In vitro relaxant and spasmolytic effects of constituents from Viburnum prunifolium and HPLC quantification of the bioactive isolated iridoids

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Abstract

Ethnopharmacological relevance

Viburnum prunifolium is a North America shrub used in ethnomedicine because of its spasmolytic, sedative, and anti-asthmatic properties.

Aim of the study

Contrasting results were reported in past literature about the active principles of this plant. Our aim was to clarify this matter by evaluating the relaxant and spasmolytic activities of the main constituents obtained from the drug.

Materials and methods

The pharmacological assays were carried out on rabbit jejunum spontaneous contractions and on guinea-pig carbachol-precontracted trachea.

Results

Cumulative concentration (1–100 μg/ml) of Viburnum prunifolium methanolic extract (MeOH extract), its purified fractions soluble in ethylacetate (EtOAc fraction) and in n-butanol (BuOH fraction), and the iridoid glucosides (2 × 10−5 to 4 × 10−4 M): 2′-O-acetyldihydropenstemide (1), 2′-O-trans-p-coumaroyldihydropenstemide (2), 2′-O-acetylpatrinoside (3), and patrinoside (4), isolated from EtOAc fraction (1 and 2) and BuOH fraction (3 and 4), induced both relaxant effect of rabbit jejunum spontaneous contractions and spasmolytic effect on guinea-pig carbachol (5.5 × 10−7 M)-precontracted trachea. Propranolol (10−6 M) antagonised all Viburnum prunifolium tested components relaxant and spasmolytic effects. At non-relaxing concentrations (0.5 μg/ml), MeOH extract and its fractions induced a potentiating effect of isoprenaline cumulative concentrations also in both isolated tissues.

Conclusion

In both tissues, the order of potency was EtOAc fraction > BuOH fraction > MeOH extract and 1 > 2 > 3 > 4 suggesting that the major iridoids of EtOAc fraction may be considered among the most active compounds.

HPLC analysis of the bioactive iridoids indicates that 1 and 2 are present for 7.38% and 14.90% in EtOAc fraction, and 3 and 4 for 18.47% and 8.86% in BuOH fraction. By comparing the values of EC50 of the fractions and compounds isolated from them, we may assume that the iridoids play a significant role in the biological activity of the corresponding fractions.

Introduction

The root and stem barks of Viburnum prunifolium L. (Caprifoliaceae), or Black Haw, a deciduous shrub of North America, are well known in folk medicine for their spasmolytic, sedative, and anti-asthmatic properties (Jarboe et al., 1966, British Herbal Pharmacopoeia, 1983, American Herbal Pharmacopoeia, 2000). Furthermore, as well as many other Viburnum species, Viburnum prunifolium is popularly used in the treatment of menstrual cramps, as an anti-abortive agent and to prevent postpartum bleeding (Osol and Farrar, 1955, Bernal and Correa, 1990, Calle et al., 1999). An aqueous infusion or decoction of the bark of roots and stems is generally used (American Herbal Pharmacopoeia, 2000).

Despite the continued use in folk medicine, contrasting results on the pharmacological properties of the plant have been reported in previous literature (Benigni et al., 1962), perhaps due to a different preparation of the extract (e.g. ethanolic extraction or aqueous decoction), various drug administrations (oral and intravenous), or to common inert adulterant in Viburnum prunifolium for biological assays (American Herbal Pharmacopoeia, 2000). Apart from a few compounds identified as being possibly responsible for the properties of Viburnum prunifolium (Mariott and Munch, 1946, Grote and Woods, 1947, Hörhammer et al., 1966, Hörhammer et al., 1967, Jarboe et al., 1966, Ojewole, 1984), several unidentified constituents showing spasmolytic activity on different isolated smooth muscles were reported (Jarboe et al., 1967). As a consequence of this situation, Black Haw is still largely unrecognized by the public and conventional medical professionals and it is absent from modern pharmacopoeia with the exception of British Herbal Pharmacopoeia (1983) and American Herbal Pharmacopoeia and Therapeutic Compendium (2000).

In the last decade, we have isolated four iridoid glucosides from Viburnum prunifolium (Fig. 1), all characterized by Valeriana-type skeleton: 2′-O-acetyldihydropenstemide (1), 2′-O-trans-p-coumaroyldihydropenstemide (2), 2′-O-acetylpatrinoside (3), and patrinoside (4). These iridoids were more easily obtained by splitting the total methanolic extract into two fractions: in fact, compounds 1 and 2 predominate in the less polar EtOAc fraction, and 3 and 4 in the fraction of medium polarity, BuOH fraction (Tomassini et al., 1999).

In a study of the same period, these two fractions proved to be active on the non-pregnant rat uterus, showing a concentration-dependent relaxant effect of spontaneous contractions (Tomassini et al., 1998). Moreover, some evidence obtained from the related species Viburnum tinus suggests that its iridoid glucosides could account for the spasmolytic and sedative activities observed for the methanolic extract of this plant (Cometa et al., 1998a).

On the basis of these considerations, the first aim of the present study was to investigate in suitable biological systems the pharmacological properties of Viburnum prunifolium in order to support the wide use of this plant in ethnomedicine.

Considering the well-known biological activities of iridoids and our preliminary experience with Viburnum species on different muscle isolated preparations, the second aim was to characterize relaxant and spasmolytic activities of these compounds through the study of the mechanism of action and the relative contribution to Viburnum prunifolium total extract activity.

In the light of the ethnobotanical use of this plant, we tested the relaxant and spasmolytic activities of the methanolic extract from the stem bark of Viburnum prunifolium, its EtOAc and n-BuOH purified fractions, and their major iridoidic compounds on isolated rabbit jejunum and guinea-pig trachea. Furthermore, in order to obtain clear information regarding the iridoidic contribution to the extract activity, an HPLC analysis of bioactive iridoids has been included.

Section snippets

Plant material, extraction and preparation of fractions

The stem bark of cultivated Viburnum prunifolium was supplied by the company A. Minardi (Bagnacavallo, Italy), which vouches for the plant identity. The plant material was also identified by the authors in the Department of Plant Biology of Sapienza University of Rome”, where a sample specimen is deposited in Herbarium Cesati (Herbarium Horti Romani—RO 2988). All solvents and reagents were purchased from Carlo Erba Reagenti (Milan, Italy).

Dried Viburnum prunifolium stem bark (300 g) was

Phytochemical analysis by HPLC

Better chromatographic profiles for iridoidic components were obtained by recording the absorbances at 210 and 310 nm. In fact, except for compound 2, which shows an absorbance maximum at 310 nm, the iridoids isolated from the extract are devoid of conjugated double-bonds and do not present any absorbance maximum at a wavelength greater than 210 nm. Due to this feature, eluents with an adequate cut-off have been used (see Section 2.3.3).

The single iridoids were identified through their retention

Discussion

Our in vitro findings extend our previous studies on Viburnum properties (Cometa et al., 1998a, Tomassini et al., 1998, Tomassini et al., 1999) and significantly expand the pharmacological evidence on Viburnum prunifolium in suitable biological systems.

We now show that Viburnum prunifolium components produce relaxant effects of rabbit jejunum spontaneous contractions and guinea-pig precontracted trachea, supporting the wide application of the plant in traditional medicine (American Herbal

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