Search for potential Angiotensin Converting Enzyme (ACE)-inhibitors from plants

Phytomedicine, Vol. 8(1), pp. 47–52 © Urban & Fischer Verlag 2001 http://www.urbanfischer.de/journals/phytomed

Phytomedicine

Search for potential Angiotensin Converting Enzyme (ACE)-inhibitors from plants M.A. Lacaille-Dubois1, U. Franck2 and H. Wagner2 1

Laboratoire de Pharmacognosie, Unité de Molécules d’Intérêt Biologique, Faculté de Pharmacie, Université de Bourgogne, Dijon, France 2 Centre of Pharma Research, Pharmaceutical Biology, University of Munich, Munich, Germany

Summary MeOH extracts, fractions and pure substances from Musanga cecropioides, Cecropia species and Crataegus oxyacantha /C. monogyna were screened by using an in vitro bio-assay based on the inhibition of Angiotensin Converting Enzyme (ACE), as measured from the enzymatic cleavage of the chromophore-fluorophore-labelled substrate dansyltriglycine into dansylglycine and diglycine. Phenolic acids showed no significant ACE-inhibition whereas flavonoids and proanthocyanidins demonstrated inhibitory activity at 0.33 mg/ml using this test system. Key words: Musanga cecropioides, Cecropia species and Crataegus oxyacantha/ C. monogyna, angiotensin converting enzyme, ACE, hypotensive agents, flavonoids, procyanidins

j Introduction Inhibition of Angiotensin I-Converting Enzyme (ACE) is currently considered to be a useful therapeutic approach in the treatment of high blood pressure. Within the enzyme cascade of the renin-angiotensin system, ACE removes histidyl-leucine from angiotensin I to form the physiologically active octapeptide angiotensin II, one of the most potent vasoconstrictors known. Angiotensin II also stimulates the synthesis and release of aldosterone from the adrenal cortex, which increases blood pressure by promoting sodium retention (and thereby water retention) in the distal tubules. Inhibition of ACE will be an effective screening method in the search for new antihypertensive agents (Wagner, 1993, Hansen et al., 1996a, 1996b, Somanadhan et al., 1999). By using an in vitro ACE-inhibition assay (Elbl and Wagner, 1991; Wagner and Elbl, 1992) for a bioguided fractionation of drugs with known antihypertensive potential, we isolated some procyanidins and flavonoids from Lespedezae capitata, Cistus clusii, and Amelanchier ovalis, with

ACE-inhibiting activities. Test compond concentrations from 0.14 to 0.33 mg/ml showed between 37 to 66% inhibition (Wagner et al., 1991). In continuation of our systematic search for antihypertensive principles in higher plants, we have screened various plant extracts and isolated compounds for ACE-inhibitory activity, using a purified enzyme from rabbit lung and dansyltriglycine as substrate (Elbl and Wagner, 1991; Hansen et al., 1995). Plant species used as diuretic, cardiotonic and antihypertensive remedies in the folk medicine in Central and South America (Cecropia species, Cecropiaceae/Moraceae) and Africa (Musanga cecropioides, Moraceae) were selected (Franck, 1998). A Crataegus extract was also chosen in this screening since it was shown to be active in cardiac insufficiency patients in comparison with captopril (as a reference ACE-inhibitor) (Tauchert et al., 1994). Furthermore ACE-inhibitory fractions were found in Crataegus fruits (Inokuchi et al., 1984). 0944-7113/01/08/01-047 $ 15.00/0

48

M. A. Lacaille-Dubois, U. Franck and H. Wagner

j Material and Methods Plant Material

Leaves of Musanga cecropioides R. Brown were collected in the North West and Central Province of Cameroon. They were provided by Dr. Kamanyi (University of Yaounde, Cameroon) and identified by Dr. G. Achundong of the National Herbarium at Obili Yaounde. Voucher specimens of the material are deposited in the National Herbarium of Yaounde. Leaves of Cecropia hololeuca Miq., C. pachystachya Tréc., C. glaziovii Sneth. were collected close to Belo Horizonte (Brazil) and were provided by Pr. A. Bragha de Oliveira and Dr. C.L. Zani, Laboratory of Chemistry of Natural Products, Centre «René Rachou» Fiocruz, Belo Horizonte (Brazil). Voucher specimens were deposited in the Herbarium of this Institute. The MeOH extract was prepared from the flowers and leaves of Crataegus oxyacantha/ monogyna and provided by the company Lichtwer (Berlin) under the batch number 461457. Reference Compounds

Isovitexin, orientin (Extrasynthese, Genay, France), (+)catechin, chlorogenic acid, (–)-epicatechin (Roth, Karlsruhe), Isoorientin, protocatechic acid, isoquercitrin (Institute of Pharmaceutical Biology, Munich). Instruments and General Methods

TLC was performed on silica gel 60 F 254 (Merck). 1 H- and 13C NMR-spectra were recorded on an AM 360 and a DRX 500 (Bruker) instrument at 24 °C and on a GSX 400 Jeol instrument. Mass spectra were obtained using a MS 80 RFA (Kratos) and a HSQ 30, Finnigan MAT instruments. HPLC analytical scale: HP 1090 A liquid chromatograph with photodiode array detection (Hewlett Packard, Walbronn). Column: Hibar column, 125 × 4 mm ID, LiChrospher 100 RP-18 (5 µm) with RP-18 precolumn (Merck). Eluent for the separation of the MeOH extracts of M. cecropioides, C. pachystachya and Crataegus-extract: Linear gradient from 10 to 30% MeCN-H2O with 1% 0.1 N H3PO4 in 0–30 min, flow rate: 1 ml/ min; detection wavelength 210 nm. Eluent for the separation of the MeOH extracts of C. hololeuca and C. glaziovii: 5 to 20% MeCN-H2O with 1% 0.1 N H3PO4 in 0–20 min, 20 to 40% MeCN-H2O with 1% 0.1 N H3PO4 in 20–35 min, 40 to 95% MeCNH2O with 1% 0.1 N H3PO4 in 35–37 min, 95% MeCNH2O with 1% 0.1 N H3PO4 isocratic in 35–40 min, flow rate: 1 ml/ min; detection wavelength: 210 nm. HPLC semi preparative scale : L-6200 A Intelligent Pump Merck-Hitachi (Merck) with a Schoeffel SF 770 Spectrophotometer (Schoeffel) and a Perkin Elmer 561 recorder (Perkin Elmer). Hibar RT column,

250 × 4 mm ID, LiChrosorb RP-18 with a RP-18 precolumn (Merck) and Hibar LiChrosorb RP-18 (7 µm), 250 × 10 mm ID, with a RP-18 precolumn (Merck). Eluent for the isolation of the proanthocyanidin C1: linear gradient from 10 to 50% MeCN-H2O in 0–20 min, 50% MeCN isocratic in 20–27 min; detection wavelength: 200 nm. Extraction and Fractionation

• Musanga cecropioides R. Brown: Powdered leaves (215 g) were defatted by n-hexane (2l) in a soxhlet and then extracted with MeOH (2l). After elimination of the solvent under reduced pressure (T < 40 °C), the residue was suspended in water and heated at 60 °C for 60 min. The chlorophyll was removed by filtration and extraction with n-hexane. The water phase was extracted with EtOAc (6 × 200 ml). After removing the solvent in vacuo (T < 40 °C), the EtOAc fraction (3.5 g) was fractionated over Sephadex LH 20 using EtOAc/CH3CH2OH/H2O (2:1:2) as an eluent. Seventeen fractions were obtained and analysed by HPLC/ UV. By comparison with reference compounds in terms of retention time and UV spectra (recorded on line by the photodiode array detection), the presence of protocatechic acid, chlorogenic acid, catechin, epicatechin, isovitexin, orientin and isoorientin was indicated. Procyanidin B2 (31 mg) and procyanidin C1 (30 mg) were isolated as pure compounds by successive chromatography on Sep-Pak and preparative HPLC (eluent: gradient of MeCN-H2O) and were identified by extensive NMR spectral data as (-)-epicatechin(4β→8)-(–)-epicatechin and (–)-epicatechin-(4β→8)(–)-epicatechin (4β→8)-(–)-epicatechin (Franck, 1998, Frank et al., 1999). • Cecropia hololeuca Miq: 6.3 g residue obtained from concentrating an ethanolic extract were suspended in water and heated at 60 °C for 60 min. The chlorophyll was eliminated by filtration and further by liquid-liquid extraction with hexane. Liquid-liquid extraction with CHCl3-MeCN-hexane-water (1:3.4:2:1) and further partitions with EtOAc (5 × 100 ml) yielded the EtOAc fraction. After evaporation of the solvent under reduced pressure, the residue (139.5 mg) was fractionated over Sephadex LH 20 using EtOAc/CH3CH2OH/ H2O (2:1:2) as an eluent. Five fractions which were obtained and analysed by HPLC/UV. The constituents were identified by comparison with authentic samples in term of retention times and UV spectra as protocatechic acid and chlorogenic acid in Fr. 1, epicatechin in Fr. 2, orientin and isoorientin in Fr. 3. Procyanidin B2 in Fr. 4 and procyanidin C1 in Fr. 5 were characterized by co-TLC with authentic samples. • Cecropia pachystachya Tréc: 250 g of the powdered leaves were extracted and fractionated according to the procedure described for Musanga cecropioides yield-

Search for potential Angiotensin Converting Enzyme-inhibitors ing 6.0 g of an EtOAc fraction. This extract, after precipitation and recristallisation of isoorientin (3.6 mg) was fractionated over Sephadex LH 20 using EtOAcEtOH-H2O (2:1:2) as an eluent. Eight fractions were obtained and analysed by HPLC/UV. The constituents were identified by comparison with authentic samples in term of retention times and UV spectra as protocatechic acid, chlorogenic acid in Fr. 2, catechin, epicatechin, isovitexin and isoquercitrin in Fr. 3. Furthermore, isoorientin was characterized by FABMS and 1H NMR spectroscopy. • Cecropia glaziovii Sneth: 14.7 g powdered stipules were extracted in a soxhlet by MeOH (100 ml). After removing the solvent under reduced pressure, the MeOH extract (1.1 g) was fractionated over Sephadex LH 20 using EtOAc/CH3CH2OH/ H2O (2:1:2) as an eluent. Six fractions were obtained and analysed by HPLC/UV. In the Fr. 2 catechin, epicatechin, and isoquercitrin were characterized by comparison with authentic samples in term of retention times and UV spectra. The presence of isoorientin, epicatechin and the procyanidin B2 was indicated in Fr.3. Procyanidin C1 was the main constituent of the Fr. 4. A methanolic extract was obtained from the leaves (12.7 g) and analysed by coTLC with authentic samples and by HPLC/UV. The presence of chlorogenic acid, catechin, epicatechin, isoorientin, procyanidin B2 and procyanidin C1 was indicated. • Crataegus-extract: An EtOAc fraction (9.8 g) was obtained from a MeOH extract (51.5 g) of Crataegus oxyacanta/ monogyna from which a portion (0.85 g) was fractionated over polyamide. Elution with 99% CH3CH2OH yielded flavonoids. Then, a mixture of CH3CH2OH/(CH3)2CO/H2O (80:14:6) eluted the last flavonoids and dimers of procyanidins. A solution of

49

(CH3)2CO/H2O (70:30) afforded a mixture of oligomeric procyanidins (62 mg). The polymeric compounds were finally eluted by DMF. The high polymers were retained on the column. ACE-Inhibition Test

The in vitro ACE-inhibitory activity was measured according to the method described by Elbl and Wagner (1991), which was later modified by Hansen et al. (1995). The chromophore- and fluorophore-labelled tripeptide dansyltriglycine is used as substrate, which is cleaved by the enzyme into dansylglycine and the peptide fragment diglycine. In presence of a specific

50

M. A. Lacaille-Dubois, U. Franck and H. Wagner

inhibitor, the product formation is partially or totally inhibited. For these studies, a commercially available angiotensin-converting-enzyme preparation from rabbit lung (EC 3.4.15.1-purchased from Sigma) has been used. The ACE solution (25 µl) was pre-incubated into microtitre plates with a test or control solution (25 µl) for 5 min at 37 °C. The enzyme reaction was started by adding a combined solution (25µl) of the substrate dansyltriglycin, and the internal standard, dansyl-Lglutamine. At the end of the incubation time (30–35 min at 37 °C), the reaction was stopped by addition of 0.1 N Na2EDTA (50 µl). The product (dansylglycine) and unreacted substrate are separated and quantified by reversed phase HPLC with UV detection at 250 nm. Instrumentation: Merck Hitachi L 6200 Intelligent Pump; Merck Hitachi AS2000 Autosampler; Lambdamax 481 LC spectrometer (Waters-Millipore); Merck Hitachi D-2500 A integrator; solvent system: Column 120 × 5.8 mm ID packed with Spherisorb S5 ODS; mobile phase: 10 mM Na2PO4 buffer (pH 7)-acetonitrile (88:12), isocratic; flow rate: 2 ml/min; detection wavelength: 250 nm. Using this isocratic elution system, the total separation is achieved within 7 min. The decreased concentration of dansylglycine in the test reaction compared with the control reaction was expressed as percentage inhibition and calculated from the equation: {dansylglycine} T Inhibition (%) = 100 – ––––––––––––––––– × 100 {dansylglycine} C where T = test reaction and C = control reaction. Determination of the Proanthocyanidin Content

According to the procedure of Hiermann et al. (1986), the powdered drug (b = 1g) was stirred at room temperature with acetone-H2O (7:3) for 15 min (4 × 15 ml). After filtration the mixture was diluted to 100 ml with 70% acetone. A portion of this solution (10 ml) was evaporated to dryness at room temperature and nBuOH-HCl 37% (95:5) was added. The mixture was heated under reflux for 2 hr. After cooling, the mixture was diluted to 100 ml and the extinction E was spectrophotometrically measured at 540 nm. The content of total procyanidins (TP%) was estimated as (TP%) = E x 4.12 / b.

j Results and Discussion The screening results of several plant methanolic extracts can be summarized as follows (see Fig. 1): A complete inhibition of the enzyme activity (100%) was obtained with the methanolic extract of Musanga ce-

cropioides leaves and a very good ACE-inhibition of 91 ± 9% (s.d.) was obtained with the methanolic extract of Cecropia glaziovii stipules at a final concentration of 0.33 mg/ml. The other extracts of Crataegus aerial parts, Cecropia hololeuca leaves, C. hololeuca bark, C. pachystachyae leaves showed a moderate activity between 33 ± 3% (C. hololeuca bark) and 42 ± 2% (C. pachystachyae leaves) at a concentration of 0.33 mg/ml. All these four extracts showed a content in procyanidins of about 11%, whereas a higher content of 13% was determined in Musanga cecropioides leaves. Since this extract was the most active, it seems as if the content in procyanidins could be partly responsible for the ACE-inhibitory activity. The ethanolic extract of C. hololeuca leaves showed an ACE-inhibition of 40 ± 4% at a concentration of 0.33 mg/ml. Through a bioguided fractionation it was possible to isolate and identify the flavone-C-glycosides orientin and isoorientin, (+)-catechin, (–)-epicatechin and two (-)-epicatechin derived oligomeric procyanidins, chlorogenic acid and other phenolic acids. Of the substances tested, isoorientin and orientin showed an inhibition of 48 ± 1% and 20 ± 2% at a concentration of 0.33 mg/ml, respectively. The most effective fraction contained mostly procyanidins with an inhibition of 94 ± 4% at a concentration of 0.33 mg/ml. The similar composition of Cecropia hololeuca and Crataegus monogyna/ C. oxyacantha suggested a similar ACE-inhibitory activity of both extracts. The Crataegus extract inhibited the enzyme by 33 ± 2% at a concentration of 0.33 mg/ml and was a little less active than the Cecropia hololeuca leaves extract (40%). The partition of the MeOH extract yielded an EtOAc extract which was separated by CC on polyamide into 3 fractions of increasing ACE-inhibitory activity: Phenolic acids and flavonoids (3–27% inhibition, flavane3-ols (15–30% inhibition), oligomeric procyanidins with low polymerisation degree (92% inhibition). The methanolic extract of the leaves of Musanga cecropioides showed an ACE-inhibition of 100% at 0.33 mg/ ml. A bioguided fractionation led to 15 fractions which showed an ACE-inhibitory activity of 22 to 57%. The most effective fraction giving an inhibition of 57 ± 1% contained chlorogenic acid, flavane-3ols (+)-catechin and (–)-epicatechin, the flavone isovitexin and the flavonol isoquercitrin. The second most effective fraction giving an inhibition of 53 ± 5% contained the procyanidins B2 and C1 and other oligomeric procyanidins with low polymerisation degree. The results of the bio-assay on the isolated compounds such as chlorogenic acid showed a low activity, whereas flavonoids and procyanidins showed inhibitory activities dependent on the type of structure (Table 1). Among the flavonoids, the C-glycosides were more active than the O-glycosides. Among the C-glyco-

Search for potential Angiotensin Converting Enzyme-inhibitors

51

Fig. 1. ACE-Inhibition of tested methanolic extracts.

sides, isovitexin and isoorientin showed about the same activity (46 ± 1% and 48 ± 1%, respectively) and were more active than their 8C-analogs, vitexin and orientin (21 ± 1% and 20 ± 2%, respectively). According to the results of Elbl and Wagner, 1991, the in vitro activity of flavonoids and procyanidins is due to the generation of chelate complexes with the

Table 1. ACE inhibitory activity of isolated compounds (tested concentration: 0.33 mg/ml) Compound

Inhibition,% ± s.d.

Chlorogenic acid Isoquercitrin Vitexin Isovitexin Orientin Isoorientin (+)-Catechin (–)-Epicatechin Procyanidin B2 Procyanidin C1

4±1 32 ± 2 21 ± 1 46 ± 1 20 ± 2 48 ± 1 16 ± 3 34 ± 1 25 ± 5 45 ± 2

The values represents means of two different experiments under standard assay conditions described in the text.

zinc atom within the active centre of the angiotensin I-converting enzyme. This chelate could be done between an heterocyclic oxygen and a phenolic hydroxyl group in its vicinity. The above results showed that the 8-C glycosylation is not favourable for the formation of a complex with the Zinc atom of the active centre of the enzyme. Among the flavan-3-ols, (–)-epicatechin was with an inhibition of 34 ± 1% twice more active than its isomer (+)-catechin (16 ± 3%). Among the proanthocyanidins, the inhibition mechanism could also be due to the complexation of the zinc atom in ACE. This was demonstrated by Wagner et al., 1991 with an Alchemy Molecular Modelling programm, which was used to calculate three-dimensional models of the energy-minimised structures of two dimeric C4/C6 and C4/C8 linked procyanidins. The results showed that the two compounds contain a putative zinc-chelating heterocyclic oxygen atom in the “upper” part of the molecule, which is positioned in a way favouring interaction with a phenolic hydroxyl group in the “lower” part of the molecule. From literature, it was assumed that the activity of the oligomeric compounds increased with the polymerisation degree. Our results were in good agreement with this conclusion. The trimeric procyanidin C1 was more active than the

52

M. A. Lacaille-Dubois, U. Franck and H. Wagner

dimeric procyanidin B2 (45 ± 2% and 25 ± 5% inhibition, respectively). A synergistic effect of flavones and procyanidins in the Musanga extract such as in Cecropiae species and Crataegus could be suggested, because none of the isolated compounds of the effective fractions reached the activity of the total fractions. ACE-inhibiting activity was previously reported for isolated compounds such as vitexin, isovitexin and (+)-catechin (Arisawa et al., 1989), isoquercitrin (Kameda et al., 1987) and procanidin B2 (Wagner et al., 1991). Orientin, isoorientin, (-)- epicatechin, and procyanidin C1 which are reported here for the first time with an ACE-inhibitory activity, need further investigations in order to establish dose-response relationship. Since a tetrameric proanthocyanidin glycoside (rats, 3 mg/kg, i.v.) (Terencio et al., 1991) showed in vivo the same ACE-inhibitory activity as captopril (0.02 mg/ kg, i.v.), further in vivo tests will have to be carried out in order to demonstrate possible therapeutic relevance of drugs containing this class of compounds. Acknowledgments

The authors are most grateful to Dr. Kamanyi and Dr. G. Achundong, (University of Yaounde, Cameroon) for supplying and identifying Musanga cecropioides from Africa. We are indebted to Pr. A. Bragha de Oliveira and Dr. C.L. Zani, University of Belo Horizonte, for supplying Cecropia species and extracts from Brazil. We also thank the company Lichtwer (Berlin) for supplying the Crataegus extract.

j References Arisawa, M., Morinaga, Y., Nishi, Y., Ueno, H., Suzuki, S., Hayashi, T., Shimizu, M., Yoshizaki, M., Morita, N., Berganza, L.H.: Chemical and pharmaceutical studies on medicinal plants in Paraguay. Constituents of angiotensin converting enzyme inhibitory fraction from “Cocu“, Allophylus edulis Radlk. Shoyakugaku Zasshi 43: 78–80, 1989. Elbl, G., Wagner, H.: A new method for the in vitro screening of inhibitors of angiotensin-converting enzyme (ACE), using the chromophore- and florophore-labelled substrate, dansyl-triglycine. Planta Med. 57: 137–141, 1991. Franck U.: Phytochemische und pharmakologische Untersuchungen der kardiovaskulären Wirkprinzipien von Cecropia hololeuca Miq., C. pachstachya Tréc., Cecropia glaziovii Sneth., Musanga cecropioides R. Brown und Crataegus L. monogyna / oxyacantha. PhD thesis, University of Munich, Germany, 1998. Franck, U., Neszmelyi, A., Wagner, H.: 2D-NMR structure elucidation of a procyanidin from Musanga cecropioides. ACH-Models in Chemistry 136: 511–517, 1999.

Hansen, K., Adsersen, A., Brogger Christensen, B., Rosendal jensen, S., Nyman, U., Wagner Smitt U.: Isolation of angiotensin converting enzyme (ACE) inhibitor from Olea europea and Olea lancea. Phytomedicine 2: 319–325, 1996a. Hansen, K., Adsersen, A., Smitt, U.W., Nyman, U., Christensen, S.B., Schwartner, C.; Wagner, H.: Angiotensin Converting Enzyme (ACE) inhibitory flavonoids from Erythroxylum laurifolium. Phytomedicine 2: 313–317, 1996b. Hansen, K., Nyman, U., Wagner Smitt, U., Adersen, A., Gudiksen, L., Rajasekharan, S., Pushpangadan, P.: in vitro screening of traditional medicines for anti-hypertensive effect based on inhibition of the angiotensin converting enzyme (ACE). J. Ethnopharmacol. 48: 43–51, 1995. Hiermann, A., Karting, T., Azzam, S.: Ein Beitrag zur quantitativen Bestimmung der Procyanidine in Crataegus. Sci. Pharm. 54: 331–337, 1986. Inokuchi, J.-K., Okabe, H., Yamauchi, T., Nagamatsu, A.: Inhibitors of angiotensin converting enzyme in crude drugs.I. Chem. Pharm. Bull. 32: 3615–3619, 1984. Kameda, K., Takaku, T., Okuda, H., Kimura, Y.: Inhibitory effects of various flavonoids isolated from leaves of persimmon on angiotensin-converting enzyme activity. J. Nat. Prod. 50: 680–683, 1987. Somanadhan B., Varughese, G., Palpu, P., Sreedharan, R., Gudiken, L., Wagner Smitt, U., Nyman, U.: An ethnopharmacological survey for potential angiotensin converting enzyme inhibitors from Indian medicinal plants. J. Ethnopharmacol. 65: 103–112, 1996. Tauchert, M., Ploch, M., Hübner, W.D.: Wirksamkeit des Weissdorn-Extractes LI 132 im Vergleich mit CaptoprilMultizentrische Doppelblindstudie bei 132 Patienten mit Herzinsuffizienz im Stadium II nach NYHA. Münch. Med. Wschr. 136 (Suppl. 1): 27, 1994. Terencio, M.C., Sanz, M.J., Paya, M. J.: Antihypertensive action of a procyanidin glycoside from Rhamnus lycioides. J. Ethnopharmacol. 31: 109–114, 1991. Wagner, H., Elbl, G., Lotter, H., Guinea, M.: Evaluation of natural products as inhibitors of agiotensin I-converting enzyme (ACE). Pharm. Pharmacol. Letters 1 : 15–18, 1991. Wagner, H., Elbl, G.: ACE-inhibitory procyanidins from Lespedeza capitata. Planta Med. 58: 297, 1992. Wagner, H.: Leading structures of plant origin for drug development. J. Ethnopharmacol. 38: 105–112, 1993.

j Address M.A. Lacaille-Dubois, Laboratoire de Pharmacognosie, Unité de Molécules d’Intérêt Biologique, Faculté de Pharmacie, Université de Bourgogne, BP 87900, 21079 Dijon Cedex, France Tel.: 0033-3-80 39 32 29; Fax: 0033-3-80 39 33 00; E-mail: [email protected]