Antolini, L

Barlocco, D

Brunetti, T

Costantino, L

El Abady, S. a.

Giannessi, F

Lins, Ap

Pessotto, P

Pirisino, Renato Giovanni Paolo

Raimondi, L

English

Pirisino, RENATO GIOVANNI PAOLO

Florence Research

Isolation and pharmacological activities of the Tecoma stansalkaloidsLuca Costantino a,*, Laura Raimondi b, Renato Pirisino b, Tiziana Brunetti c,Pompeo Pessotto c, Fabio Giannessi c, Arlete Paulino Lins a, Daniela Barlocco d,Luciano Antolini e, Samia A. El-Abady fa Dipartimento di Scienze Farmaceutiche, Universita` di Modena e Reggio Emilia, Via Campi 183, I-41100 Modena, Italyb Dipartimento di Farmacologia Preclinica e Clinica, Universita` di Firenze, Viale Pieraccini 6, I-50134 Firenze, Italyc Departments of Chemical and Endocrinology and Metabolism Research, Sigma-Tau Pharmaceutical Industries S.p.A., Pomezia (Rome), Italyd Istituto Chimico Farmaceutico e Tossicologico, Viale Abruzzi 42, 20131 Milan, Italye Dipartimento di Chimica, Universita` di Modena e Reggio Emilia, Via Campi 183, I-41100 Modena, Italyf Department of Chemistry, University College for Girls, Ain Shams University, Heliopolis, Cairo, EgyptReceived 22 November 2002; accepted 4 February 2003Il Farmaco 58 (2003) 781/785www.elsevier.com/locate/farmacAbstractTecoma stans is a plant traditionally used in Mexico for the control of diabetes. Amongst the alkaloids isolated from the plantharvested in Egypt, Tecomine was shown to be one of the compounds responsible for the hypoglycemic action. Given the interest insubstances able to treat type II diabetes, we isolated the main alkaloids present in the plant growing in Egypt and Brazil and testedthem in vivo on db/db mice. Contrary to previous literature reports on different animal models, Tecomine was unable to modifyglycemia; the only effect seen being a decrease in plasma cholesterol levels. On the contrary, when tested in vitro on glucose uptakein white adipocytes, the compound showed a marked effect. The two other alkaloids isolated, namely 5b-Hydroxyskitanthine, earlycalled Base C, and Boschniakine were inactive both in vivo and in vitro assays.# 2003 E´ditions scientifiques et me´dicales Elsevier SAS. All rights reserved.Keywords: Tecoma stans ; Alkaloids; Hypoglycaemic activity1. IntroductionTecoma stans , a bush common to Latin America, is aplant traditionally used in Mexico for the control ofdiabetes. According to these folk reports, it has beenshown recently that the plant extract is able to reducethe area under glucose tolerance curve in normal rabbits[1]. Moreover, intravenous administration of Tecomastans leaves infusion in normal dogs was shown toproduce an early hyperglycemic response followed by aslow decrease of the glucose blood values with aconcomitant hypertriglyceridemia; no importantchanges in insuline levels were noted [2]. It is knownthat this plant contains monoterpene alkaloids, and twoof these are apparently responsible for the hypoglycae-mic activity of the plant. In fact, Tecomine 1 andTecostanine 2, isolated from Tecoma stans growing inEgypt, were shown to produce a marked reductions inthe blood sugar of fasting rabbits (normal or alloxan-treated animals) both orally or i.v.; the mechanism ofaction of these substances was not elucidated, but theyshowed to be inactive in the absence of pancreas [3,4].Recently, Tecostanine 2 was shown to be inactive ashypoglycemic agent in normoglycemic and hyperglyce-mic rats [5]. Given the great interest in novel agents ableto treat diabetes, we have now investigated the effect onan in vivo model of genetic diabetes (db/db mouse) ofTecomine 1 and of the two other alkaloids that arepresent in large quantities in both the plants we collectedfrom Egypt and Brazil, namely 5b-Hydroxyskitanthine(3), early called Base C, and Boschniakine (4), which atthe best of our knowledge, had never been tested asantidiabetic agents. A parallel in vitro study on adipo-* Corresponding author.E-mail address: costantino.luca@unimo.it (L. Costantino).0014-827X/03/$ - see front matter # 2003 E´ditions scientifiques et me´dicales Elsevier SAS. All rights reserved.doi:10.1016/S0014-827X(03)00133-2cyte glucose uptake was also performed on compounds1, 3 and 4.2. Results and discussionIt is known that Tecoma stans possesses a greatvariability in its alkaloidic composition depending onthe place of growth, Tecomine 1 being present in all thesamples examined [6]. We determined the compositionof the alkaloidic fraction of the plant collected in Egyptand in another country, Brazil, in order to findsubstance(s) that could be useful for the treatment oftype II diabetes. The plant collected in Egypt showed agas chromatography (GC) of the alkaloidic fraction inwhich 3 is 11.37% of the area of the entire chromato-gram (RT 16.95 min), 4 is 17.30% (RT 18.59 min), 1 is10.40% (RT 18.93 min), and Tecostanine 2 is 21.88%(RT 19.13 min) (total 60.90% area of the entirechromatogram); the plant from Brazil shows for 311.77%, for 4 24.89%, for 1 11.48% and Tecostanine 2is 3.69% (total 51.83% of the entire chromatogram), thusconfirming the variability of the composition of thealkaloidic fraction of this plant. According to the GCanalysis, we were able to isolate from the crudealkaloidic fraction in appreciable quantity only com-pounds 1, 3 and 4 (besides Tecostanine 2 from the plantcollected in Egypt [5]).All the isolated alkaloids were tested on db/db mice, agenetic model of type II diabetes characterized byinsulin resistance and obesity. In particular, the Teco-mine hydrochloride (1a), the 5b-Hydroxyskitanthineoxalate (3b) and the Boschniakine oxalate hemihydrate(4a) were administered at the following doses: 1a and 4aat the dose of 50 mg/kg p.o.; 3b at the dose of 63.4 mg/kg p.o. Contrary to what previously reported for 1 ondifferent animal models, none of the compounds provedable to significantly reduce plasma glycemia or insulinlevels after 2 or 7 h from the treatment. In the sameexperimental setting, Rosiglitazone, at the dose of 5 mg/kg p.o., was able to reduce glycemia (372.69/21.6 mg/dlversus control 621.09/36.1). However, it should benoted that compound 1a reduced plasma cholesterollevels (72.96 mg/dl9/4.08 (SE) versus control (94.489/3.63 (SE), PB/0.02) while body weight, free fatty acids,triglycerides and urea, as well as food and water intakedid not change following the treatment.It is possible that pharmacokinetic factors or adifferent sensitivity of the animal species consideredcan affect the in vivo activity of 1.This hypothesis seems to be supported by the resultsof a parallel in vitro study which showed that Tecomine1 exerts a potent stimulating effect on the basal glucoseuptake rate in rat adipocytes from normoglycemic rats,with an EC50 value of 6.79/109 M. On the contrary,the two other alkaloids tested (3, 4) were inactive up to100 mM.More studies are thus needed in order to assessdefinitively the therapeutic interest of Tecomine forthe treatment of diabetes.3. Experimental3.1. ChemistryMelting points (m.p.) were determined on a Buchimelting point apparatus and are uncorrected. Opticalrotation was obtained on a Perkin/Elmer mod. 241spectropolarimeter. 1H (200 MHz) and 13C (50 MHz)NMR spectra were recorded on a Bruker AMX-200spectrophotometer. Chemical shifts are shown in ppmwith TMS as internal standard; J values are given in Hz.IR spectra were recorded on a Perkin/Elmer FT-IRspectrophotometer mod. 1600. X-ray crystallographydata were collected on a Enraf/Nonius CAD4 diffract-ometer. The GC analyses were carried out on a Perkin/Elmer mod sigma 3b gas chromatograph, equipped witha flame ionisation detector, connected to a Perkin/Elmer nelson integrator mod 1022, using a capillarycolumn (Hewlett-Packard, 25 m/0.31 mm i.d., filmthickness 1.03 mm) cross-linked with 5% phenyl methylsilicone under the following conditions carrier gas H2,split ratio 90:10, injector and detector temperature250 8C and column temperature programmed from50 8C (5 min) to 200 8C at 10 8C/min. Mass spectra ofeluted peaks were obtained in the above experimentalconditions by means of a mass spectrometer FinniganMat SSQ 710 equipped with a gas chromatographVarian 3400 (Centro Interdipartimentale Grandi Stru-menti, Modena University). Microanalyses were carriedout in the Microanalysis Laboratory of the Diparti-mento di Scienze Farmaceutiche, Modena University.L. Costantino et al. / Il Farmaco 58 (2003) 781/785782Elemental analyses were within9/0.4% of the theoreticalvalues. Silica gel (Merck, 70/230 mesh) was used forcolumn chromatography. Rosiglitazone was synthesizedaccording to a published procedure [7] (Table 1).3.2. Plant materialLeaves of Tecoma stans (Juss) (Bignoniaceae) grow-ing in II Cairo, Egypt, or in Brazil (from San Pauloprovince) collected during July 1999 and November1998, respectively, were used. A voucher specimen(LC2002/01 and LC2002/02, respectively) was depositedin the Herbarium of the Dipartimento di ScienzeFarmaceutiche, Universita` di Modena e Reggio Emilia.3.3. Extraction procedureThe dried leaves (500 g) were extracted (Soxhlet) withEt2O/NH3 15% (10/90 v/v) (3 l). The organic layer wasthen extracted with 4 N HCl (1000 ml); the aqueousphase was then washed with ether (3/200 ml), basifiedwith NH3 30% and extracted with CH2Cl2 (2/500 ml).The organic layer was then dried (Na2SO4) and thesolvent removed under reduced pressure. The crudealkaloidic fraction thus obtained (3 g from the plantsharvested in Brazil or Egypt) was then chromato-graphed on silica gel with as mobile phase CH2Cl2 andincreasing quantity of CH3OH (from 0 to 40%). 4 waseluted at 2% CH3OH (TLC mobile phase CH2Cl2/CH3OH/NH3 89.5/10/0.5, Rf 0.9), 1 at 5% (TLC, Rf0.80), 3 at 10% (TLC, Rf 0.29).3.3.1. Tecomine (1)Yield 0.2 g, oil; purity (GC) /90%, [a]25D (CHCl3):/1448 (c/1) ([a ]D (CHCl3) /1608 [8];1H-NMR(CDCl3): 1.17 (3H, d, J 6.7), 1.19 (3H, d, J 7.6), 1.78(2H, t, J 11.1), 1.97 (1H, qd, J 7.6, 3.1), 2.35 (3H, s),2.50/2.65 (1H, m), 2.70/2.80 (1H, m), 3.05 (1H, ddd, J11.0, 5.8, 1.5), 3.28 (1H, ddd, J 11.0, 6.0, 2.0), 5.90 (1H,m), in accordance to Ref. [9]. MS m/z 179 (M, 100),164 (22), 150 (20), 136 (22), 121 (22), 111 (30), 93 (27), 77(12), 57 (70), in accordance to Ref. [9].3.3.2. Tecomine HCl (1a)M.p. 175/6 8C (CH3OH/Et2O);1H NMR (DMSO-d6): 1.11 (3H, d, J 7.40), 1.18 (3H, d, J 6.58), 2.10 (1H,ddd, J 10.03, 7.31, 2.69), 2.77 (3H, s), 2.78/3.30 (4H,m), 3.55 (1H, m), 3.80 (1H, m), 6.06 (1H, m), 11.71 (1Hbroad s). Anal . C11H17NO /HCl C, H, N.3.3.3. 5b-Hydroxyskitanthine (3)Yield 0.4 g, purity (GC) /90%; 1H NMR (CDCl3):0.92 (3H, d, J 6.83), 1.18 (3H, d, J 6.89), 1.48/1.75 (m,7H), 1.90/2.10 (m, 2H), 2.24 (s, 3H), 2.65 (ddd, J 10.2,4.5, 2.2), 2.79 (1H, ddd, J 11.4, 4.5, 2.3). 13C NMR:13.1, 23.9, 30.4, 33.1, 36.9, 39.6, 45.6, 55.0, 60.3, 62.2,83.0. MS m/z 183 (M, 90), 182 (100), 166 (30), 164(32), 150 (80), 122 (30), 108 (28), 98 (20), 84 (70), 74 (40),58 (38), 57 (60), 55 (35). IR (Nujol) (cm1): 3400. (thespectra are in accordance with Ref. [10]). In order toconfirm the structure, 3 was converted in its methiodide3a and subjected to X-ray crystallography.3.3.4. 5b-Hydroxyskitanthine methiodide (3a)5b-Hydroxyskitanthine 3 (0.20 g, 1.10 mmol) wasdissolved in anh. Et2O (5 ml); CH3I was added (0.93 ml,1.50 mmol) at room temperature (r.t.) under stirring. Awhite precipitate appeared immediately; the suspensionwas stirred for 3 h, then the precipitate was collected,washed with Et2O and dried. Yield 0.22 g (62%), m.p.293/5 8C (CH3OH);1H NMR (DMSO-d6): 0.90 (3H, d,J 6.85), 1.17 (3H, d, J 6.83), 1.35/2.05 (6H, m), 2.43(1H, m), 2.90 (1H, t), 3.11 (3H, s), 3.12 (3H, s), 3.45 (3H,m), 4.45 (1H, s). Crystals suitable for X-ray analysiswere grown from a methanolic solution. The crystalstructure is the same as reported [11], with the same cellparameters.3.3.5. 5b-Hydroxyskitanthine (COOH)2 (3b)M.p. 158/60 8C (CH3OH/Et2O);1H NMR (DMSO-d6): 0.89 (3H, d, J 6.85), 1.17 (d, 3H, J 6.83), 1.35/2.05(m, 6H), 2.15 (m, 1H), 2.42/2.60 (m, 4H), 2.67 (s, 3H),3.23 (m, 2H), 8.40 (2H, broad s). Anal . C13H23NO5: C,H, N.3.3.6. Boschniakine (4)Yield 0.7 g, oil; purity (GC) /96%, [a]25D (CHCl3)/22.58 (c/1) ([a ]D /218 [8]; IR (Nujol) (cm1): 1697;1H NMR (CDCl3): 1.38 (3H, d, J 6.91), 1.60/1.84 (1H,m), 2.36/2.54 (1H, m), 3.00/3.48 (3H, m), 8.62 (1H, s),8.83 (1H, s), 10.23 (1H, s), in accordance to Ref. [12];Table 1Elemental analysesComp. Formula C Calc./found H Calc./found N Calc./found1a C11H17NO /HCl 61.25/61.58 8.41/8.33 6.49/6.113b C11H21NO /(COOH)2 57.13/57.51 8.48/8.33 5.12/5.404a C10H11NO /(COOH)2 /0.5 /H2O 55.38/55.33 5.42/5.18 5.38/5.50L. Costantino et al. / Il Farmaco 58 (2003) 781/785 783MS m/z 161 (M, 100), 160 (30), 146 (80), 132 (40), 117(57), 91 (22), 77 (19), 65 (10), 51 (10).3.3.7. Boschniakine (COOH)2 /0.5 /H2O (4a)M.p. 113/6 8C (CH3OH/Et2O);1H NMR (DMSO-d6): 1.31 (3H, d, J 6.90), 1.55/1.70 (m, 1H), 2.30/2.45(m, 1H), 3.00/3.15 (1H, m), 3.19 (1H, s), 3.20/3.30 (2H,m), 8.68 (1H, s), 8.86 (1H, s), 10.21 (1H, s). Anal .C12H13NO5.0.5 /H2O: C, H, N.3.4. Pharmacology3.4.1. Glucose uptake in Wistar rats white adipocytes3.4.1.1. Animals. Male Wistar rats (180/200 g) wereobtained from Charles-River Italy breeding colonies.The animals were housed at 239/1 8C with a 12 h light/dark cycle, light on at 07:00 h and were fed a standardlaboratory diet with water ad libitum. All the experi-ments were carried out in accordance with the EuropeanCommunities Council Directive of 24 November 1986(86/609/EEC) for experimental animal care.3.4.1.2. Adipocyte preparation. Adipocytes were pre-pared according to Rodbell [13]. The epidydymal fatpads were removed and placed in oxygenated coldKrebs bicarbonate buffer pH 7.5 of the followingcomposition (mM): NaHCO3 (25), glucose (11.1),NaCl (118), KCl (4.7), KH2PO4 (1.2), MgSO4 (0.6).The tissue was then minced with scissors and digestedfor 40/60 min at 37 8C in Krebs buffer containingcollagenase (0.5 mg/ml; type I, Sigma) and 2% free fattyacid bovine serum albumin (SIGMA). After digestionthe tissue was filtered on a nylon gauze and washedextensively with warm Krebs buffer. Adipocytes werethen concentrated and counted. An average amount of300.000/400.000 cells/tube were used.3.4.1.3. Glucose uptake. Glucose uptake was measuredin floating, viable, adipocytes using the non metaboli-sable analogue of glucose, the 3H-2-deoxy-D-glucose(100 mM, 0.2 mCi/ml).Cells were first pre-incubated for 10 min at 37 8C inatmosphere of 5% CO2 before the addition of thepharmacological stimuli. Insulin (10 nM, Sigma) orthe tested compounds were then added to cell suspen-sions and incubated for 45 or 10 min, respectively beforethe addition of the labelled compound.Glucose uptake was evaluated after 10 min from theaddition of the 3H-2-deoxy-D-glucose (100 mM, 0.2 mCi/ml). At that time Phloretin (200 mM; Sigma) was addedto each tube. Cells were then filtered on nitrocellulosefilter (8 mM; Sartorius) under light vacuum. After threewashings with cold Krebs buffer, the filters were driedup at r.t. and placed in a vials containing Filter-Count(Packard). The radioactivity was measured using a b-counter. Basal uptake was completely abolished by theuse of Phloretin (200 mM), whereas the insulin-depen-dent uptake was inhibited by more than 80% by usingCytocalasin (20 mM).Results are expressed as nmoles of deoxy-glucose/106cells or as the ratio between the uptake measured instimulated cells over that measured in cells without anytreatment (basal uptake).Stock solutions (1 mM) of the compounds 1, 3 and 4(as free bases) were prepared in DMSO. At theconcentrations used, DMSO (maximum tested 0.01%)had no effect on glucose uptake. Results are the mean9/SE of four different exp. run in duplicate. Basal uptake:1.279/0.22 nmol/106 cells/10 min.3.4.2. In vivo antidiabetic assaysMale C57BL/KsJ db/db mouse (8 weeks old) wereused. Six animals were used for control and for thecompounds to be assayed. Mouse were treated p.o. twiceeach day (at 08:30 and 18:30 h) for 7 days by gavage atthe dose of 50 mg/kg in C.M.C. (1%) and Tween 80(0.5%) (10 ml/kg) for 1a and 4a; 63.4 mg/kg were usedfor 3b; Rosiglitazone was used as a reference compoundat the dose of 5 mg/kg (10 ml/kg), p.o.; then they weresacrificed at 7th day after 7 h from the last treatmentand the following parameters were determined: plasmaglucose (evaluated by Kit glucose GDH, Roche),triglycerides, cholesterol and urea (by Kits Roche),free fatty acids (by Kit NEFA C, Wako). Plasma insulinwas assessed by Kit 125I-Rat insulin (Amersham).Controls received only vehicle (10 ml/kg).AcknowledgementsWe thank Professor Dr M. Hammouda, Departmentof Botany, University College for Girls, Ain ShamsUniversity, Heliopolis, Cairo, Egypt, for kindly provid-ing the plant material.References[1] R. Roman Ramos, J.L. Flores Saenz, G. Partida Hernandez, A.Lara Lemus, F. Alarcon Aguillar, Experimental study of thehypoglycemic effect of some antidiabetic plants, Arch. Invest.Med.(Mex) 22 (1991) 87/93.[2] M. Loyoza Meckes, V. Mellado-Campos, Is the Tecoma stanstnfusion an antidiabetic remedy?, J. Ethnopharmacol. 14 (1985)1/9.[3] Y. Hammouda, M.S. Amer, Antidiabetic effect of Tecomine andTecostanine, J. Pharm. Sci. 55 (1966) 1452/1454.[4] Y. Hammouda, A.K. Rashid, M.S. Amer, Hypoglycaemicproperties of Tecomine and Tecostanine, J. Pharm. Pharmacol16 (1964) 833/835.[5] L. Costantino, A.P. Lins, D. Barlocco, F. Celotti, S.A. El-Abady,T. Brunetti, R. Maggi, L. Antolini, Characterization and phar-L. Costantino et al. / Il Farmaco 58 (2003) 781/785784macological actions of Tecostanine, an alkaloid of Tecoma stans ,Die Pharmazie, in press.[6] E.M. Dickinson, G. Jones, Pyrindane alkaloids from Tecomastans , Tetrahedron 25 (1969) 1523/1529.[7] B.C.C. Cantello, M.A. Cawthorne, D. Haigh, R. Hindley, S.A.Smith, P.L. Thurlby, The synthesis of BRL 49653/A novel andpotent antihyperglycaemic agent, Bioorg. Med. Chem. Lett. 4(1994) 1181/1184.[8] G.A. Cordell, The Monoterpene Alkaloids, in: R.H.F. Manske(Ed.), The Alkaloids, vol. 16, Academic Press, 1977, pp. 431/510.[9] G. Vidari, M. Tripolini, P. Novella, P. Allegrucci, L. Garlaschelli,Desymmetrization of bicyclo[3.3.0]octane-3,7-dione by theSchmidt reaction: an easy synthesis of Tecomanine, Tetrahedron:Asymmetry 8 (1997) 2893/2903.[10] J. Cossy, D. Belotti, C. Leblanc, Total synthesis of (///)-Actinidine and of (///)-Isooxyskytanthine, J. Org. Chem. 58(1993) 2351/2354.[11] G. Ferguson, W.C. Marsh, Crystal structures and absolutestereochemistry of Tecomanine methoperchlorate and ‘alkaloidC’ methiodide: two monoterpene alkaloids from Tecoma stans , J.Chem. Soc. Perkin II (1975) 1124/1128.[12] Y. Imakura, S. Kobayashi, Y. Yamahara, M. Kihara, M.Tagawa, F. Murai, Studies on costituents of bignoniaceae plants.IV. Isolation and structure of a new iridoid glucoside, campsiside,from Campsis chinensis , Chem. Pharm. Bull. 32 (1985) 2220/2227.[13] M. Rodbell, Metabolism of isolated fat cells, J. Biol. Chem. 239(1964) 375/380.L. Costantino et al. / Il Farmaco 58 (2003) 781/785 785

Isolation and pharmacological activities of the Tecoma stans alkaloids.

https://flore.unifi.it/bitstream/2158/1060145/1/Il%20farmaco-%20tecoma.pdf

Isolation and pharmacological activities of the Tecoma stans alkaloids.

Abstract

Similar works

Full text

Available Versions

Florence Research