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Antispasmodic activity of an extract from Plantago lanceolata L. and some isolated compounds.

Abstract

An ethanolic spissum extract of the aerial parts of Plantago lanceolata L. was examined for antispasmodic activity on isolated ileum and trachea of the guinea-pig. Isolated constituents were investigated as well. The P. lanceolata extract inhibited the contractions of the guinea-pig ileum that were induced by various agonists such as acetylcholine (ACh), histamine, potassium and barium ions. Additionally the trachea contractions induced by barium ions were inhibited. The compounds luteolin, acteoside, plantamajoside an catalpol peracetate but not catalpol, isoacteoside, lavandulifolioside and aucubin inhibited the ACh-induced contractions of the guinea-pig ileum. Luteolin and acteoside reduced the barium-induced contractions of the guinea-pig trachea. Two recently isolated compounds did not show antispasmodic activity: luteolin-3', 7-diglucuronide and [beta]-hydroxy-acteoside.

[c] 2006 Elsevier GmbH. All rights reserved.

Keywords: Plantago lanceolata; Antispasmodic effect; Ileum; Trachea

Introduction

Several effects have been described for Plantago lanceolata L. such as bactericide effects, anti-inflammatory, phagocytose induction and increased interferon production (reviewed by Brautigam, 1992; Sasaki et al., 1989; Murai et al., 1995). The aerial parts of P. lanceolata L. are used to treat bronchial catarrh and inflammation of the mucous membrane of the pharynx (N.N. Monography of the commission E, 1985). For this indication 3-6 g/day are usually used which can be reduced by evaluating the relevant constituents and enriching the extracts. A recent clinical study revealed broncholytic activity of an extract of P. lanceolata (Kraft, 1996). A clear pharmacological proof of antispasmodic effects, however, is still missing.

The aim was to perform a pharmacological antispasmodic testing (two models) of an ethanolic extract of P. lanceolata L. and of fractionated well known and newly isolated compounds of this plant.

Methods and substances

Plant material

An ethanolic (20%) spissum extract of P. lanceolata L. (voucher 6822791) with a drug/extract ratio of 1 and a thereof produced fluid extract (voucher 6822900) were tested (details in Fleer, 2000). The material was supplied by Finzelberg, Andernach, Germany and Serturner, Gutersloh, Germany. The Plantago extract was tested in 10 and 20 [micro]l/ml concentrations (final organ bath concentration). The pure compounds were tested at concentrations equivalent to extract concentration specified in the figures; the phenylethanoids were tested: acteoside corresponded to 30 [micro]l/ml extract, plantamajoside to 200 [micro]l/ml, lavandulifolioside to 470 [micro]l/ml and isoacteoside to 140 [micro]l/ml. The tested compounds were easily water soluble or dissolved in 4 [micro]l/ml ethanol; the extract maximally contained 4 [micro]l/ml ethanol; the same ethanol concentration was used in control experiments. An ultrasonic bath helped to dissolve the compounds and extracts. As described earlier (Fleer, 2000), the content of the investigated compounds in the extract was determined by capillary electrophoresis method: luteolin 0.063 mg/g, acteoside 4.5 mg/g, plantamajoside 0.7 mg/g, lavandulifolioside 0.3 mg/g, isoacteoside 1.0 mg/g.

Isolated compounds and their identification by HPLC

The characterization of the isolated compounds was performed by using a Waters 600 Multi-solvent Delivery System with a RP-18 column hypersil ODS (250 mm, 5 [micro]m) and a Waters 990, photodiode array detector (Manchester, England). The pump flow was 2 ml/min and the detection was done at 330nm. All the investigated compounds were detected (Fig. 1) by this method except the iridoid glucosides aucubin and catalpol. Acteoside was most prominent (Fig. 1).

[FIGURE 1 OMITTED]

The phenylethanoids acteoside, plantamajoside and lavandulifolioside, the iridoid glucoside catalpol and the flavonoids luteolin and luteolin-7-glucuronide were previously described for P. lanceolata and were identified by comparison with published data (Ofterdinger-Daegel, 1993; Franzyk et al., 1998; Ravn et al., 1990; Basaran et al., 1988; Morota et al., 1989; Youssef and Frahm, 1995; Markham and Geiger, 1994; Klimik, 1995). [beta]-Hydroxy-acteoside and luteolin-3', 7-diglucuronide used from P. lanceolata were recently isolated for the first time; procedures for identification of the compounds have been described (Fleer, 2000).

Guinea-pig ileum

Under a protocol approved by the Animal Care and Use Committee of Munster, Germany, male and female guinea pigs (Charles River, Sulzfeld, Germany) weighing 250-450 g were used. The guinea pigs were anesthesized (C[O.sub.2]) and sacrificed by cervical displacement followed by exsanguination. The proximal ileum was removed, washed and placed in Krebs-Henseleit solution. Segments of 1 cm were placed in 10 ml organ baths with a resting tension of 0.5 g (preload). One end was attached to a force displacement transducer (lever transducer B40, Typ 373) combined with a two-channel amplifier (type 301, Hugo Sachs Elektronik, March, Germany); for the recording of tension changes a multi-pen recorder was used (Rikadenki Kogyo, Tokio, Japan). The composition of Krebs-Henseleit solution was (mM): NaCl 118.1, KCl 4.7, Ca[Cl.sub.2] 2.5, MgS[O.sub.4] 1.2, NaHC[O.sub.3] 25 and glucose 5.6. The solution was kept at 37[degrees]C, pH 7.4 and gassed with carbogen (a 95% [O.sub.2]/5% C[O.sub.2] mixture). Equilibration time for the ileum incubation was 30 min. After the tissues had been pretreated with the extract or compounds cumulative concentration response curves using the agonists ACh-chloride, histamine hydrochloride, potassium chloride or barium chloride were recorded isotonically in the organ bath, and the effect was allowed to reach a steady state at each concentration.

Guinea-pig trachea

From the same animals two zig-zag tracheal strips were prepared (Emmerson and Mackay, 1979) and placed in a 10 ml organ bath of Krebs-Henseleit solution (see above). The tissue was suspended with resting tension of 0.5 g and allowed to equilibrate for at least 1.5 h with washing every 15 min. The extracts and substances were allowed to act on the preparations for 5 min, and afterwards the agonists such as barium chloride were cumulatively added and were allowed to reach a steady state at each concentration.

Statistics

After fitting the concentration-response curves, the [EC.sub.50] and [E.sub.max] values were calculated. The results are expressed as mean[+ or -]SEM of a given number of independent experiments.

For statistical evaluation multiple comparisons of means were carried out by one-way analysis of variance followed by a post hoc test (Newman-Keuls test).

Results

In this study, the antispasmodic activities of an ethanolic spissum extract and of isolated compounds were examined in two pharmacological models, the contracted guinea-pig ileum and trachea.

Ileum

Fig. 2 shows the overall effect of the Plantago extract on contractions of guinea-pig ileum induced by acetylcholine (ACh). The extract exhibited an antispasmodic effect at both concentrations (10 and 20 [micro]l/ml) used whereas ethanol (solvent) had only a marginal effect. Atropine completely abolished the ACh effect.

Rather the same results were obtained when histamine, potassium ions or barium ions were used as stimulators of contraction (data not shown); in this case compounds such as diphenhydramine (weakly effective) and papaverine (highly effective) were used as antagonistic controls (data not shown).

[FIGURE 2 OMITTED]

Fig. 3 shows the ACh (A) and histamine-dependent (B) contraction curve of guinea-pig ileum in the absence and presence of Plantago extract. Both extract concentrations (10 and 20 [micro]l/ml) shifted the ACh-response curves to the right and down (Fig. 3A). The lower concentration of the extract (10 [micro]l/ml) reduced the ACh-induced contraction to approx. 60%, the higher one (20 [micro]l/ml) to approx. 45%. Atropine (100 nM) was even more effective (Fig. 3A).

The same holds for histamine as an agonist for ileum concentration (Fig. 3B). Both extract concentrations (10 and 20 [micro]l/ml) shifted the concentration-response curve to the right and down. The lower concentration of the extract (10 [micro]l/ml) reduced the histamine-induced contraction to approx. 75%, the higher one (20 [micro]l/ml) to approx. 32% (Fig. 3B). Diphenhydramine (170 nM) was less effective at this concentration (data not shown).

Fig. 4 shows the potassium chloride (A) and barium chloride induced (B) contraction curve of guinea-pig ileum in the absence and presence of Plantago extract. Both extract concentrations (10 and 20 [micro]l/ml) shifted the potassium-response curves to the right and down (Fig. 4A). The lower concentration of the extract (10 [micro]l/ml) reduced the potassium-induced contraction to approx. 80%, the higher one (20 [micro]l/ml) to approx. 57% (Fig. 4A). Papaverine (10 [micro]M) was less effective (data not shown).

[FIGURE 3 OMITTED]

The same holds for barium chloride as an agonist for ileum contraction (Fig. 4B). Both extract concentrations (10 and 20 [micro]l/ml) shifted the concentration-response curve to the right and down. The lower concentration of the extract (10 [micro]l/ml) tended to reduce (not significantly) the barium-induced contraction to approx. 83%, the higher one (20 [micro]l/ml) to approx. 74% (Fig. 4B). Papaverine (10 [micro]M) was more effective (data not shown).

[FIGURE 4 OMITTED]

Since the potency of extracts was rather similar irrespective of the stimulator used, further experiments with respect to ileum are only shown for ACh.

Fig. 5 shows the antispasmodic effect of luteolin, catalpol and catalpolperacetate on the concentration-dependent contraction of guinea-pig ileum mediated by ACh. At the high luteolin concentration (corresponding to 100 [micro]l/ml Plantago extract), the concentration--response curve was shifted to the right and down (Fig. 5A). The higher concentration of luteolin reduced the ACh-induced contraction to approx. 45%; the lower concentration (equivalent to 10 [micro]l/ml Plantago extract) had no effect (Fig. 5A). Catalpol peracetate (100 [micro]M) but not catalpol (552 [micro]M) shifted the concentration--response curve to the right and down. Catalpolperacetate reduced the ACh-induced contraction to approx. 37%.

[FIGURE 5 OMITTED]

Other isolated compounds such as isoacteoside and lavandulifolioside did not show any effect (data not shown). Other phenylethanosides such as plantamajoside and acetoside had a small effect (curve shifted down by maximally 13%; data not shown). The iroidglucoside aucubin was without effect (data not shown).

In Table 1 data on above-mentioned compounds are summarized with respect to ACh as agonist for ileum contraction. The maximum contractions ([E.sub.max]) of the guinea-pig ileum induced by ACh were significantly reduced by several compounds as indicated. [EC.sub.50]s are indicated as well and in most cases did not change.

Trachea

Relaxation experiments on trachea were performed, too. [Ba.sup.2+] turned out to be the most effective stimulator and data only for this ion are shown.

Fig. 6 shows the effect of the Plantago extract on contractions of guinea-pig trachea induced by barium ions. Both extracts reduced the barium-induced contraction whereas ethanol (solvent) had no effect. Papaverine of 0.5 [micro]M inhibited the barium ion effect to 40%, and 10 [micro]M was even more effective (data not shown). The lower concentration of the extract (10 [micro]l/ml) decreased the barium-induced contraction to approx. 76%, the higher one (20 [micro]l/ml) to approx. 59%.

Fig. 7 shows the effect of acetoside and of luteolin on the concentration-dependent contraction of guinea-pig trachea induced by barium ion. Luteolin (corresponding to 285 [micro]l/ml Plantago extract) and acteoside (corresponding to 30 [micro]l/ml Plantago extract) shifted the concentration--response curve to the right and down. When ACh instead of barium ion was used, the outcome of effects with luteolin, catalpol and catalpolperacetate was rather the same (data not shown).

In Table 2 data on the above-mentioned extract and compounds are summarized with respect to barium as agonist for trachea contraction. The maximum contractions ([E.sub.max]) of the guinea-pig trachea induced by barium were significantly reduced by many compounds as indicated.[EC.sub.50]s are indicated as well and are not modified to a major degree.

[FIGURE 6 OMITTED]

Discussion

Plantago extracts had antispasmodic activity irrespective of the system (ileum or trachea) and the stimulator (ACh, histamine, [Ba.sup.2+], [K.sup.+]) used. The extract, therefore, is a valuable remedy. The question is to whether eliminate or concentrate specific constituents (compounds) in the extract.

The recently described fractionated compounds (Fleer, 2000) (flavonoids, phenylethanoids and an iridoid glucoside) were investigated. Luteolin and the phenylethanoids acteoside and plantamajoside exhibited antispasmodic activity. Acteoside significantly reduced the maximum contraction ([E.sub.max]) of the guinea-pig ileum induced by ACh to 87% and plantamajoside significantly reduced the contractions to 91%. Additionally acteoside significantly decreased the maximum contraction of the guinea-pig trachea to 42% when induced by barium. The [EC.sub.50] values of ACh or barium ions were not affected by these compounds indicating that the antagonism of these phenylethanoids is non-competitive. In contrast, the other tested phenylethanoids isoacteoside and lavandulifolioside exhibited no antispasmodic activity. An antihistaminic activity of acteoside was shown previously in the guinea-pig ileum model (Schapoval et al., 1998). In this context, it is of interest that the tested ethanolic extract of P. lanceolata also slightly inhibited histamine-induced contractions (data not shown).

[FIGURE 7 OMITTED]

Luteolin exhibited significant antispasmodic activity at a concentration of 19 [micro]g/ml that corresponds to 285 [micro]l/ml P. lanceolata fluidextract. The ACh-induced contractions of the guinea-pig ileum and the barium-induced contractions of the trachea were reduced to 45%. The antispasmodic activity of luteolin has been reported previously (van den Broucke and Lemli, 1983). Contrary to the aglycon the glycosides luteolin-7-glucuronide and luteolin-3', 7-diglucuronide were isolated from an inactive fraction, showing that both compounds have no antispasmodic activity.

The already described constituents catalpol and its synthesized peracetate were tested. Neither the genuine iridoid glucoside nor the fractions that contain the iridoid glucoside exhibited antispasmodic activity. However, catalpolperacetate, which was prepared from catalpol by acetylation, inhibited the ACh-induced contractions of the guinea-pig ileum at a concentration of 100 [micro]M, whereas catalpol was not effective even at the high concentration tested (550 [micro]M). The antispasmodic activity of the peracetates of some iridoid glucosides on rat uterus was published previously (Ortiz de Urbina et al., 1994).

Acknowledgements

Thanks are due to Fa. Serturner, Gutersloh for providing the extract. Thanks are also due to I. Kaiserling-Buddemeier for technical assistance. The help of Dr. Nahrstedt improving the manuscript is greatly acknowledged.

References

Basaran, A., Calis, I., Anklin, C., Nishibe, S., Sticher, O., 1988. Lavandulifolioside: a new phenylpropanoid glycoside from Stachys lavandulifolia. Helv. Chim. Acta 71, 1483-1490.

Brautigam, M., 1992. Plantago lanceolata Hagers Handbuch der pharmazeutischen Praxis. In: Hansel, R., Keller, K., Rimpler, H., Schneider, G. (Eds.), Drogen A-Z, 5 Aufl. Springer, Berlin.

Emmerson, J., Mackay, D., 1979. The zig-zag trachea strip. J. Pharm. Pharmacol. 31, 798.

Fleer, H., 2000. Spasmolytische und antiemetische Wirkung. Phytochemische und pharmakologische Untersuchungen an Plantago lanceolata L. und Cynara scolymus L. Thesis, Munster, Germany.

Franzyk, H., Husum, T., Jensen, S., 1998. A caffeoly phenylethanoid glycoside from Plantago myosuros. Phytochemistry 47, 1161-1162.

Klimik, B., 1995. Flavoniod glucuronides from Verbascum lychnitis L. and nigrum L. Acta Polon. Pharm. 52, 53-56.

Kraft, K., 1996. Therapeutic profile of a Plantaog lanceolata fluid extract (PLFE) in acuta respiratory disease (ARD). Forsch. Komplementarmed. 3, 319.

Markham, K., Geiger, H., 1994. [.sup.1.H] nuclear magnetic resonance spectroscopy of flavonoids and their glycosides in hexadeuterodimethylsulfoxide. In: Harborne, J.B. (Ed.), The flavonoids. Advances in research since 1986, first ed. Hall, London, pp. 450-451.

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Murai, M., Tamayama, Y., Nishibe, S., 1995. Phenylethanoids in the herb of Plantago lanceolata and inhibitory effect on arachidon acid induced mouse ear edema. Planta Med. 61, 479-480.

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Ortiz de Urbina, A., Martin, M., Fernandez, B., San Roman, L., Cubillo, L., 1994. In vitro antispasmodic activity of peracetylated penstemonoside, aucubin and catalpol. Planta Med. 60, 512-515.

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Sasaki, H., Nishimura, H., Morata, T., Chin, M., Mitsuhashi, H., Komatsu, Y., Maruyama, H., Guo-rui, T., Wie, H., Yu-lang, X., 1989. Immunosuppressive principles of Rehmannia glutinosa var. hueichingensis. Planta Med. 55, 458-462.

Schapoval, E., Winter de Vargas, M., Chaves, C., Bridi, R., Zuanazzi, J., Henriques, A., 1998. Antiinflammatory and antinociceptive activities of extracts and isolated from Stachytarpheta cayennensis. J. Ethnopharm. 60, 53-59.

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H. Fleer (a), E.J. Verspohl (b,*)

(a) Institut fur Pharmazeutische Biologie und Phytochemie der Westfalischen Wilhelms-Universitat Munster, Hittorfstr. 56, 48149 Munster, Germany

(b) Abteilung fur Pharmakologie, Institut fur Pharmazeutische und Medizinische Chemie der Westfalischen Wilhelms-Universitat Munster, Hittorfstr. 58-62, 48149 Munster, Germany

*Corresponding author.

E-mail address: verspoh@uni-muenster.de (E.J. Verspohl).
Table 1. Antispasmodic activity of the extract and the compounds of P.
lanceolata on guinea-pig ileum against the agonist acetylcholine,
[E.sub.max]: contraction in relation to control (%)

 Concentration [E.sub.max] (%) [EC.sub.50] (nM)
Antagonist (organ bath) mean [+ or -] SEM mean [+ or -] SEM

Acetylcholine -- 100 83 [+ or -] 10
 (control)
P. lanceolata 10 [micro]l/ 63 [+ or -] 6.7* 100 [+ or -] 19
 fluid extract ml
Acteoside 240 [micro]M 87 [+ or -] 3.7* 114 [+ or -] 29
Plantamajoside 234 [micro]M 91 [+ or -] 1.2* 64 [+ or -] 6
Lavandulifolioside 200 [micro]M 96 [+ or -] 1.0 66 [+ or -] 12
Isoacteoside 240 [micro]M 98 [+ or -] 2.6 56 [+ or -] 19
Luteolin 68 [micro]M 45 [+ or -] 5.1* 551 [+ or -] 88*
Catalpol 550 [micro]M 98 [+ or -] 3.5 70 [+ or -] 2
Catalpolperacetate 100 [micro]M 37 [+ or -] 3.6* 63 [+ or -] 12
Atropine 100 nM 40 [+ or -] 19.8* 3578 [+ or -] 601

[EC.sub.50]: concentration of the agonist acetylcholine (nM) in the
presence or absence of the agonist acetylcholine, *p<0.05.

Table 2. Antispasmodic activity of the extract and the compounds of P.
lanceolata on guinea-pig trachea against the agonist barium,
[E.sub.max]: contraction in relation to control (%)

 [EC.sub.50]
 Concentration [E.sub.max] mean ([micro]M)
Antagonist (organ bath) [+ or -] SEM mean [+ or -] SEM

Barium ion 100 2.71 [+ or -] 0.30
 (control)
P. lanceolata 10 [micro]l/ml 76 [+ or -] 8.9* 4.57 [+ or -] 0.71
 fluidextract
Acteoside 240 [micro]M 42 [+ or -] 9.9* 7.34 [+ or -] 3.31
Luteolin 68 [micro]M 45 [+ or -] 2.4* 7.86 [+ or -] 0.51*

[EC.sub.50]: concentration of the agonist ([micro]M) in the presence or
absence of the agonist barium, *p<0.05.
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Author:Fleer, H.; Verspohl, E.J.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:1USA
Date:Jun 1, 2007
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