PROSTAGLANDINSLEUKOTRIENES ANDESSENTIALFATTYACIDS Prcxtaglandma Leukofriene\ and E%wnial Fatty Acids (1992) 47. 85.Yl C’ Longman GroupUK Ltd 1942
Review
Some Secondary Plant Metabolites in Desmodium adscendens and Their Effects on Arachidonic Acid Metabolism M. E. Addy Department
of Biochemistry, University oj’Ghana, PO Bos 54, Legon. Ghana (Reprint requests to MEA)
The effects of three chemically different groups of compounds, (triterpenoid saponins, hetaphenylethylamines and tetrahydroisoquinolines), known to be present in Desmodium adscendens, on plasma membrane ion channel, cytochrome P450 NADPH-dependent oxygenation of arachidonic acid, and production of prostaglandins by the cyclooxygenase enzyme system, are described. The very high-conductance calciumactivated potassium ion channel, which is responsible for the maintenance of tone in smooth muscles, was activated by the saponins. The cytochrome P450 NADPH-dependent monooxygenase reaction, which produces epoxy- and hydroxylated eicosanoids from arachidonic acid metabolism, was inhibited by an analogue of the tetrahydroisoquinoline present in the plant. This analogue also acted as a reductant in the prostaglandin synthesizing system using microsomes from ram seminal vesicles. The same system was activated by the betaphenylethylamines found in the plant material, with the formation of more prostaglandins, the type being dependent on the amount of cyclooxygenase enzyme used and the presence or absense of coenzyme.
ABSTRACT.
INTRODUCTION
able cell such as smooth muscle, one of these ion channels is the very high conductance calcium-activated potassium channel which has a single channel conductance, a high potassium ion selectivity, and can be activated by cytoplasmic Ca++ concentration as well as membrane depolarization. All smooth muscle are known to have these calcium-activated potassium (Ca++-K+) channels in their membranes. The activation or opening of these channels causes hyperpolarization of the membrane due to K+ efflux (5). Influx of extracellular Ca++ is prevented and contraction is abolished. The channels are therefore responsible for maintaining and restoring the basic or resting tone in these muscles. Charybdotoxin, a peptidyl toxin and a minor component of the crude venom of the scorpion Leiurus quinquestriatus var. hebraeus has been shown to block the activity of this Ca++-K+ channel by binding to the external pore of the channel protein (6). The binding, which is reversible, closes the channel, prevents K+ conduction which is responsible for the maintenance of membrane polarity. and causes depolarization leading to muscle contraction. The channel stays open for much longer periods of time (that is, charybdotoxin binds less frequently to the external pore) when an extract of D. adscendens or its saponin, is present inside the cell (G. J. Kaczorowski,
Desmodium adscendens, var. adscendens, (Papilionaceae), which is used in Ghana for the management of asthma, has been shown to inhibit smooth muscle contractions due to antigen and other more specific contractile stimuli, and to relax precontracted tissues (l-3). Four chemically distinct groups of secondary plant metabolites, triterpenoid saponins, tetrahydroisoquinolines, beta-phenylethylamines and indole-3-alkylamines, have been isolated from the plant (3, 4). This paper describes how three of these compounds affect different aspects of arachidonic acid (AA) metabolism and generation of different second messenger systems, and discusses how such effects could explain the plant’s anti-asthmatic property.
CALCIUM ACTIVATED POTASSIUM CHANNEL Plasma membranes of cells have ion channels which operate to maintain a potential gradient across the membrane. The membrane is therefore polarized. In an excitDate received 14 February 1992 Date accepted 3 1 March 1992 85
86
Prostaglandins
Leukotrienes
and Essential Fatty Acids
rham hose
HO OH R’z
Soyasaponin
I
R:
‘cy4H
9
Soyasaponin
III
R :
4.
89
90
Prostaglandins
Leukotrienes
and Essential Fatty Acids
Table A comparison of PG synthesis in response to n-butanol extract of D. udscendens (nBF) and compounds or analogues of compounds known to be present in the plant Addition
AA
PGF
PGE
References
PGH
A: No GSH None 0.1 nBF 1.O nBF Tyramine Hordenine Salsolinol
27.9 + 17.0f 26.1 !c 11.7+ 25.3 f 15.8 +
5.4 6.8 7.1 8.7 12.1 8.1
9.4 !c 2.3 6.0 a! 0.9 8.4 i 1.7 12.8 5 2.7 8.0 + 0.9 13.3 + 3.3 13.9k3.1 12.05 1.6 6.6 f 3.4 10.9 ?I 1.7 3.4 * 0.8 22.1 f 2.2
3.1 4.2 4.4 9.4 10.2 20.1
f + f + * f
0.6 0.3 0.9 3.0 2.5 2.9
29.3 + 20.0 f 30.6 i. 9.5 + 14.5 + 5.4?
7.9 6.9 7.9 6.0 10.3 3.5
4.1 f0.3 3.8 ?I 0.0 4.6 + 0.8 4.2k1.9 13.9 f 4.7 32.8 k 0.6
3.4 f 0.3 2.3 k 0.3 2.4 f 0.3 10.8i:4.0 10.8 f 4.0 21.7 + 3.7
B: 0.5 mM GSH None 0.1 nBF 1.O nBF Tyramine Hordenine Salsolinol
19.8f30 27.3 + 4.7 17.1 + 4.7 41.8f5.1 15.9 ?I 6.4 5.1 + 1.8
Figures are means f SEM of percentage I-‘“C in AA and its metabolites. N = 4. *Addition = material added during pre-incubation.
phenylethylamines and the tetrahydroisoquinolines present in D. adscendens could modulate the cyclooxygenase reaction by acting as reductant coenzymes in the redox enzyme systems in this pathway of AA metabolism, producing PGE? or PGFZu, different second messengers, depending on the microenvironment of the cell. Leukotriene D4 (LTDJ-induced contraction in lung tissue is through AA metabolism via cyclooxygenase activity and thromboxane (TX) synthesis (21). One subfraction of D. adscendens inhibited such contractions dose-dependently (2), indicating modulation of cyclooxygenase activity. In vascular tissue, PGH,, the common TX which causes contraction, or to prostacyclin, which causes relaxation. PGH, itself contracts smooth muscle. The plant could prevent the contraction of lung tissue by directing AA metabolism towards formation of relaxant prostanoids.
CONCLUSIONS Three structurally different types of compounds present in one plant, D. adscendens have been shown to affect different processes, i.e. opening and closing of an ion channel and enzymatic pathways for the production of second messengers from AA. These reactions all have the effect of preventing the contraction of, or relaxing, pre-contracted smooth muscle. Some of the second messengers that are affected also mediate responses other than smooth muscle contraction/relaxation. Calcium movement across plasma membranes is responsible for secretion occuring in response to stimuli, and eicosanoids participate in the mucus seretion, cell infiltration and other inflammatory processes associated with asthma. The effects of the compounds in D. adscendens on AA metabolism which are described here could contribute to its reported anti-asthmatic property.
1. Addy, M E and Dzandu, W K. Dose-response effects of Desmodiltm adscetldens aqueous extract on histamine response. content and an aphylactic reactions in guinea-pig. Journal of Ethnopharmacology 18: 13-20. 1986. 2. Addy. M E and Burka. J F. Effect of Desrnodirrm adsc,enderu fraction Fl, (DAFl), on tone and agonistinduced contraction of guinea-pig airway smooth muscle. Phytotherapy Research 3: 85-90. 1989. .3 Addy, M E. Several Chromatographically distinctcomponents from Desmodiunr adscendens inhibit smooth muscle contractions. International Journal of Crude Drug Research 27: 8 l-9 1. 1989. 4. Asante-Poku, S, Sakakibara. J. and Addy, M E. Alkaloids of Desmodium adscendens. Ghana Medical Journal 22: 111-115, 1988. Ca++5. Singer. J J and Walsh, J V Jr. Large-conductance activated K’ channels in freshly dissociated smooth muscle cells. Membrane Biochemistry 6: 83-l 10. 1986. 6. Vazquez, J. Feigenbaum, P, Katz, G, King, V F, Reuben, J P, Roy-Contancin. L, Slaughter. R S. Kaczorowski, G J, and Garcia. M L. Characterization of high affinity binding sites for charybdotoxin in sarcolemmal membranes from bovine aortic smooth muscle. Journal of Biological Chemistry 264: 20902-20909, 1989. 7. Walsh J V Jr. and Singer J J. Calcium action potentials in single freshly isolated smooth muscle cells. American Journal of Physiology 239: C 162-C174, 1980. 8. Addy, M E and Burka J F. Effect of Desmodium adscendens fraction 3 on contractions of respiratory smooth muscle. Journal of Ethnophannacology 29: 225-325, 1990. 9. Addy, M E and Burka. J B. Effect of Desmodium adscendens fractions on antigen- and arachidonic acidinduced contractions of guinea-pig airways. Canadian Journal of Physiology and Pharmacology 66: 820-825, 1988. 10. Addy. M E and Burka, J F. Dose-response effect of one subfraction of Desmodium adscendens aqueous extract on antigen- and arachidonic acid-induced contractions of guinea-pig airways. Phytotherapy Research 1: 180-186. 1987. 11. White. R E and Coon, M J. Oxygen activation by cytochrome P450. Annual Review of Biochemistry 49: 315-356, 1980. 12. Capdevila, J Mamett, L J, Chacos, N. Prough R A and Estabrook R W. Cytochrome P-450.dependent oxygenation of arachidonic acid to hydroxyeicosatetraenoicacids. Proceedings of the National Academy of Sciences USA 79: 767-770, 1982. 13. Schwartzman. M, Ferreri, N R, Carroll, MA, SonguMize. E and McGiff J C. Renal cytochrome P450-related arachidonate metabolite inhibits (Na++K) ATPase. Nature 314: 620-622. 1985. 14. Postnov, Y U, Reznikova M. and Boriskina G. Na-Kadenosine triphosphatase in the kidney of rats with renal hypertension and spontaneously hypertensive rats. Pflugers Archiv, European Journal of Physiology 362: 95-99, 1976. 15. Addy, M E and Schwartzman, ML. Extracts of Desmodium adscertdens inhibit NADPH-dependent oxygenation of arachidonic acid by kidney cortical microsomes. Phytotherapy Research. Accepted for publication. 1991. 16. Stecher, P G (Ed), Merck Index. (8th Edition). Merck & Co Inc. Rahway N J. p 91. 1968. 17. King, V F, Garcia, M L, Himmel, D, Reuben, J P, Lam Y T. Pan, J. Han, G and Kaczorowski, G J. Interaction of tetrandrine with slowly inactivating calcium channels. Journal of Biological Chemistry 263: 2238-2244. 1988. 18. Lands. W, Lee. R and Smith, W. Factors regulating the biosynthesis of various prostaglandins. Annals New York Academy of Sciences. 180: 107-122, 1971. 19. Addy, M E and Schwartzman, M L. An extract of
Some Secondary
Plant Metabolites
Desmodium adscenderu activates cyclooxyenase and increases prostaglandin synthesis by ram seminal vesicle microsomes. Submitted to Prostaglandins, 1992. 20. Castellani, S, Scarti. L, Masotti G, Del Rosso, A. Chen. J L and Semeri. G G N. Selective stimulation of renal PGE, by potassium canrenoate in essential hypertension.
in Desmodium adscendensand Their Effects on Arachidonic
Acid Metabolism
p 297 in Prostaglandins in Clinical Research: Cardiovascular System Alan R. Liss Inc.. 1989. 2 1. Piper. P J and Samhoun. M N. Stimulation of arachidonic acid metabolism and generation of thromboxane A, by leukotrienes B. C, and D4 in guinea-pig lung i?z vitro. British Journal of Pharmacology 77: 267-275. 19%.
9
Review
Some Secondary Plant Metabolites in Desmodium adscendens and Their Effects on Arachidonic Acid Metabolism M. E. Addy Department
of Biochemistry, University oj’Ghana, PO Bos 54, Legon. Ghana (Reprint requests to MEA)
The effects of three chemically different groups of compounds, (triterpenoid saponins, hetaphenylethylamines and tetrahydroisoquinolines), known to be present in Desmodium adscendens, on plasma membrane ion channel, cytochrome P450 NADPH-dependent oxygenation of arachidonic acid, and production of prostaglandins by the cyclooxygenase enzyme system, are described. The very high-conductance calciumactivated potassium ion channel, which is responsible for the maintenance of tone in smooth muscles, was activated by the saponins. The cytochrome P450 NADPH-dependent monooxygenase reaction, which produces epoxy- and hydroxylated eicosanoids from arachidonic acid metabolism, was inhibited by an analogue of the tetrahydroisoquinoline present in the plant. This analogue also acted as a reductant in the prostaglandin synthesizing system using microsomes from ram seminal vesicles. The same system was activated by the betaphenylethylamines found in the plant material, with the formation of more prostaglandins, the type being dependent on the amount of cyclooxygenase enzyme used and the presence or absense of coenzyme.
ABSTRACT.
INTRODUCTION
able cell such as smooth muscle, one of these ion channels is the very high conductance calcium-activated potassium channel which has a single channel conductance, a high potassium ion selectivity, and can be activated by cytoplasmic Ca++ concentration as well as membrane depolarization. All smooth muscle are known to have these calcium-activated potassium (Ca++-K+) channels in their membranes. The activation or opening of these channels causes hyperpolarization of the membrane due to K+ efflux (5). Influx of extracellular Ca++ is prevented and contraction is abolished. The channels are therefore responsible for maintaining and restoring the basic or resting tone in these muscles. Charybdotoxin, a peptidyl toxin and a minor component of the crude venom of the scorpion Leiurus quinquestriatus var. hebraeus has been shown to block the activity of this Ca++-K+ channel by binding to the external pore of the channel protein (6). The binding, which is reversible, closes the channel, prevents K+ conduction which is responsible for the maintenance of membrane polarity. and causes depolarization leading to muscle contraction. The channel stays open for much longer periods of time (that is, charybdotoxin binds less frequently to the external pore) when an extract of D. adscendens or its saponin, is present inside the cell (G. J. Kaczorowski,
Desmodium adscendens, var. adscendens, (Papilionaceae), which is used in Ghana for the management of asthma, has been shown to inhibit smooth muscle contractions due to antigen and other more specific contractile stimuli, and to relax precontracted tissues (l-3). Four chemically distinct groups of secondary plant metabolites, triterpenoid saponins, tetrahydroisoquinolines, beta-phenylethylamines and indole-3-alkylamines, have been isolated from the plant (3, 4). This paper describes how three of these compounds affect different aspects of arachidonic acid (AA) metabolism and generation of different second messenger systems, and discusses how such effects could explain the plant’s anti-asthmatic property.
CALCIUM ACTIVATED POTASSIUM CHANNEL Plasma membranes of cells have ion channels which operate to maintain a potential gradient across the membrane. The membrane is therefore polarized. In an excitDate received 14 February 1992 Date accepted 3 1 March 1992 85
86
Prostaglandins
Leukotrienes
and Essential Fatty Acids
rham hose
HO OH R’z
Soyasaponin
I
R:
‘cy4H
9
Soyasaponin
III
R :
4.
89
90
Prostaglandins
Leukotrienes
and Essential Fatty Acids
Table A comparison of PG synthesis in response to n-butanol extract of D. udscendens (nBF) and compounds or analogues of compounds known to be present in the plant Addition
AA
PGF
PGE
References
PGH
A: No GSH None 0.1 nBF 1.O nBF Tyramine Hordenine Salsolinol
27.9 + 17.0f 26.1 !c 11.7+ 25.3 f 15.8 +
5.4 6.8 7.1 8.7 12.1 8.1
9.4 !c 2.3 6.0 a! 0.9 8.4 i 1.7 12.8 5 2.7 8.0 + 0.9 13.3 + 3.3 13.9k3.1 12.05 1.6 6.6 f 3.4 10.9 ?I 1.7 3.4 * 0.8 22.1 f 2.2
3.1 4.2 4.4 9.4 10.2 20.1
f + f + * f
0.6 0.3 0.9 3.0 2.5 2.9
29.3 + 20.0 f 30.6 i. 9.5 + 14.5 + 5.4?
7.9 6.9 7.9 6.0 10.3 3.5
4.1 f0.3 3.8 ?I 0.0 4.6 + 0.8 4.2k1.9 13.9 f 4.7 32.8 k 0.6
3.4 f 0.3 2.3 k 0.3 2.4 f 0.3 10.8i:4.0 10.8 f 4.0 21.7 + 3.7
B: 0.5 mM GSH None 0.1 nBF 1.O nBF Tyramine Hordenine Salsolinol
19.8f30 27.3 + 4.7 17.1 + 4.7 41.8f5.1 15.9 ?I 6.4 5.1 + 1.8
Figures are means f SEM of percentage I-‘“C in AA and its metabolites. N = 4. *Addition = material added during pre-incubation.
phenylethylamines and the tetrahydroisoquinolines present in D. adscendens could modulate the cyclooxygenase reaction by acting as reductant coenzymes in the redox enzyme systems in this pathway of AA metabolism, producing PGE? or PGFZu, different second messengers, depending on the microenvironment of the cell. Leukotriene D4 (LTDJ-induced contraction in lung tissue is through AA metabolism via cyclooxygenase activity and thromboxane (TX) synthesis (21). One subfraction of D. adscendens inhibited such contractions dose-dependently (2), indicating modulation of cyclooxygenase activity. In vascular tissue, PGH,, the common TX which causes contraction, or to prostacyclin, which causes relaxation. PGH, itself contracts smooth muscle. The plant could prevent the contraction of lung tissue by directing AA metabolism towards formation of relaxant prostanoids.
CONCLUSIONS Three structurally different types of compounds present in one plant, D. adscendens have been shown to affect different processes, i.e. opening and closing of an ion channel and enzymatic pathways for the production of second messengers from AA. These reactions all have the effect of preventing the contraction of, or relaxing, pre-contracted smooth muscle. Some of the second messengers that are affected also mediate responses other than smooth muscle contraction/relaxation. Calcium movement across plasma membranes is responsible for secretion occuring in response to stimuli, and eicosanoids participate in the mucus seretion, cell infiltration and other inflammatory processes associated with asthma. The effects of the compounds in D. adscendens on AA metabolism which are described here could contribute to its reported anti-asthmatic property.
1. Addy, M E and Dzandu, W K. Dose-response effects of Desmodiltm adscetldens aqueous extract on histamine response. content and an aphylactic reactions in guinea-pig. Journal of Ethnopharmacology 18: 13-20. 1986. 2. Addy. M E and Burka. J F. Effect of Desrnodirrm adsc,enderu fraction Fl, (DAFl), on tone and agonistinduced contraction of guinea-pig airway smooth muscle. Phytotherapy Research 3: 85-90. 1989. .3 Addy, M E. Several Chromatographically distinctcomponents from Desmodiunr adscendens inhibit smooth muscle contractions. International Journal of Crude Drug Research 27: 8 l-9 1. 1989. 4. Asante-Poku, S, Sakakibara. J. and Addy, M E. Alkaloids of Desmodium adscendens. Ghana Medical Journal 22: 111-115, 1988. Ca++5. Singer. J J and Walsh, J V Jr. Large-conductance activated K’ channels in freshly dissociated smooth muscle cells. Membrane Biochemistry 6: 83-l 10. 1986. 6. Vazquez, J. Feigenbaum, P, Katz, G, King, V F, Reuben, J P, Roy-Contancin. L, Slaughter. R S. Kaczorowski, G J, and Garcia. M L. Characterization of high affinity binding sites for charybdotoxin in sarcolemmal membranes from bovine aortic smooth muscle. Journal of Biological Chemistry 264: 20902-20909, 1989. 7. Walsh J V Jr. and Singer J J. Calcium action potentials in single freshly isolated smooth muscle cells. American Journal of Physiology 239: C 162-C174, 1980. 8. Addy, M E and Burka J F. Effect of Desmodium adscendens fraction 3 on contractions of respiratory smooth muscle. Journal of Ethnophannacology 29: 225-325, 1990. 9. Addy, M E and Burka. J B. Effect of Desmodium adscendens fractions on antigen- and arachidonic acidinduced contractions of guinea-pig airways. Canadian Journal of Physiology and Pharmacology 66: 820-825, 1988. 10. Addy. M E and Burka, J F. Dose-response effect of one subfraction of Desmodium adscendens aqueous extract on antigen- and arachidonic acid-induced contractions of guinea-pig airways. Phytotherapy Research 1: 180-186. 1987. 11. White. R E and Coon, M J. Oxygen activation by cytochrome P450. Annual Review of Biochemistry 49: 315-356, 1980. 12. Capdevila, J Mamett, L J, Chacos, N. Prough R A and Estabrook R W. Cytochrome P-450.dependent oxygenation of arachidonic acid to hydroxyeicosatetraenoicacids. Proceedings of the National Academy of Sciences USA 79: 767-770, 1982. 13. Schwartzman. M, Ferreri, N R, Carroll, MA, SonguMize. E and McGiff J C. Renal cytochrome P450-related arachidonate metabolite inhibits (Na++K) ATPase. Nature 314: 620-622. 1985. 14. Postnov, Y U, Reznikova M. and Boriskina G. Na-Kadenosine triphosphatase in the kidney of rats with renal hypertension and spontaneously hypertensive rats. Pflugers Archiv, European Journal of Physiology 362: 95-99, 1976. 15. Addy, M E and Schwartzman, ML. Extracts of Desmodium adscertdens inhibit NADPH-dependent oxygenation of arachidonic acid by kidney cortical microsomes. Phytotherapy Research. Accepted for publication. 1991. 16. Stecher, P G (Ed), Merck Index. (8th Edition). Merck & Co Inc. Rahway N J. p 91. 1968. 17. King, V F, Garcia, M L, Himmel, D, Reuben, J P, Lam Y T. Pan, J. Han, G and Kaczorowski, G J. Interaction of tetrandrine with slowly inactivating calcium channels. Journal of Biological Chemistry 263: 2238-2244. 1988. 18. Lands. W, Lee. R and Smith, W. Factors regulating the biosynthesis of various prostaglandins. Annals New York Academy of Sciences. 180: 107-122, 1971. 19. Addy, M E and Schwartzman, M L. An extract of
Some Secondary
Plant Metabolites
Desmodium adscenderu activates cyclooxyenase and increases prostaglandin synthesis by ram seminal vesicle microsomes. Submitted to Prostaglandins, 1992. 20. Castellani, S, Scarti. L, Masotti G, Del Rosso, A. Chen. J L and Semeri. G G N. Selective stimulation of renal PGE, by potassium canrenoate in essential hypertension.
in Desmodium adscendensand Their Effects on Arachidonic
Acid Metabolism
p 297 in Prostaglandins in Clinical Research: Cardiovascular System Alan R. Liss Inc.. 1989. 2 1. Piper. P J and Samhoun. M N. Stimulation of arachidonic acid metabolism and generation of thromboxane A, by leukotrienes B. C, and D4 in guinea-pig lung i?z vitro. British Journal of Pharmacology 77: 267-275. 19%.
9
