Choleretic effects of yarrow (Achillea millefolium s.l.) in the isolated perfused rat liver.

Abstract

Different species from the Achillea millefolium aggregate are used against gastrointestinal and hepato-biliary disorders in traditional European medicine. In this work, a fraction enriched in dicaffeoylquinic acids (DCCAs) and luteolin-7-O-[beta]-D-glucuronide was investigated on its choleretic effect in the isolated perfused rat liver (IPRL) compared to cynarin (1,3-DCCA), the main choleretic compound of Cynara scolymus L. A fraction containing 3,4-, 3,5- and 4,5-DCCA and luteolin-7-O-[beta]-D-glucuronide was prepared by solid phase extraction from a 20% methanolic extract of yarrow. A total amount of 48.8% DCCAs and 3.4% luteolin-7-O-[beta]-D-glucuronide was determined by HPLC analysis with cynarin as internal standard. IPRL experiments revealed a dose-dependant increase in bile flow (23-44-47%) by the Achillea fraction. Choleresis was two- to three-fold higher than that of cynarin. The combined effect of DCCAs and luteolin-7-O-[beta]-D-glucuronide stimulated bile flow more effectively than the single compound cynarin. Due to their polar structure, these compounds are quantitatively extracted into teas and tinctures; hence, they seem to be the choleretic active principles in the traditional application forms of yarrow.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Achillea millefolium s.l.; Dicaffeoylquinic acids; Choleresis; Isolated perfused rat liver

Introduction

Different species from the Achillea millefolium aggregate are used in traditional European medicine as infusions and tinctures against gastrointestinal and hepato-biliary disorders due to their antiphlogistic, spasmolytic and antimicrobial properties (Willuhn, 2002; Jurenitsch, 1992). While the antiphlogistic activity is mediated by sesquiterpenes (Kastner et al., 1993; Sosa et al., 2001), flavonoids are described as the antispasmodic compounds (Lemmens-Gruber et al., 2005), and the essential oil and sesquiterpenes exhibit antimicrobial effects (Simic et al., 2002). However, the choleretic principles of yarrow are still unknown.

Extracts from artichoke leaves are widely used in the treatment of hepato-biliary dysfunction and digestive complaints. Mono- and dicaffeoylquinic acids (DCCAs, Fig. 1) and flavonoids as the main phenolic constituents of Cynara scolymus L. are reported to have choleretic, hepatoprotective and cholesterol-lowering effects (Wegener and Fintelmann, 1999). A standardised artichoke extract showed a considerable increase in bile secretion in a randomised placebo-controlled double-blind study (Kirchhoff et al., 1994). Furthermore, in the isolated perfused rat liver (IPRL) cynarin (1,3-DCCA) and other DCCAs besides monocaffeoylquinic acids and flavonoids were reported to be the choleretic active principles in artichoke leaves (Matuschowski, 1998).

[FIGURE 1 OMITTED]

As yarrow is traditionally applied similar to artichoke and contains a considerable amount of DCCAs as well, a fraction enriched in those phenolcarboxylic acids and luteolin-7-O-[beta]-D-glucuronide was prepared and tested in the IPRL in order to examine its effect on bile flow. As a positive control we used cynarin, the main choleretic constituent of C. scolymus L. (Fig. 1).

Material and methods

Plant material

A commercial sample of the aerial parts of yarrow (A. millefolium s.l.) supplied by Aboca, Sansepolcro, Italy (sample 1D5787), was used for the preparation of the plant extract. A voucher specimen (Aboca 1) is deposited at the Department of Pharmacognosy, University of Vienna.

Preparation of the extract

Twenty-five grams of the powdered drug were extracted with 1500 ml 20% (v/v) methanol p.a. for 30 min under reflux, followed by centrifugation. For exhaustive extraction, the residue was treated in the same way for another 20min. Methanol was removed from the combined extracts by evaporation under reduced pressure at 40 [degrees]C. The remaining aqueous solution was lyophilisated and yielded 9.1 g crude extract, giving a drug-extract ratio of 2.7:1.

In order to obtain a fraction enriched in DCCAs, the extract was dissolved in 270 ml 20% (v/v) methanol in an ultrasonic bath for 10 min. Twenty millilitres of the solution were applied to a C18-cartridge (Varian, Mega BE-C18, 10 g, 60 ml), previously conditioned with 2 reservoir volumina (RV) methanol and 2 RV deionised water. After purging with air for 10 min, the extract was eluted at a flow rate of 8ml/min with 4 RV deionised water, followed by 4 RV 20% (v/v) methanol and 4 RV 80% (v/v) methanol, respectively. This procedure was repeated several times in order to fractionate the whole extract. From the combined 20% (v/v) methanol fractions methanol was evaporated under reduced pressure at 40 [degrees]C, and the remaining aqueous solution was lyophilisated, affording 1.3 g of a fraction.

Analysis of the extract

To 4.2 mg of the fraction obtained by solid phase extraction 200 [micro]l of internal standard solution (1.04 mg in 1ml methanol) were added and diluted to 1.00 ml with 20% (v/v) methanol. The solution was centrifuged, and 10 [micro]l were injected repeatedly onto the HPLC column.

Analysis of the fraction was performed by HPLC on a Perkin Elmer Series 200 instrument equipped with a column oven and an UV/VIS detector set at a wavelength of 345 nm. As stationary phase a 5[micro] Hypersil BDS C18 (250 x 4 mm) column was used, the mobile phase consisted of water adjusted to pH 2.8 with acetic acid (A) and acetonitrile (HPLC-grade, Riedel-de Haen, purchased from Sigma-Aldrich) containing 0.8% acetic acid (B). Gradient elution was performed starting with 16% B at 0.1%/min for 20min at a flow rate of 1.2 ml/min and a temperature of 25 [degrees]C (Fig. 2).

Quantification of the DCCAs and the flavonoid luteolin-7-O-[beta]-D-glucuronide was carried out by internal standardisation with cynarin (Sequoia Research Products, Oxford, UK; purity > 96%). As the DCCAs contained in the sample are structural isomers of the internal standard cynarin, we used a calibration factor of 1 for all of them. As luteolin-7-O-[beta]-D-glucuronide is not commercially available, a calibration curve with the internal standard cynarin and luteolin-7-O-[beta]-D-glucoside (Extrasynthese, Genay, France; purity > 98%) instead of the glucuronide was established. We found linear correlation from 0.059 to 0.297 mg/ml with a correlation coefficient of 0.9993. The calibration factor was calculated, taking into account the molecular weight of luteolin-7-O-[beta]-D-glucoside and -glucuronide, as 0.877.

[FIGURE 2 OMITTED]

Liver perfusion

Male Sprague-Dawley rats (body weight: 180-270g; liver weight: 11.4-15.4 g) were purchased from the Department of Animal Research and Genetics of the Medical University of Vienna. Animals were housed in a temperature- and humidity-controlled room under a 12 h light-dark cycle with free access to water and food.

Liver perfusion experiments were carried out as described before (Jager et al., 2003) on animals anaesthetised with phenobarbital according to the protocol approved by the committee of the institution. Before the excision of the liver, glass canulas for the influent perfusate were applied in the portal vein, for the effluent perfusate in the vena cava inferior. For the collection of bile, a canula was inserted into the bile duct.

The excised liver was transferred to a chamber where it was kept constantly at 37 [degrees]C. Perfusion was carried out in a single pass system at a constant flow rate of 35ml/min using freshly prepared Krebs Henseleit bicarbonate-C[O.sub.2] buffer (KHB) pH 7.4 (118 mM NaCl, 4.5 mM KCl, 2.75 mM Ca[Cl.sub.2], 1.19 mM K[H.sub.2]P[O.sub.4], 1.18 mM MgS[O.sub.4] and 25mM NaHC[O.sub.3]). The buffer was equilibrated with 95% [O.sub.2] and 5% C[O.sub.2] during the whole procedure. The perfusion pressure was constantly monitored by measuring the water pressure. Single bile drops falling from the bile duct canula were weighed (approximately 8 mg) in order to determine the bile flow by measuring the time interval between drops. The bile flow rate is expressed in [micro]l/[g.sub.liver]/min.

The basal flow was determined by equilibrium perfusion with pure KHB for 30min. Then 5, 10 and 20 mg of the Achillea fraction were diluted in KHB giving final concentrations of 10, 20 and 40 mg/l. Livers were perfused with those solutions for 10 min each, followed by intervals of 10 min purging with pure KHB. The increase in bile flow was expressed in percentage of the basal value.

Cynarin was used as a positive control. A stock solution of 120mg in 6 ml ethanol was prepared and added to the perfusion medium to give final concentrations of 10, 20 and 40 mg/l. The perfusion procedure was carried out under identical conditions as mentioned above for the Achillea fraction.

Statistics

For statistical analysis, the arithmetic mean and standard error of the mean (SEM) of three individual experiments were calculated. Differences of p < 0.01 were regarded as statistically significant.

Results and discussion

As shown in Fig. 2, HPLC analysis of the fraction prepared by eluting the Achillea extract with 20% (v/v) methanol from C18-cartridges revealed a total amount of 48.8% DCCAs (7.9% 3,4-DCCA, 24.2% 3,5-DCCA and 16.7% 4,5-DCCA) and 3.4% luteolin-7-[beta]-D-O-glucuronide besides other minor compounds that could not be identified because of their small amount in the fraction. Due to its polar structure the flavonoid luteolin-7-[beta]-D-O-glucuronide eluted together with the DCCAs. The remaining flavonoids, present in the crude plant extract, were eluted with 80% (v/v) methanol from the C18-cartridges, whereas chlorogenic acid, also a major compound of the crude extract, was eluted with water, thus not being present in the tested fraction.

As shown previously for the phenolic constituents from artichoke (Matuschowski, 1998), the DCCAs are the main choleretic active principles. In particular, cynarin revealed an increase in bile flow of 20% at a concentration of 5.7 mg and a perfusion rate of 1 ml/[g.sub.liver]/min which corresponds to our results. Monocaffeoylquinic acids contribute to the choleretic activity to a minor extent whereas the flavonoid luteolin-7-[beta]-D-O-glucoside exhibited no significant effect on bile flow which explains that this study focussed on the DCCAs from yarrow.

In the liver perfusion experiments both, the Achillea fraction and cynarin, showed a dose-dependant increase in bile flow in the tested concentration range. Fig. 3 shows the course of bile flow after 30 min equilibrium perfusion with pure KHB followed by application of increasing concentrations (10, 20, 40mg/l; 10 min each) of Achillea fraction with intervals of 10 min purging with pure KHB. A dose-dependant increase in bile flow of 23.1% ([+ or -]6.9), 44.1% ([+ or -]17.2) and 47% ([+ or -]12.2) by the Achillea fraction was observed. Compared to the single compound cynarin, which was applied in the same concentrations, showing an increase in bile flow of 5.1% ([+ or -]2.0), 15.9% ([+ or -]3.6) and 21.6% ([+ or -]8.9), the Achillea fraction exhibited a two- to three-fold higher increase (p < 0.01) in bile flow (Fig. 4).

[FIGURE 3 OMITTED]

Candidates for the observed choleretic effect of the Achillea fraction are the above-mentioned DCCAs, amounting to 48.8% in the tested fraction, due to their structural similarity to the choleretic active DCCAs from C. scolymus L. Since they are not commercially available and difficult to isolate, no experiments with the pure compounds could be carried out. The DCCAs from yarrow seem to act synergistically, thus stimulating bile flow much more effective than the single compound cynarin. The flavonoid luteolin-7-[beta]-D-O-glucuronide being also present in the fraction might also contribute to the choleretic effect.

Due to their polar structure, mono- and DCCAs are extracted almost quantitatively into infusions and tinctures, the traditional application forms of yarrow in traditional European medicine. While the results obtained in this study indicate the choleretic effect of the DCCAs, it is also known that chlorogenic acid exerts an increase in bile flow (Matuschowski, 1998). Since all those phenolcarbonic acids are present in yarrow with a total amount of about 2.5%, we strongly believe that they are the main choleretic active principles in yarrow, thus justifying the traditional application in case of hepato-biliary disorders.

[FIGURE 4 OMITTED]

Moreover, a randomised placebo-controlled double-blind cross-over study revealed an increase in choleresis up to 51.5% after oral intake of a standardised artichoke extract containing 0.38% caffeoylquinic acid derivatives, therefrom 0.16% DCCAs and 0.06% cynarin which corresponds to a daily intake of 7mg caffeoylquinic acids in total and 1.1 mg cynarin, respectively (Kirchhoff et al., 1994). In contrary, we investigated a tea preparation from yarrow and found a caffeoylquinic acid content of 10 mg per cup, therefrom 7.5 mg DCCAs. Considering the recommended dosage of three cups of tea, a daily intake of 22.5 mg DCCAs is reached, thus leading to the conclusion that tea preparations from yarrow might exert a considerable increase in bile flow.

Furthermore, we suggest a standardisation of phyto-pharmaceuticals containing yarrow with regard to the content of DCCAs, the compounds responsible for the choleretic effect. As described above, we established a HPLC method that allows a rapid analysis of the DCCAs by internal standardisation with cynarin.

In conclusion, this is the first time an in vitro choleretic effect of yarrow has been shown. Hence, a choleretic activity can be considered also in humans after oral intake of preparations containing yarrow due to the phenolcarbonic acid content. Taking into account the low risk of side effects and the further beneficial therapeutical effects like spasmolytic and antiphlogistic activity, yarrow preparations might be appropriate especially for long-term treatment of digestive disorders.

References

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B. Benedek (a), N. Geisz (a), W. Jager (b), T. Thalhammer (c), B. Kopp (a,*)

(a) Department of Pharmacognosy, University of Vienna, PharmaCenter Vienna, Althanstrasse 14, A-1090 Vienna, Austria

(b) Department of Clinical Pharmacy and Diagnostics, University of Vienna, PharmaCenter Vienna, Althanstrasse 14, A-1090 Vienna, Austria

(c) Department of Pathophysiology, Medical University of Vienna, Wahringer Gurtel 18-20, A-1090 Vienna, Austria

*Corresponding author. Tel.: + 43 1 42 77 55 255; fax: + 43 1 42 77 95 52.

E-mail address: [e-mail] (B. Kopp).