in the content of arachidonic acid and prostaglandins within the various periods of the annual growth cycle was proved by the calculation of correlation dependence of their content in the sample investigated. The correlation coefficient was found to be 0.95. The interrelationship between the content of arachidonic acid and prostaglandins seems to be regular. September is considered to be the beginning of the dormant state period for Populus balsamfira which restrains the growth of sprouts,
leaves
and
physiological
activity
in general.
There is a low content or lack of biologically active substances in the buds. Subsequent months which coincide with the period of restrained physiological activity are characterized by the increase in the content (i.e. accumulation) of various substances. The lowest content of arachidonic acid and prostaglandins was in February and March when all the physiological functions of Populus balsamifera are minimized. The minimum tioned above is needed
quantity
of the substances
8” min-‘. 2 m x 2.5 mm glass column packed with Chromaton N-AW-DMCS with 5% SE-30 (phase I); (ii) temp. programmed at 80-240” for 6” min.- ‘, 3.5 m x 3 mm glass column packed with Chromaton N-Super with 3% OV-225 (phase II); temp. 85”. 2 m x 2.5 mm glass column packed with Apeizon L. (phase III). The determination of iodine number was carried out by the Ganus method [143. ‘HNMR (2OOMHz CDCI,, TMS as int. standard). Arachidonic acid: 6 0.87 (3H, t, H-20); 1.28(6H. m. H-17. H-18 and H-19); 1.69 (2H, m, H-3); 2.07 (4H, m, H-4 and H-16); 2.31 (2H, t, H-2); 2.81 (6H, m, H-7, H-10 and H-13); 3.65 (3H. s, H-l); 5.35 (8H, m, H-5, H-6, H-8, H-9, H-11, H-12, H-14 and H-15). Double bond location in the aruchidonic ucid. The arachidonic acid was oxidized with periodate-permanganate and the acid fragments produced were methylated with CH,N,. CC analysis (phase III) identified the methyl and dimethyl derivatives of glutaric, capronic and malonic acids.
men-
for the vital functions of the tree in a dormant state. In April all the reproductive functions regenerate and there is a sharp increase in the content of biologically active substances because of the growth requirements. Neither arachidonic acid nor prostaglandins were detected in the leaves. Thus, we suggest that arachidonic acid is the substrate for the synthesis of prostaglandins in the plants as well as in animal organisms. EXPERIMENTAL
The buds of Populus halsamifera were selected from the trees of the same age growing in one location over a three year period with samples taken during the final week each month. Bud samples were averaged according to the tree height. The extraction of arachidonic acid was performed by essentially the same method employed for its extraction from animal tissue [13]. TLC was carried out with hexane-ester (7: 3) (system I) and on silica gel KSK-gypsum (20: 1) with petrol-Et&HOAc (90: 10: 1) (system II). Detection was achieved with 5% alcohol soln of phosphomolybdic acid and I, vapour. GLC w&s carried out with a FID detector and He as carrier gas. pres. 3 kgcm- *. (i) temp. programmed at 13&280” for
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. II. 12. 13. 14.
Hartly. P. (1909) J. Physiol. 38, 355. Pzhavskaya, F. M. (1976) Zhir. Ryb Morsk. Mlekopitay., 470. Light, J. and Samuelsson, B. (1972) Eur. J. Biochem. 28,232. Tyutyunnikov, V. N. (1966) Khim. Zhir. 632. Gellerman, J. Ci. and Anderson, W. H. (1975) J. Biochem. Biophys. Acta 388, 277. Janistyn, B. (1982) J. Plantu 1.55, 342. Bandyukova. V. A., Deineko, G. I. and Shapiro, D. K. (1983) Chem. Nat. Compd 101. Look, M. and Kuc, J. (1987) Metah.. Srruc. Func. 75. Rubchevskaya, L. P. and Levin, E. D. (1984) Wood Chem. 4. 109. Alautdinov. Sh. T. and Levin, E. D. (1979) Wood Chem. 3, 110. Levin, E. D., Alautdinov, Sh. T. and Cherepanova, V. E. (1984) Nat. Chem. Compd 5, 567. Levin, E. D., Cherepanova, V. E., Zimovtseva, I. A. and Sedlova, T. 0. (1988) Phytochemistry 27, 3241. Herb, S. F., Ricmenschneider, R. W. and Donaldson, J. (1951) J. Am. Oil Chem. Sot. 28. 55. Horwitz, W.. Editor, P. C. and Reynolds, H. (1970) Official Methods of Analysis of the Association of Official Analytical Chemists, p. 425.