Effect of flavonoids from various Mediterranean plants on enzymatic activity of intestinal carboxylesterase

Biochimie 86 (2004) 919–925 www.elsevier.com/locate/biochi

Effect of flavonoids from various Mediterranean plants on enzymatic activity of intestinal carboxylesterase P. Stocker a,*, M. Yousfi b, O. Djerridane b, J. Perrier a, R. Amziani b, S. El Boustani c, A. Moulin a a

Institut Méditerranéen de Recherche en Nutrition, Faculté des Sciences de St-Jérôme, Université d’Aix-Marseille, 13397 Marseille cedex 20, France b Laboratoire des Sciences fondamentales, Université Amar Telidji Laghouat, BP37G Laghouat, Algeria c Faculté des Sciences Semlalia, Université Cadi Ayyad, BP 2390, Marrakech, Maroc Received 8 March 2004; accepted 13 September 2004 Available online 01 October 2004

Abstract Flavonol compounds of three Mediterranean plants from the Algerian Atlas used traditionally in Arab folk medicine, Arenaria serpyllifolia, Rhamnus alaternus and Thapsia garganica, were found to inhibit the enzymatic activities of both rat intestine and purified porcine liver carboxylesterase in a concentration-dependent manner. Results indicate that the flavonol compounds from the aerial part of these plants lead to the inactivation of the CE pI = 5.1 with Ki of micromolar range. These results encourage us to perform further biological investigation. © 2004 Elsevier SAS. All rights reserved.

1. Introduction A wide variety of plant-derived compounds, including flavonoid compounds, is present in the human diet or is consumed for medicinal reasons. Modern authorised physicians are increasing their use of pure flavonoids to treat many important common diseases, due to their proven ability to inhibit specific enzymes which include hydrolases, oxidoreductase, DNA synthases, RNA polymerases, lipoxygenase, gluthation S-transferase and a number of digestive enzymes including a-amylase, trypsin and lipase [1–6]. Carboxylesterase (CE) isoenzymes (EC 3.1.1.1) are known to be serine esterases, which are widely distributed, in animal tissue [7]. They are essential enzymes in the catabolism of numerous xenobiotics, such as carboxylesters, thioesters and aromatic amides, and are involved in the detoxication of ester and amide prodrugs [8]. The central biological role of CE appears to be drug and xenobiotic metabolism necessary for the chemopro* Corresponding author. Institut Méditerranéen de Recherche en Nutrition, Faculté des sciences de St-jérôme, Université d’Aix-Marseille, IMRN, service 342, avenue escadrille Normandie Niemen, 13397 Marseille cédex 20, France. Tél. : +33-4-91-28-88-37; Fax : +33-4-91-28-84-40. E-mail address: [email protected] (P. Stocker). 0300-9084/$ - see front matter © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.biochi.2004.09.005

tective functions of proteins in the liver and other front-line tissues. A glycerol-ester hydrolase (GEH) pI = 5.1 was recently purified to homogeneity from the porcine intestinal mucosa, and its complete amino acid sequence was deduced from that of the corresponding cDNA. The GEH and liver CE shared 97% and 98% sequence identity at the amino acid and nucleotide levels, respectively [9]. The pI 5.1 CE isoenzyme was also found in the rat intestinal mucosa [10] and the amino acid sequence of several CE isoenzymes of rat liver are now available [11–15]. The high specificity of intestinal CE towards exogenous ester containing substrate along with the presence of the enzyme in the microsomal and cytosolic fractions [16] suggests that the enzyme may be involved in the cell signal transduction via diacylglycerol and protein kinase [17,18]. It is therefore important to study the catalytic mechanism responsible for CE-induced hydrolysis, which can be accomplished through the use of potent and selective inhibitors. In the present study, we examined the inhibitory effects of phenolic extracts from various Mediterranean plants used traditionally in Arab folk medicine for treatment of digestive system affection, respiratory system troubles, fever, skin disease, rheumatism and other inflammatory diseases, on both intestinal rat and purified porcine liver CE.

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2. Materials and methods 2.1. Tissues and reagents Adult male Sprague–Dawley rats (250 g) were killed by CO2 inhalation. Rat organs were dissected and immediately homogenised in 20 mM Tris/HCL buffer, pH 7.4. The homogenates were centrifuged at 10,000 g for 15 min at 4 °C. Plants were collected from a local herbalist in flowering times between March and May in the area of the Algerian Saharan Atlas (at 40 km at the south of Laghouat). The samples were identified at the Agronomic National Institute of Alger, and the voucher specimens were deposited at the laboratory of the Fundamental Sciences, University of Laghouat. Purified porcine liver CE and all reagents were from Sigma-Aldrich. Standards polyphenol compounds were purchased from Extra Synthese (Lyon-France). 2.2. Extraction and purification of phenolic compounds Freeze-dried plants (aerial parts) were finely powdered. Ten grams of powder were homogenised in 100 ml cold aqueous ethanol (80%). Three successive extractions with ethanol (80%) were carried out at room temperature for 1 h. After removing the alcohol under vacuum at 40 °C, ammonium sulfate (20%) and metaphosphoric acid (2%) were added to the aqueous phase. Pigments were removed by three successive extractions with petroleum ether (2:1, v/v). Phenolic compounds were then extracted three times with ethyl acetate (1:1, v/v). The three organic phases were combined, filtered through Whatman paper to eliminate the aqueous phase, and evaporated to dryness under vacuum at 40 °C. The residue was dissolved in methanol (2 ml). Methanol extracts were filtered before analysis by HPLC. 2.3. Analyses of phenolics The concentration of total phenols in plant extracts was estimated by the Folin–Ciocalteu procedure [19] and expressing results in micrograms per milliliter or molar equivalents (µM) of quercetin, a naturally occurring polyphenol. Gelatin-salt block test described in Ref. [20] was used for detection of tannins. Separation and determination of phenolics were performed by HPLC (waters 2690 connected to a diode array detector waters 996) using a Purosphere RP-18 5 µm (250 mm × 4 mm i.d.; Merck) column. The solvent system used was a gradient of A (water, pH 2.6 made with H3PO4) and B (acetonitrile). The gradient was from 10% to 30% B in 20 min followed by an increase from 30% to 65% B in 10 min. This condition was kept for 1 min before a return to the initial condition. The solvent flow rate was 1 ml min–1, and the separation was performed at 30 °C. The diode array detector was used to record the UV spectra of the different

peaks (spectra to 240 to 600 nm) to identify the different classes of phenolics and test their purity. Chromatogram and spectrum were treated with Millenium software. 2.4. Enzyme and protein assays Enzyme activity determination was performed by titration on tributyrin (30 mM) and methyl butyrate (40 mM) at 37 °C using a Metrohm pH-stat and 0.01 M NaOH as described previously [9]. One unit of enzymatic activity corresponds to 1 µmol of fatty acid released per min. All the activities were measured at pH = 8.0 including that towards p-nitrophenylacetate [21]. 2.5. Peptide synthesis and experiments The peptide KMNLIKLSSQRDNKE, corresponding to the amino acid sequence from position K267-E281 on the rat intestinal CE polypeptide chain, was synthesised and purified by the Marseille CNRS-CIML. The preparation of the polyclonal peptide antibodies and protein concentrations from various tissues were determined by the immunoprecipitation experiments. Immunoblot analyses were carried as described in Ref. [16]. 2.6. Enzyme inhibition assay Determination of Ki values was performed for the CE using p-nitrophenyl acetate as a substrate. Inhibition constants were calculated by assessment of the reduction in the formation of p-nitrophenol, monitored by a spectrophotometric assay at 414 nm, using purified porcine CE and intestine rat extract. Reactions were performed at 37 °C in 96wells plates in a total volume of 250 µl of 20 mM (Tris pH 7.4), and data were recorded at 15 s intervals for up to 5 min. Carboxylesterase specific activity was expressed as µmol/min/mg protein (rat intestine extract) using three concentrations of substrate. Enzyme velocity values versus inhibitor concentrations were plotted, and Ki values were calculated from curve fits using GraphPad Prism software. 3. Results and discussion The phenolic extracts of 15 plants known for their therapeutic properties in traditional Arab medicine (Table 1) were tested on the enzymatic activity of rat intestinal and purified porcine liver CE. The total phenol content of each fraction was estimated by the Folin–Ciocalteu procedure, and the amount of polyphenols in plants was expressed in milligrams per grams of fresh matter. These phenolic extracts were evaluated for their inhibition of CE in two systems, rat intestinal and commercial purified porcine liver CE. 3.1. Inhibition of purified porcine liver CE (in the 4.5–6.5 pI range) The enzyme specific activity performed by titration on tributyrin and methyl butyrate was 30 and 35 µmol/min/mg

P. Stocker et al. / Biochimie 86 (2004) 919–925 Table 1 Medicinal plants commonly used in Arab folk medicine Plant name

Popular use in Arab folk medicine Digestive Anti Other inflammatory *

Caryophyllaceae A. serpyllifolia Rhamnaceae * R. alaternus Apiaceae * T. garganica Lamiaceae T. serpyllum Anacardiaceae P. lentiscus Lamiaceae A. iva Asteraceae Artemisia campestris Lamiaceae * T. polium Globulariaceae Globularia alypum Asteraceae Artemisia herba halba Brassicaceae Oudneya africana Asteraceae Artemisia arboresens Zygophyllaceae Zygophyllum Cornutum coss Cupressaceae Juniperus oxycedrus Asteraceae Anthemis arvensis

*

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the extract. Data from this plot indicate that the inhibition by A. serpyllifolia was competitive with a Ki value of 8 µM (Fig. 1a), and the inhibition of R. alaternus was non competitive with a Ki value of about 24 µM (Fig. 1b). The results obtained for the other phenolic extract were reported in Table 2. Ajuga iva and Thymus serpyllum showed poor inhibition with Ki values of 44 and 130 µM, respectively. Other plants did not show significant inhibition (Ki > 200 µM) or were not inhibitors (Ki > 1000 µM). 3.2. Inhibition of the pI 5.1 rat intestine CE isoenzyme

* * *

*

*

* * * * *

*

*

protein, respectively. The experiments using p-nitrophenylacetate as substrate were performed spectrophotometrically as described in materials and methods with a microplate reader. The CE specific activity was 60 µmol/ min/mg protein. For each extract, the IC50 was estimated from the plot of the inhibition of enzyme activity as a function of phenolics concentration arbitrary expressed in molar equivalents (µM) of quercetin, gallic acid or caffeic acid, according to the phenolic class of the extract. The influence of incubation time on the inhibition of the enzyme activity by polyphenol fractions was tested up to 30 min. Preincubation of the CE with fractions did not significantly cause a time dependent inhibition. Among all plant phenolic extracts, only three (Arenaria serpyllifolia, Thapsia garganica and Rhamnus alaternus) were found to inhibit significantly, in a concentrationdependent manner, the CE activity. The best inhibitor was the phenolic extract of A. serpyllifolia. To investigate the type of enzyme inhibition, we assayed the CE activity in the presence of different concentrations of the substrate PNPA (0.25– 1.0 mM) and polyphenol extracts. The Ki value was obtained from the plot 1/vi = f [I] as described by Dixon [22] where vi is the rate of the enzyme and I the phenolic concentration of

We further examined the inhibiting effect of some extracts on the enzymatic activity of the pI 5.1 rat intestine CE isoenzyme. Its specific activity on p-nitrophenyl acetate was 480 µmol/min/mg protein. According to the previous results, only the best and some bad inhibiting extracts were tested. We observed that the best inhibitor of porcine liver CE inhibited also dramatically rat intestinal CE activity in a concentration-dependent manner, confirming that these extracts were a potent inhibitor on CE. Inhibition kinetics revealed that both A. serpyllifolia and T. garganica extracts inhibited rat intestinal CE in a competitive manner with Ki values of 15 and 17 µM, respectively, showing a large increase in affinity of the inhibitors compared to the normal substrate PNPA (KM = 4.5 mM). This indicated that the binding sites of both inhibitors are different from the PNPA binding pocket. Except R. alaternus which inhibited significantly rat intestinal CE pI = 5.1 with Ki = 34 µM, and Teucrium which are poor inhibitor (Ki = 200 µM), other phenolic extracts did not show any enzyme inhibition effect. In addition, it is interesting to note that the inhibitor to enzyme molar ratio values was roughly 3–5 times higher for the rat (Table 3). There are mainly four groups of isoenzymes of the CE in the 4.5–6.5 pI range in the rat liver [23–26] and as much in the porcine liver [27], whereas only isoenzyme Table 2 Ki values and inhibition type for total polyphenol extracts of Arab folk medicinal plants performed for the commercial porcine CE Name of the plants A. serpyllifolia T. garganica R. alaternus A. iva T. polium P. lentiscus Anthemis arvensis Globularia alypum Artemisia campestris T. serpyllum Artemisia herba halba Oudneya africana Artemisia arboresens Zygophyllum cornutum coss Juniperus oxycedrus

Ki (µM) 8 12 24 44 130 210 500 514 588 >1000 >1000 >1000 >1000 >1000 >1000

Type of inhibition Competitive Competitive Non competitive Non competitive Non competitive Competitive Non competitive Non competitive Competitive Non inhibitor Non inhibitor Non inhibitor Non inhibitor Non inhibitor Non inhibitor

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Fig. 1. 1/vi plots of porcine corboxylesterase according to the total phenol concentration of A. serpyllifolia (a) and R. Alaternus L (b) plants. Activities were determined by formation of PNP by CE at several substrate concentrations: 0.025 mM (S1), 0.5 mM (S2) and 0.1 mM (S3). The graph represents the means of three experiments. Table 3 Ki and inhibitor to enzyme molar ratio values for some total polyphenol extracts obtained for commercial porcine and rat intestinal CE Name of the plants A. serpyllifolia T. garganica R. alaternus T. polium Anthemis arvensis T. serpyllum Juniperus oxycedrus

Ki (µM) porc 8 12 24 130 500 >1000 >1000

I/E (M/M) porc 600 900 1700 10,000 38,000

Ki (µM) rat 15 17 34 200 >1000 >1000 >1000

I/E (M/M) rat 2400 2600 5200 32,000

pI = 5.1 was detected in the intestine [9]. This suggests that the extracts would be specifically inhibiting CE pI = 5.1. We sought to elucidate the relationship between the structure of phenolic compounds and their enzyme inhibition effect. Each extract was analysed by HPLC using a diode array detector for simultaneous recording of chromatographic analysis at several wavelengths. Because there are a significant number of compounds in different phenolic classes, each of which has a different absorption maximum, it is common to quantify each class at the wavelength of its absorption maximum. Flavones and flavonols are flavonoid compounds. Flavones such as luteolin

and flavonols such as quercetin exhibit a maximum at about 340 and 360 nm, respectively [28], and red anthocyanins have a maximum at about 520 nm. Hydroxycinnamic derivatives DHC, such as caffeic acid, have a maximum at about 320 nm and most all phenolics such as hydroxybenzoic derivatives DHB absorb light at 280 nm [29]. The shape of the spectral curve of each chromatographic peak provides information about the type of phenolic compounds. Retention times and spectral characteristics were compared to typical commercial standards, although identification of phenolic compounds is a delicate problem since standards for complex forms are not always available. Seven chromatographic peaks were detected in A. serpyllifolia extract. Two of them showed a UV spectrum characteristic to a hydroxycinnamic derivative (with absorption maxima at 323 and 318 nm), the others showed a flavonol characteristic spectrum (with absorption maxima at 358, 360, 366, 362, and 363 nm). In R. alaternus extract only flavonol derivatives were detected (five peaks with absorption maxima around 360 nm). Two chromatographic peaks with flavonol derivative (absorption maxima at 364 and 365 nm) and one with hydroxycinnamic derivative (maximum at 323 nm) were detected in T. garganica extract. All the peaks detected in these three

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Table 4 Dominant phenolic compounds expressed in molar percentage of each plant, according to the HPLC analysis Name of the plants A. serpyllifolia R. alaternus T. garganica T. serpyllum P. lentiscus A. iva Artemisia campestris T. polium Globularia alypum Artemisia herba halba Oudneya africana Artemisia arboresens Zygophyllum cornutum coss Juniperus oxycedrus Anthemis arvensis

Phenolic content (mg/100 g) 150 600 245 758 5050 250 4623 700 1915 1270 410 573 334 1263 11523

Flavonoids 91 100 98

Dominant phenolic compounds (molar percentage) Hydroxycinamic derivatives Hydroxybenzoic derivatives 9 2 80

20

100 100 100 100 nd

nd nd

extracts did not absorb light at 280 nm, and no red anthocyanins compounds were detected. Moreover, the extracts gave a negative test for tannins which were analysed by gelatin-salt block test. According to the HPLC analysis and the spectral characteristic, the concentration of phenolics of each plant was expressed in percentage (w/w) of caffeic acid equivalent for hydroxycinnamic, of gallic acid for hydroxybenzoic and quercetin for flavonoids (Table 4). Our results highlighted that extracts including flavonoid derivatives show potent CE inhibition activity, while extracts containing only hydroxycinnamic and/or hydroxybenzoic derivatives did not exhibit any inhibitory effect. Although effective compounds remain to be identified, we can conclude that the inhibitory effect could be attributed to the flavonoids alone, although Pistacia lentiscus and Teucrium polium extracts containing only flavone derivatives did not exhibit strong inhibitory activity. But according to the HPLC analysis, flavonol pattern of the inhibiting extracts was not observed in P. lentiscus and T. polium extracts which contained mainly luteolin. The inhibitory effect could be attributed to flavonoids even though some standard such as quercetine, rutin or naringin alone were reported to be inactive. Only few studies are reported on phytochemical investigations of T. garganica and R. alaternus and no report was found on Arenaria species. In T. garganica, polyoxygenated guaianolide (named thapsigargins) and other terpenoids isolated from the plant roots were recognised with various biological activities [30–32]. However, previous studies on the secondary metabolites of the genus Thapsia have shown clear variations between, and also within the species [33]. In this work, we report the investigation of flavonoid derivatives identified in the aerial part of an Algerian Thapsia species which had not been examined before.

100 nd 4 100 nd nd

nd 96 nd nd 100

Rhamnus alaternus is known to be rich with anthraquinone and flavone heterosides [34–36]. Marzouk et al. report the structural elucidation of several phenolic metabolites from the aerial part of an Egyptian Rhamnus species in which flavonoids such as kaempferol and quercetin were identified [37]. In our work, we have tentatively identified flavonoids as a class of compounds present in the plant extracts of T. garganica, R. alaternus and A. serpyllifolia species which showed a potent inhibitory activity of the pI 5.1 CE. This result could be related to the better stability of the active flavonoids when placed in a more favourable chemical environment. In this study, it is difficult to be conclusive about the most active flavonoid compounds and the observed effect of A. serpyllifolia, R. alaternus and T. garganica extracts is probably due to the direct effect of some flavonoid compounds that are maintained in their active form through the presence of other flavonoids with protective structure. Investigating the real contribution of a particular class of compounds present in the extracts to the observed effects would need a particular strategy. Studying the effects of various extracts specifically enriched in one class of compounds could represent an interesting way to achieve this purpose. Indeed, it has been shown in the past that intestinal mucosa pI 5.1 CE showed significant hydrolysing activity towards phorbol ester and diacylglycerol [18] which has often been used as an activator of protein kinase C [38]. This suggests that CE prevent the accumulation of diacylglycerols or other phorbol-like activators and indirectly block the activation of protein kinase C. This would affect the series of cellular responses induced by protein kinase C and enterocyte proliferation. This CE inhibitory properties of flavonoid compounds from A. serpyllifolia, R. alaternus and T. garganica showed a novel interest of these plants in the study of the catalytic mechanism responsible for CE-induced hydrolysis.

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In addition among the 15 quoted species, only these three plants were recognised to treat pathologies of the intestinal tract such as constipation or intestinal pains [39]. In several cases, the uses of theses plants and their respective therapeutic prescription in popular medicine are not easy to understand, and we can put the question if there is a relation between the therapeutic properties of these plants and their inhibiting effects on the CE activity.

References

4. Conclusion

[4]

The diversity and complexity of the phenolic compounds depend on at least two factors: first, the variety of aglycones and the number of glycosides possible, sometimes in acylated form, and second, the possibility of condensation into complex molecules, e.g., condensed tannins. In this work, we studied the effects of phenolic compounds extracted from various plants used in Arab folk medicine on specific intestinal mucosa pI 5.1 CE activity. From the tested plants, we observed that hydroxycinnamic and/or hydroxybenzoic derivatives did not exhibit any inhibitory effect on CE, while some unidentified flavonoids show potent CE inhibition activity. In summary, our data show that the phenolic extracts of three medical plants, A. serpyllifolia, T. garganica and R. alaternus, lead to the inactivation of the CE pI = 5.1 specifically, with Ki of 8, 15 and 24 µM, respectively. The degree of activity was not related to tannin content. The inhibitor to enzyme molar ratio was found to be 2400, 2600 and 5200, respectively. Then the I/E ration from the active compounds should decrease strongly suggesting that they could act as competitor on the active site of the CE. This is of interest, because CEs have showed to hydrolyse phorbol esters which cause various effects in different tissues in different species by activation of protein kinase C [40,41]. Since protein kinase C has been suggested to be involved in carcinogenesis and cell proliferation [38,42], it is important that changes in CE with phorbol-hydrolyzing activity should be clarified. We suggest that flavonoids as those contained in the extracts of A. serpyllifolia, R. alaternus and T. garganica with CE inhibitory properties can regulate the enterocyte cellular expression via biochemical mechanism related to signal transduction. Although the so-called flavonoids remain to be separated and their structure elucidated, although the mechanism of action remains to be elucidated, this work is the first report on the inhibitory activity of the pI = 5.1 CE by flavonoids, which may explain the beneficial folk medical utilisation of these plants for the treatment of some intestinal disorder. These flavonoid compounds remain to be identified and further biological investigation should be done to allow to study their structure–function interactions with the enzyme. Purification and characterisation of the compounds from the active extracts are in process.

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