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Influence of epilobium extracts on prostaglandin biosynthesis and carrageenin induced oedema of the rat paw
Journal of Ethnopharmacology, Elsevier Scientific Publishers
17 (1986) Ireland Ltd.
161
161-169
INFLUENCE OF EPZLOBZUM EXTRACTS ON PROSTAGLANDIN BIOSYNTHESIS AND CARRAGEENIN INDUCED OEDEMA OF THE RAT PAW
A. HIERMANNa,
H. JUANb
and W. SAMETZb
aInstitut fiir Pharmakognosie, bInstitut fiir Experimentelle und Klinische Universitiit Graz, Universitiitsplatz 4, A-801 0 Graz (Austria) (Accepted
Pharmakologie,
May 20, 1986)
Summary
Epilobium species have been used as remedies in folk-medicine for the treatment of pathophysiological processes of the prostata. In this paper the influence of extracts of Herba Epilobii angustifolii L. and Herba Epilobii paruiflori Schreb. on prostaglandin biosynthesis and the carrageenin rat paw oedema is described. Aqueous extracts of Herba E. angustifolii reduced the release of prostaglandins I*, Ez and Dz (in the perfused rabbit ear) approximately 5 times more effectively than did similar extracts of Herba E. paruiflori. Methanolic extracts were inactive. The aqueous extract of E. angustifolium strongly reduced the carrageenin-induced rat paw oedema whereas that of E. paruiflorum was inactive. The chemical nature of the active compound(s) is as yet unknown but flavonoids and sitosterol derivatives can be excluded.
Introduction
Extracts of the species of the genus Epilobium L. (Fam. Onagraceae) have traditionally been used in folk-medicine for the treatment of prostata adenoma (benign prostata-hyperplasia) and inflammations of the prostata. However, few reports have concerned the pharmacology of these plants (Pukhalskaya et al., 1975; Syrkin et al., 1977; Chantrill et al., 1952). Previous work has revealed the existence of several flavonoids and sitosterol derivatives in Epilobium species (Hiermann, 1984; Hiermann and Mayr, 1985). Both classes of compounds are known to inhibit prostaglandin (PG) biosynthesis (Baumann et al., 1980; Walker, 1977) and the formation of rat paw oedema (Gabor, 1979; Gupta et al., 1980). We have therefore studied the effect of crude aqueous extracts of E. angustifolium L. and E. parviflorum Schreb. on PG biosynthesis and the carrageenin rat paw oedema, an experimental model of acute inflammation. 0378-C741/86/$03.50 Published and Printed
o 1986 in Ireland
Elsevier
Scientific
Publishers
Ireland
Ltd.
162
Methods Extracts
Five-gram samples of E. angustifolium or E. parviflorum (herba), collected in Styria, Austria and identified botanically (Saukel, 1982), were extracted for 10 min in 250 ml boiling water (or 150 ml for rat paw oedema experiments) and were subsequently filtered (aqueous extract). Five grams of each herb were stirred three times with 100 ml methanol at 60°C filtered, evaporated in vacuum to dryness and thereafter dissolved in 250 ml hot water (or 150 ml for rat paw oedema experiments; methanolic extract). Before testing, all extracts were made blood-isotonic by addition of appropriate amounts of sodium chloride measured with an osmometer (Knaur). Release of labelled PGs
The influence of the extracts on PG release was determined using the perfused rabbit ear (perfusion rate, 3 ml Tyrode solution/min) into which the labelled PG precursor [ 14C] arachidonic acid (AA) was incorporated according to the basal experiments of Isakson et al. (1977) with some modifications (Juan and Sametz, 1980, 1983; Sametz and Juan, 1982). In brief, 3.5 h after labelling with [14C]AA (56 Bq), extracts of E. angustifolium or E. parviflorum were infused into the ear for 50 min. Calculated as equivalents to the weight of the dry herb, the final concentrations of E. angustifolium, extracts in the perfusion solution were 1.25,2.5 and 5.0 mg/ml and that of E. parviflorum extract was 5.0 mg/ml. The contralateral ear served as control (infusion of saline). Four hours after labelling, the calcium ionophore A 23187 (10 pg) was injected intraarterially as bolus in both ears to stimulate the biosynthesis of PGs (in the presence of bovine serum albumin, 0.5 mg/ml). The effluent was collected for 20 min before (basal release) and 20 min after the injection of A 23187 (stimulated release), extracted for fatty acids and PGs and developed in a TLC-system (Juan and Sametz, 1980; 1983). Estimation of radioactive PGs (comigrating with authentic PGs) was carried out by liquid scintillation counting of each zone of 0.4 cm into which the plate was scraped (Juan and Sametz, 1983). Radioimmunoassay
(RIA)
PGEz and PGIz (as 6-keto-PGFla!) were measured in the ear perfusate by means of specific RIA exactly as described by Egg (1984). The experimental procedure (basal release, stimulated release, time) was exactly the same as described for release of labelled PGs. Paw oedema
Female Sprague-Dawley rats, 170-200 g (Versuchstierzucht und -haltung, Himberg, Austria), were used after being deprived of food for 20 h prior to the experiments. Oedema was measured by the mercury displacement
163
method. The paw of the anaesthetized rat was dipped into a cylinder filled with mercury, exactly up to a reference mark on the paw. The mercury cylinder was connected via saline-filled tube to a Statham pressure transducer P23Db. Changes in pressure were recorded by a Multi Pen Recorder (Rikadenki). Two hours before the injection of carrageenin, groups of rats were given the aqueous extracts of E. angustifolium or E. parviflorum (1 ml corresponding to 33 mg/ml dry herb per 100 g body weight) by oral tube. Under the same conditions, 2 mg/kg indomethacin (as sodium salt in saline, 0.9%) was applied for the comparison of the anti-inflammatory effect. Controls received the same volume of the vehicle. Before the experiments, the rats were anaesthetized with phenobarbitone (50 mg/kg, i.p.). Thirty minutes prior to injection of carrageenin (0.1 ml, 2%) into the plantar region of the right hind paw, the volumes of the paws were measured. The left paw (control) was injected with 0.1 ml of sodium chloride (0.9%) immediately after the injection of carrageenin. Oedema was determined from the treated paw. The volumes of the control paws were designated as 100%. Significance of differences between means (against controls) was assessed by two-sample t-test, with a significance level of P < 0.05.
PGI 2
C Ep
PGE2
Ea
C Ep Ea
PGD2
C Eq Ea
[ ‘Y!]AA. Influence of aqueous extracts of (Ea) (5 mg/ml based on dry herb) on the release of labelled PGI, (measured as 6-keto-PGF,,), PGE, and PGD, stimulated by bolus injection of 10 Mg A 23187 (small basal release subtracted) C, controls. Values are given as means f S.E.M. (n = 4); P < 0.05, P < 0.01. Fig.
1. Perfused
E.poruiflorum
rabbit ear labelled and E. angustifolium
with
164
Results Inhibition of prostaglandin release The aqueous extracts of both plants reduced the A 23187-stimulated release of labelled PGIz (measured as 6-keto-PGF,), PGEB and PGDz at the high dose level (5 m&ml based on dry herb). The extract of E. ang~stifo~~~m was far more potent (Fig. 1) and dose-dependently reduced the release of PGs (Fig. 2). The small basal release of labelled PGs, which was not significantly higher than the background on the plate (Juan and Sametz, 1980), was not influenced by the extracts (n = 4, P < 0.1). In contrast to the aqueous extract, the methanolic extract of E. angustifolium did not reduce the release of any labelled Pg (Fig. 3). The inhibitory influence of E. angustifoEium (2.5 mgjml based on dry herb) on the A 23X37-stimulated release of PGIz and PGEz was also investjgated by RIA (Fig. 4). inhibition of the ~arrag~enin rat paw oedema The aqueous extract of E. angustifo~ium strongly reduced the development of the oedema whereas the methanolic extract had a distinctly lower effect (Fig. 5). The extracts of E. paruiflorum were ineffective (Fig. 5). The influence of E. ang~st~foli~m on rat paw oedema was compared with the anti-in~ammatory effect of 2 mg/kg indomethacin (p.0,) {Fig. 6).
POE
2
PGD2
Fig. 2. Perfused rabbit ear labelled with [‘VIAA. Influence of three concentrations ([l] = 1.25 mg/ml; [Z] = 2.5 mg/ml; [3] = 5 mg/ml) of the aqueous extract of E. ung~s~~~o~~~~on release of labelled PGs stimulated by 10 gg A 23187 (basal release subtracted). 0, control. Values are given as means r S.E.M. (n = 4); P < 0.05, P < 0.01
165
PGl*
PGE2
PGD2
Fig. 3. Perfused rabbit ear Iabelled with [14C]AA. Influence of aqueous and methanolic extracts of 1.25 mg/ml (based on dry herb) E. angustifolium (Ea) on the release of labelled PGI, (measured as 6-keto-PGF,,), PGE, and PGD, stimulated by bolus injection of 10 pg A 23187 (small basal release subtracted) C, controls. Values are given as means + S.E.M. (n = 4);P < 0.05, P < 0.01.
Discussion The results show that aqueous extracts of E. angustifolium strongly reduced the release of PGs, whereas extracts of E. parviflorum were far less potent. Both extracts were investigated for an anti-inflammatory effect. The extract of E. angustifolium strongly depressed the formation of the rat paw carrageenin oedema whereas that of E. parviflorum was inactive (Fig. 5). This effect may correlate to some extent with inhibition of release of proinflammatory PGs as shown in the perfused rabbit ear. However, additional mechanisms are obviously of importance, since the extract of E. parviflorum was completely inactive with regard to oedema inhibition but somewhat reduced the release of PGs (the potency of E. parviflorum was about l/5 that of E. angustifolium). Indomethacin (2 mg/kg, p.o.) depressed the oedema formation to a similar extent as the extract of E. angustifolium
166
120-
2 loo0,
PGE 2
!
E ," z 80-
-i 0 tD 2
60-
F
LO -
tl 20-
C
Ea
Fig. 4. Stimulated release (by a bolus injection of 10 gg A 23187) of PGE, (measured as 6-l&o-PGF,,) from isolated perfused rabbit ear measured by ence of aqueous extract of 2.5 mgiml (based on dry herb) E. angustifolium columns). C, control (open columns). Values are given as means r S.E.M. P< 0.01.
and PGI, RIA. Influ(Ea, hatched (n = 4),
(Figs. 5 and 6), probably due to inhibition of cyclooxygenase (Van Arman, 1978). Dexamethasone also reduced the carrageenin effect (Bonta et al., 1977), probably not only by inhibiting the biosynthesis of PGs, although corticoids have been found to depress the release of arachidonate (Blackwell and Flower, 1978). The experiments do not give a clear answer to the question of the mechanism of action of the extract of E. augustifolium. There are no indications as to the chemical nature of the active principle(s) of the extracts. Since the methanolic extract of E. angustifokum in contrast to the aqueous extract does not reduce PG release and has a lower inhibitory
167
% 180
160
140
120
100
0,5 0
;
’
’
2
4
,I
6
24
hours Fig. 5. Rat paw oedema. Influence of 33 mg/ml (based on dry herb) aqueous extracts g p.o.) of E. parviflorum (a), E. angustifolium (P) and methanolic extracts of E. angustifolium (0 ) on percentage increase in paw volume following injection of carrageenin (2%, 0.1 ml) at time. Oedema was quantified by subtracting the volume of the control paw from that of the treated paw. The volumes of the control paws were set at 100%. Control groups (4). Values are given as means * S.E.M. (n = 12-15); *P < 0.05,
(1 ml/l00
***p
< 0.001.
effect on the rat paw oedema, the main active components appear to be hydrophilic. On phytochemical investigation of the methanolic extracts major amounts of flavonoids (Hiermann, 1984) and sitosterol derivatives (Hiermann and Mayr, 1985) were found to be present. Furthermore, the aqueous extracts of E. angustifolium exhibited the same pharmacological activity on PG release and rat paw oedema after extraction with ethyl acetate. Flavonoids and sitosterol derivatives were detected in the ethyl acetate phase. We therefore conclude that flavonoids and sitosterol derivatives can be excluded as the main candidates of the observed effect. Experiments are in progress to investigate the chemical nature of the active component(s).
168 “lo 180
160
I
,
-05
0
1
!
1
2
I
4
6
hours
Fig. 6. Rat paw oedema. Influence of 2 mg/kg indomethacin (0) (1 ml/100 g p.o.) on percentage increase in paw volume following injection of carrageenin (2%, 0.1 ml) at time. Oedema was quantified by subtracting the volume of the control paw from that of the treated paw. The volumes of the control paws were set at 100%. Control groups (3). Values are given as means +_S.E.M. (n = 6) *P < 0.05, **P < 0.01.
Acknowledgements The authors wish to thank Dr. E.A. staglandins and Dr. Egg for providing supported by the Austrian Scientific animal experiments were carried out Science and Research.
Schijlkens for the kind gift of proPG antisera. The experiments were Research Funds (grant 4991). The with permission of the Ministry of
References Baumann, J., Wurm, G. and v.Bruchhausen, tase durch Flavonoide und Phenolderivate Archiv
der Pharmazie
313,
330-337.
F. (1980) Hemmung der Prostaglandinsynthemit deren O;‘-RadikalfS~gereigenschaften.
169
Blackwell, G.J., Flower, R.J., Nijkamp, F.P. and Vane, J.R. (1978) Phospholipase A, activity of guinea pig isolated perfused lungs: stimulation and inhibition by antiinflammatory steroids. British Journal of Pharmacology 62, 79-89. Bonta, I.L., Bult, H., Vincent, J.E. and Zijlstra, F.J. (1977) Acute inflammatory effects of aspirin and dexamethasone in rats deprived of endogenous prostaglandin precursors. Journal
of Pharmacy
and Pharmacology
29, 1-7.
Chantrill, B.H., Coultland, C., Dickinson, L. Inkley, G.W., Morris, W. and Pyle, A.H. (1952) The action of plant extracts on a bacteriophage. The Journal of General Microbiology
6, 74-84.
Egg, D. (1984) Concentrations of prostaglandins D,, E,, F,, 6-keto-F, and thromboxane B, in synovial fluid from patients with inflammatory joint disorders and osteoarthritis. Zeitschrift
fur Rheumatologie
43,
89-96.
Gabor, M. (1979) In: J.R. Vane and S.H. Ferreira (Eds.), Anti-Znflammatory Drugs, Vol. 50/11, Springer-Verlag, Berlin-Heidelberg, pp. 698-739. Gupta, M.B., Nath, R., Scrivastava, N., Shanker, K., Kishor, K. and Bhargava, K.P. (1980) Anti-inflammatory and antipyretic activities of p-sitosterol. Planta Medica 39, 157163. Hiermann, A. (1983) Die Untersuchung potentieller Wirkstoffe in Epilobium-Arten. 1. Mitteilung: Aufkhirung der Flavonoidmuster. Scientia Pharmaceutics 51, 158-167. Hiermann, A. and Mayr, K. (1985) Die Untersuchung potentieller Wirkstoffe in Epilobium-Arten. 3. Mitteilung: Uber das Vorkommen von p-Sitosterolderivaten in Epilobium angustifolium L. und Epilobium parviflorum Schreb. Scientia Pharmaceutica 53,39-44.
Isakson, P.C., Raz, A., Denny, S.E., Wyche, A. and Needleman, P. (1977) Hormonal stimulation of arachidonic release from isolated perfused organs. Relationship to prostaglandin biosynthesis. Prostaglandins 14, 853-861. Juan, H. and Sametz, W. (1980) Histamine-induced release of arachidonic acid and of prostaglandins in the peripheral vascular bed. Naunyn-Schmiedeberg’s Archives of Pharmacology 324, 183-190. Juan, H. and Sametz, W. (1983) Uptake, stimulated release and metabolism of (l-l%)eicosapentaenoic acid in a perfused organ of the rabbit. Naunyn-Schmiedeberg’s Archives
of Pharmacology
324,
207-211.
Pukhalskaya, E.Kh., Chernyakhovskaya, I.Yu., Petrova, M.F., Denisova, S.I. and Alieva, T.A. (1975) Macromolecular antitumor agents from Chamaenerium angustifolium. Neoplasma
22, 29-37.
Sametz, W. and Juan, H. (1982) Release of different prostaglandins from vascular tissue by different stimulators. Prostaglandins, Leukotriens and Medicine 9, 593-602. Syrkin, A.B., Yuskov, S.F. and Postolnicov, S.F. (1977) Antineoplastic and toxic effects of new phytohemagglutinin chanerol from Chamaenerium angustifolium. Proceedings of the American Association of Cancer Research 18, 44. Van Arman, C.G. (1979) In: J.R. Vane and S.H. Ferreira (Eds.) Anti-Znflammatory Drugs (Vol. 50/H), Springer-Verlag, Berling-Heidelberg, pp. 75-91. Walker, H. (1977) In: G.L. Bach (1978) Wirksamkeit von Sitosterin beim Prostataadenom und bei entziindlichem Rheumatismus. Euromed 8, 585.
17 (1986) Ireland Ltd.
161
161-169
INFLUENCE OF EPZLOBZUM EXTRACTS ON PROSTAGLANDIN BIOSYNTHESIS AND CARRAGEENIN INDUCED OEDEMA OF THE RAT PAW
A. HIERMANNa,
H. JUANb
and W. SAMETZb
aInstitut fiir Pharmakognosie, bInstitut fiir Experimentelle und Klinische Universitiit Graz, Universitiitsplatz 4, A-801 0 Graz (Austria) (Accepted
Pharmakologie,
May 20, 1986)
Summary
Epilobium species have been used as remedies in folk-medicine for the treatment of pathophysiological processes of the prostata. In this paper the influence of extracts of Herba Epilobii angustifolii L. and Herba Epilobii paruiflori Schreb. on prostaglandin biosynthesis and the carrageenin rat paw oedema is described. Aqueous extracts of Herba E. angustifolii reduced the release of prostaglandins I*, Ez and Dz (in the perfused rabbit ear) approximately 5 times more effectively than did similar extracts of Herba E. paruiflori. Methanolic extracts were inactive. The aqueous extract of E. angustifolium strongly reduced the carrageenin-induced rat paw oedema whereas that of E. paruiflorum was inactive. The chemical nature of the active compound(s) is as yet unknown but flavonoids and sitosterol derivatives can be excluded.
Introduction
Extracts of the species of the genus Epilobium L. (Fam. Onagraceae) have traditionally been used in folk-medicine for the treatment of prostata adenoma (benign prostata-hyperplasia) and inflammations of the prostata. However, few reports have concerned the pharmacology of these plants (Pukhalskaya et al., 1975; Syrkin et al., 1977; Chantrill et al., 1952). Previous work has revealed the existence of several flavonoids and sitosterol derivatives in Epilobium species (Hiermann, 1984; Hiermann and Mayr, 1985). Both classes of compounds are known to inhibit prostaglandin (PG) biosynthesis (Baumann et al., 1980; Walker, 1977) and the formation of rat paw oedema (Gabor, 1979; Gupta et al., 1980). We have therefore studied the effect of crude aqueous extracts of E. angustifolium L. and E. parviflorum Schreb. on PG biosynthesis and the carrageenin rat paw oedema, an experimental model of acute inflammation. 0378-C741/86/$03.50 Published and Printed
o 1986 in Ireland
Elsevier
Scientific
Publishers
Ireland
Ltd.
162
Methods Extracts
Five-gram samples of E. angustifolium or E. parviflorum (herba), collected in Styria, Austria and identified botanically (Saukel, 1982), were extracted for 10 min in 250 ml boiling water (or 150 ml for rat paw oedema experiments) and were subsequently filtered (aqueous extract). Five grams of each herb were stirred three times with 100 ml methanol at 60°C filtered, evaporated in vacuum to dryness and thereafter dissolved in 250 ml hot water (or 150 ml for rat paw oedema experiments; methanolic extract). Before testing, all extracts were made blood-isotonic by addition of appropriate amounts of sodium chloride measured with an osmometer (Knaur). Release of labelled PGs
The influence of the extracts on PG release was determined using the perfused rabbit ear (perfusion rate, 3 ml Tyrode solution/min) into which the labelled PG precursor [ 14C] arachidonic acid (AA) was incorporated according to the basal experiments of Isakson et al. (1977) with some modifications (Juan and Sametz, 1980, 1983; Sametz and Juan, 1982). In brief, 3.5 h after labelling with [14C]AA (56 Bq), extracts of E. angustifolium or E. parviflorum were infused into the ear for 50 min. Calculated as equivalents to the weight of the dry herb, the final concentrations of E. angustifolium, extracts in the perfusion solution were 1.25,2.5 and 5.0 mg/ml and that of E. parviflorum extract was 5.0 mg/ml. The contralateral ear served as control (infusion of saline). Four hours after labelling, the calcium ionophore A 23187 (10 pg) was injected intraarterially as bolus in both ears to stimulate the biosynthesis of PGs (in the presence of bovine serum albumin, 0.5 mg/ml). The effluent was collected for 20 min before (basal release) and 20 min after the injection of A 23187 (stimulated release), extracted for fatty acids and PGs and developed in a TLC-system (Juan and Sametz, 1980; 1983). Estimation of radioactive PGs (comigrating with authentic PGs) was carried out by liquid scintillation counting of each zone of 0.4 cm into which the plate was scraped (Juan and Sametz, 1983). Radioimmunoassay
(RIA)
PGEz and PGIz (as 6-keto-PGFla!) were measured in the ear perfusate by means of specific RIA exactly as described by Egg (1984). The experimental procedure (basal release, stimulated release, time) was exactly the same as described for release of labelled PGs. Paw oedema
Female Sprague-Dawley rats, 170-200 g (Versuchstierzucht und -haltung, Himberg, Austria), were used after being deprived of food for 20 h prior to the experiments. Oedema was measured by the mercury displacement
163
method. The paw of the anaesthetized rat was dipped into a cylinder filled with mercury, exactly up to a reference mark on the paw. The mercury cylinder was connected via saline-filled tube to a Statham pressure transducer P23Db. Changes in pressure were recorded by a Multi Pen Recorder (Rikadenki). Two hours before the injection of carrageenin, groups of rats were given the aqueous extracts of E. angustifolium or E. parviflorum (1 ml corresponding to 33 mg/ml dry herb per 100 g body weight) by oral tube. Under the same conditions, 2 mg/kg indomethacin (as sodium salt in saline, 0.9%) was applied for the comparison of the anti-inflammatory effect. Controls received the same volume of the vehicle. Before the experiments, the rats were anaesthetized with phenobarbitone (50 mg/kg, i.p.). Thirty minutes prior to injection of carrageenin (0.1 ml, 2%) into the plantar region of the right hind paw, the volumes of the paws were measured. The left paw (control) was injected with 0.1 ml of sodium chloride (0.9%) immediately after the injection of carrageenin. Oedema was determined from the treated paw. The volumes of the control paws were designated as 100%. Significance of differences between means (against controls) was assessed by two-sample t-test, with a significance level of P < 0.05.
PGI 2
C Ep
PGE2
Ea
C Ep Ea
PGD2
C Eq Ea
[ ‘Y!]AA. Influence of aqueous extracts of (Ea) (5 mg/ml based on dry herb) on the release of labelled PGI, (measured as 6-keto-PGF,,), PGE, and PGD, stimulated by bolus injection of 10 Mg A 23187 (small basal release subtracted) C, controls. Values are given as means f S.E.M. (n = 4); P < 0.05, P < 0.01. Fig.
1. Perfused
E.poruiflorum
rabbit ear labelled and E. angustifolium
with
164
Results Inhibition of prostaglandin release The aqueous extracts of both plants reduced the A 23187-stimulated release of labelled PGIz (measured as 6-keto-PGF,), PGEB and PGDz at the high dose level (5 m&ml based on dry herb). The extract of E. ang~stifo~~~m was far more potent (Fig. 1) and dose-dependently reduced the release of PGs (Fig. 2). The small basal release of labelled PGs, which was not significantly higher than the background on the plate (Juan and Sametz, 1980), was not influenced by the extracts (n = 4, P < 0.1). In contrast to the aqueous extract, the methanolic extract of E. angustifolium did not reduce the release of any labelled Pg (Fig. 3). The inhibitory influence of E. angustifoEium (2.5 mgjml based on dry herb) on the A 23X37-stimulated release of PGIz and PGEz was also investjgated by RIA (Fig. 4). inhibition of the ~arrag~enin rat paw oedema The aqueous extract of E. angustifo~ium strongly reduced the development of the oedema whereas the methanolic extract had a distinctly lower effect (Fig. 5). The extracts of E. paruiflorum were ineffective (Fig. 5). The influence of E. ang~st~foli~m on rat paw oedema was compared with the anti-in~ammatory effect of 2 mg/kg indomethacin (p.0,) {Fig. 6).
POE
2
PGD2
Fig. 2. Perfused rabbit ear labelled with [‘VIAA. Influence of three concentrations ([l] = 1.25 mg/ml; [Z] = 2.5 mg/ml; [3] = 5 mg/ml) of the aqueous extract of E. ung~s~~~o~~~~on release of labelled PGs stimulated by 10 gg A 23187 (basal release subtracted). 0, control. Values are given as means r S.E.M. (n = 4); P < 0.05, P < 0.01
165
PGl*
PGE2
PGD2
Fig. 3. Perfused rabbit ear Iabelled with [14C]AA. Influence of aqueous and methanolic extracts of 1.25 mg/ml (based on dry herb) E. angustifolium (Ea) on the release of labelled PGI, (measured as 6-keto-PGF,,), PGE, and PGD, stimulated by bolus injection of 10 pg A 23187 (small basal release subtracted) C, controls. Values are given as means + S.E.M. (n = 4);P < 0.05, P < 0.01.
Discussion The results show that aqueous extracts of E. angustifolium strongly reduced the release of PGs, whereas extracts of E. parviflorum were far less potent. Both extracts were investigated for an anti-inflammatory effect. The extract of E. angustifolium strongly depressed the formation of the rat paw carrageenin oedema whereas that of E. parviflorum was inactive (Fig. 5). This effect may correlate to some extent with inhibition of release of proinflammatory PGs as shown in the perfused rabbit ear. However, additional mechanisms are obviously of importance, since the extract of E. parviflorum was completely inactive with regard to oedema inhibition but somewhat reduced the release of PGs (the potency of E. parviflorum was about l/5 that of E. angustifolium). Indomethacin (2 mg/kg, p.o.) depressed the oedema formation to a similar extent as the extract of E. angustifolium
166
120-
2 loo0,
PGE 2
!
E ," z 80-
-i 0 tD 2
60-
F
LO -
tl 20-
C
Ea
Fig. 4. Stimulated release (by a bolus injection of 10 gg A 23187) of PGE, (measured as 6-l&o-PGF,,) from isolated perfused rabbit ear measured by ence of aqueous extract of 2.5 mgiml (based on dry herb) E. angustifolium columns). C, control (open columns). Values are given as means r S.E.M. P< 0.01.
and PGI, RIA. Influ(Ea, hatched (n = 4),
(Figs. 5 and 6), probably due to inhibition of cyclooxygenase (Van Arman, 1978). Dexamethasone also reduced the carrageenin effect (Bonta et al., 1977), probably not only by inhibiting the biosynthesis of PGs, although corticoids have been found to depress the release of arachidonate (Blackwell and Flower, 1978). The experiments do not give a clear answer to the question of the mechanism of action of the extract of E. augustifolium. There are no indications as to the chemical nature of the active principle(s) of the extracts. Since the methanolic extract of E. angustifokum in contrast to the aqueous extract does not reduce PG release and has a lower inhibitory
167
% 180
160
140
120
100
0,5 0
;
’
’
2
4
,I
6
24
hours Fig. 5. Rat paw oedema. Influence of 33 mg/ml (based on dry herb) aqueous extracts g p.o.) of E. parviflorum (a), E. angustifolium (P) and methanolic extracts of E. angustifolium (0 ) on percentage increase in paw volume following injection of carrageenin (2%, 0.1 ml) at time. Oedema was quantified by subtracting the volume of the control paw from that of the treated paw. The volumes of the control paws were set at 100%. Control groups (4). Values are given as means * S.E.M. (n = 12-15); *P < 0.05,
(1 ml/l00
***p
< 0.001.
effect on the rat paw oedema, the main active components appear to be hydrophilic. On phytochemical investigation of the methanolic extracts major amounts of flavonoids (Hiermann, 1984) and sitosterol derivatives (Hiermann and Mayr, 1985) were found to be present. Furthermore, the aqueous extracts of E. angustifolium exhibited the same pharmacological activity on PG release and rat paw oedema after extraction with ethyl acetate. Flavonoids and sitosterol derivatives were detected in the ethyl acetate phase. We therefore conclude that flavonoids and sitosterol derivatives can be excluded as the main candidates of the observed effect. Experiments are in progress to investigate the chemical nature of the active component(s).
168 “lo 180
160
I
,
-05
0
1
!
1
2
I
4
6
hours
Fig. 6. Rat paw oedema. Influence of 2 mg/kg indomethacin (0) (1 ml/100 g p.o.) on percentage increase in paw volume following injection of carrageenin (2%, 0.1 ml) at time. Oedema was quantified by subtracting the volume of the control paw from that of the treated paw. The volumes of the control paws were set at 100%. Control groups (3). Values are given as means +_S.E.M. (n = 6) *P < 0.05, **P < 0.01.
Acknowledgements The authors wish to thank Dr. E.A. staglandins and Dr. Egg for providing supported by the Austrian Scientific animal experiments were carried out Science and Research.
Schijlkens for the kind gift of proPG antisera. The experiments were Research Funds (grant 4991). The with permission of the Ministry of
References Baumann, J., Wurm, G. and v.Bruchhausen, tase durch Flavonoide und Phenolderivate Archiv
der Pharmazie
313,
330-337.
F. (1980) Hemmung der Prostaglandinsynthemit deren O;‘-RadikalfS~gereigenschaften.
169
Blackwell, G.J., Flower, R.J., Nijkamp, F.P. and Vane, J.R. (1978) Phospholipase A, activity of guinea pig isolated perfused lungs: stimulation and inhibition by antiinflammatory steroids. British Journal of Pharmacology 62, 79-89. Bonta, I.L., Bult, H., Vincent, J.E. and Zijlstra, F.J. (1977) Acute inflammatory effects of aspirin and dexamethasone in rats deprived of endogenous prostaglandin precursors. Journal
of Pharmacy
and Pharmacology
29, 1-7.
Chantrill, B.H., Coultland, C., Dickinson, L. Inkley, G.W., Morris, W. and Pyle, A.H. (1952) The action of plant extracts on a bacteriophage. The Journal of General Microbiology
6, 74-84.
Egg, D. (1984) Concentrations of prostaglandins D,, E,, F,, 6-keto-F, and thromboxane B, in synovial fluid from patients with inflammatory joint disorders and osteoarthritis. Zeitschrift
fur Rheumatologie
43,
89-96.
Gabor, M. (1979) In: J.R. Vane and S.H. Ferreira (Eds.), Anti-Znflammatory Drugs, Vol. 50/11, Springer-Verlag, Berlin-Heidelberg, pp. 698-739. Gupta, M.B., Nath, R., Scrivastava, N., Shanker, K., Kishor, K. and Bhargava, K.P. (1980) Anti-inflammatory and antipyretic activities of p-sitosterol. Planta Medica 39, 157163. Hiermann, A. (1983) Die Untersuchung potentieller Wirkstoffe in Epilobium-Arten. 1. Mitteilung: Aufkhirung der Flavonoidmuster. Scientia Pharmaceutics 51, 158-167. Hiermann, A. and Mayr, K. (1985) Die Untersuchung potentieller Wirkstoffe in Epilobium-Arten. 3. Mitteilung: Uber das Vorkommen von p-Sitosterolderivaten in Epilobium angustifolium L. und Epilobium parviflorum Schreb. Scientia Pharmaceutica 53,39-44.
Isakson, P.C., Raz, A., Denny, S.E., Wyche, A. and Needleman, P. (1977) Hormonal stimulation of arachidonic release from isolated perfused organs. Relationship to prostaglandin biosynthesis. Prostaglandins 14, 853-861. Juan, H. and Sametz, W. (1980) Histamine-induced release of arachidonic acid and of prostaglandins in the peripheral vascular bed. Naunyn-Schmiedeberg’s Archives of Pharmacology 324, 183-190. Juan, H. and Sametz, W. (1983) Uptake, stimulated release and metabolism of (l-l%)eicosapentaenoic acid in a perfused organ of the rabbit. Naunyn-Schmiedeberg’s Archives
of Pharmacology
324,
207-211.
Pukhalskaya, E.Kh., Chernyakhovskaya, I.Yu., Petrova, M.F., Denisova, S.I. and Alieva, T.A. (1975) Macromolecular antitumor agents from Chamaenerium angustifolium. Neoplasma
22, 29-37.
Sametz, W. and Juan, H. (1982) Release of different prostaglandins from vascular tissue by different stimulators. Prostaglandins, Leukotriens and Medicine 9, 593-602. Syrkin, A.B., Yuskov, S.F. and Postolnicov, S.F. (1977) Antineoplastic and toxic effects of new phytohemagglutinin chanerol from Chamaenerium angustifolium. Proceedings of the American Association of Cancer Research 18, 44. Van Arman, C.G. (1979) In: J.R. Vane and S.H. Ferreira (Eds.) Anti-Znflammatory Drugs (Vol. 50/H), Springer-Verlag, Berling-Heidelberg, pp. 75-91. Walker, H. (1977) In: G.L. Bach (1978) Wirksamkeit von Sitosterin beim Prostataadenom und bei entziindlichem Rheumatismus. Euromed 8, 585.