Ciencia-bio-comp.vp:corelventura 7.0

Leishmanicidal activity of alkaloids from
Hamelia patens
Alírica I. Suárez*, Beth Diaz, Stephen Tillett, Elizabeth Valdivieso B.
and Reinaldo S. Compagnone!
1Facultad de Farmacia. 2Instituto de Biología Experimental, Facultad de Ciencias. 3Escuela de Química, Facultad de Ciencias. Universidad Central de Venezuela, Abstract
The crude ethanolic extract from the leaves of Hamelia patens Jacq. was active in vitro against Leishmania mexicana. Partition of the extract, showed that the activity was confined to
the dichloromethane extract. The four alkaloids isolated from this extract: isopteropodine (1),
palmirine (2), rumberine (3) and mitrajavine (4) were evaluated in vitro for antileishmanial acti-
vity. Palmirine (2), one of the main alkaloids, showed the highest leishmanicidal activity with
IC = 56 µM, the alkaloid mitrajavine (4) and the two flavonoids isolated from the ethyl acetate ex-
tract: kaempferol-3-O-rutinoside (5), and (-) epicatechine (6) are reported for the first time in this
Key words: Rubiaceae, Hamelia patens, leishmanicidal, alkaloids, flavonoids.
Actividad leishmanicida de alcaloides de Hamelia patens El extracto etanólico obtenido de las hojas de Hamelia patens Jacq. fue activo en pruebas in vitro contra Leishmania mexicana. La partición del extracto mostró que la actividad estaba
confinada al extracto de diclorometano. Cuatro alcaloides aislados de ese extracto, isopteropo-
dina (1), palmirina (2), rumberina (3) y mitrajavina (4), fueron evaluados in vitro en actividad
leishmanicida. Palmirina (2) mostró la más alta actividad con un IC = 56 µM. El alcaloide mi-
trajavine (4) y los dos flavonoides aislados del extracto de acetato de etilo, kaempferol-3-O-ruti-
nosido (5) y (-) epicatequina (6), son reportados por primera vez en esta planta.
Palabras claves: Rubiaceae, Hamelia patens, leishmanicida, alcaloides, flavonoides.
tions such as: malaria, rheumatism, gastri-tis, dysentery, ulcers, and as an antihemo- Hamelia patens, a plant belonging to
rragic (1). As part of our search for bioactive the Rubiaceae, is a species distributed throughout tropical America. The plant is number of plants used in Venezuela to treat known commonly as “coralito” and, is widely chloromethane extract of the leaves from Ha- treat different diseases and health condi- melia patens exhibited strong in vitro activity * Autor para la correspondencia. E-mail: [email protected]
Scientific Journal of the Experimental Faculty of Sciences, at the Universidad del Zulia Volume 16 Nº 2, April-June 2008 A.I. Suárez et. al. / Ciencia Vol. 16, Nº 2(2008) 148 - 155 against Leishmania mexicana. For that rea- MeOD using TMS as internal reference, em- pounds isolated from the mentioned extract.
The aerial parts of Hamelia patens were in- vestigated previously by Aquino et ál. (2) who UCR Mass Spectrometry Facility at Riversi- reported the presence of 5,7,2,5´-tetrahidr- de, California. Isolation procedures were oxyflavone-7-rutinoside; Prabir et ál. (3) iso- monitored by employing thin-layer chroma- lated ephedrine and rosmarinic acid, Borges et ál. (4) reported three of the alkaloids here mentioned: isopteropodine (1), palmirine (2),
and rumberine (3), and more recently Rios &
Plant material
Aguilar-Guadarrama (5) isolated cycloarta- Hamelia patens Jacq. was collected in nols and other triterpenes. This paper deals San Diego de los Altos, Miranda State, Vene- with the isolation, structural determination, zuela, and identified by Dr. Stephen Tillett. A and the leishmanicidal activity of the alka- voucher specimen was deposited in the Her- loids found in our research (figure 1).
barium “Victor Manuel Ovalles” of the Facul-tad de Farmacia, Universidad Central de Ve- Materials and methods
nezuela, with the accession number MYF11810.
General procedure
Extraction and isolation
a Kofler hot-stage instrument and were un- were exhaustively extracted with MeOH in a Perkin-Elmer 1320 spectrometer. 1H and 13C Figure 1. Structures of alkaloids tested for leishmanicidal activity: isopteropodine (1), palmirine (2), Scientific Journal of the Experimental Faculty of Sciences, at the Universidad del Zulia Volume 16 Nº 2, April-June 2008 Leishmanicidal activity of alkaloids from Hamelia patens (28.5 g) was dissolved in MeOH-H O 1:1, and Isolates
partitioned between hexane, CH Cl , EtOAc, Isopteropodine (1). Yellow needles
and H O. The antileishmanial activity was tracts showed the highest values, these were (NH), 1730 (COOR), 1690 (CONH). 1H (CDCl , 270 MHz), d: 0.86 (1H, dd, J =12.1, 12.3 Hz, H-14a), 1.37 (3H, d, J = 6.2 Hz. H-18), 1.84 (1H, m, H-20), 1.97 (1H, m, H-15), 2.36-2.43 the CH Cl six fractions were collected to yield compounds 1 (85 mg), 2 (43 mg), 3 (32
mg), and 4 (72 mg). From the EtOAc extract,
H-5b), 3.58 (3H, s, OCH ), 4.34 (1H, m, H-3), compounds 5 (75 mg), and 6 (162 mg) were
4.40 (1H, m, H-19), 6.85 (1H, d, J = 8.2 Hz, H-12), 6.99 (1H,ddd, J = 8.2, 8,1, 1.1 Hz, graphy eluted with EtOAc-MeOH (5-40%).
H-10), 7.16 (1H,ddd, J = 8.2, 8,1, 1.1 Hz, All the isolated were further purified by crys- H-11), 7.26 (1H, d, J = 8.1 Hz, H-9), 7.39 (1H, tallization in appropriate solvents. Structu- s, H-17), 8.04 (1H, brs, NH). 13C NMR (CDCl , 67 MHz) ä: 181.5 (C-2), 72.1 (C-3), 53.9 (C-5), 30.2 (C-6), 56.7 (C-7), 134.1 (C-8), 109.6 (C-12), 140.4 (C-13), 34.6 (C-14), 29.9 their physical and spectroscopic data with (C-18), 71.3 (C-19), 37.6 (C-20), 53.3 (C-21), Palmirine (2). Colorless amorphous so-
lid, m.p. 103-104 oC; [A] 20 -48 (c, 0.40 1.45 (3H, d, J = 6.5 MHz, H-18), 3.58 (3H, s,OCH ), 3.75 (3H, s, OCH ), 4.35 (1H, m, H-3), 6.87 (1H, d, J = 7.6 Hz, H-12), 7.15 (1 H,dd, J= 7.5, 0.8 Hz, H-11), 7.30 (1H, d, J= 1.3 Hz,H-9), 9.30 (1H, brs, NH). 13C NMR (CDCl , 67 MHz) d: 181.0 (C-2), 71.3 (C-3), 54.1 (C-5),30.3 (C-6), 57.6 (C-7), 133.8 (C-8), 111.9(C-9), 156.0 (C-10), 111.2 (C-11), 109.9 (C-15), 110.0 (C-16), 155.1 (C-17), 18.6(C-18), 72.3 (C-19), 38.2 (C-20), 53.6 (C-21), 167.6 (C-22), 51.0 (C-23), 56.7 (CH OAr).
Rumberine (3). Amorphous yellow solid,
m.p. 185-188 oC; [a] 20 -52 (c, 023 CHCl ), MS , 384.32 m/z , IR (KBr) g cm-1: 3300 cm-1 (NH, OH), 1730 (COOR), 1700 (CONH). 1H(CDCl , 270 MHz), d: 1.25 (1H, m, H-20), 1.43 (3H, d, J = 6.5 MHz, H-18), 3.56 (3H, s, Figure 2. Structures of flavonoids from Hamelia OCH ), 4.42 (1H, m, H-3), 6.80 (1H, d, J = 7.6 Hz, H-12), 7.18 (1H,dd, J = 7.5, 0.8 Hz, Scientific Journal of the Experimental Faculty of Sciences, at the Universidad del Zulia Volume 16 Nº 2, April-June 2008 A.I. Suárez et. al. / Ciencia Vol. 16, Nº 2(2008) 148 - 155 H-11), 7.37 (1H, d, J = 1.3 Hz, H-9), 8.50 (-)Epicatechine (6) 1 H NMR (metha-
nol-d , 270 MHz) d; 2.80 (2H, dd, J = 4.2, 182.0 (C-2), 71.2 (C-3), 53.9 (C-5), 30.2 16.3 Hz, H-4), 4.18 (1H, brs, H-3), 4.85 (1H, (C-6), 56.8 (C-7), 134.0 (C-8), 124.8 (C-9), brs, H-2), 5.90 (1H, d, J = 2.3 Hz, H-6), 140.5 (C-10), 127.7 (C-11), 133.9 (C-12), 6.01(1H, d, J = 2.3 Hz, H-8), 6.76 (1H, d, J = 134.0 (C-13), 34.5 (C-14), 30.2 (C-15), 109.9 8.2 Hz, H-5’), 6.83 (1H, dd, J = 1.8, 8.2 Hz, H-6’), 7.02 (1H, d, J = 1.8 Hz, H-2’). 13C NMR (methanol-d , 67 MHz) d: 78.5 (C-2), 66.2 (C-3), 27.9 (C-4), 156.3 (C-5), 94.6 (C-6),156.6 (C-7), 95.0 (C-8), 156.0 (C-9), 98.8 Mitrajavine (4). White solid, mp 113-
(C-10), 130.9 (C-1’), 114.6 (C-2’), 143.4 115oC [a]20 -45 (c, 0.30 CHCl ), IR (KBr) g (C-3’), 144.8 (C-4’), 114.8 (C-5’), 118.1 (C-6’).
cm-1: 3400 (NH), 1710 (COOR), 1615 (aro-matic ring). FAB-MS: 382.1860. C 1H NMR Assay for leishmanicidal activity
(CDCl , 270 MHz) d: 1.37 (3H, d, J = 6.5 Hz, H-18), 1.49 -1.68 (2H, m, H-14), 2.04 (2H, Leishmania mexicana, strain NR, cha- m, H-20, H-21), 3.26 (1H, d, J = 11.6 Hz, racterized by Ramirez and Guevara (6), was H-3), 2.60 (1H, m, H-5), 3.06 (2H, d, J = 12.12 Hz, H-6), 3.72 (3H, s, OCH ), 3.78 (3H, 10% inactivated fetal bovine serum, at 26 °C.
s, OCH ), 4.71 (1H, m, H-19), 6.76 (1H, dd, J The bioassays were carried out in duplicate = 2.48, 8.64 Hz), 6.87 (1H, d; J = 2.21 Hz, ), with the promastigote form of the parasite, 7.14 (1H, d, J = 8.64 Hz), 7.54 (1H, s, H-17), MHz) d: 131.2 (C-2), 59.9 (C-3), 53.6 (C-5), was dissolved in DMSO and added to cultu- 21.7 (C-6), 127.6 (C-7), 109.5 (C-8), 154.1 res of L. mexicana in the log phase at a den- sity of 2 X 106 cells/mL (figures 3, 4, 5 y 6).
(C-12), 131.2 (C-13), 34.5 (C-14), 31.3(C-15), 107.9 (C-16), 155.8 (C-17), 18.6 Results and discussion
(C-18), 72.5 (C-19), 38.5 (C-20), 53.6 (C-21), graphy the CH Cl extract of the leaves affor- Kaempferol-3-O-rutinoside (5). 1H
ded isopteropodine (1), palmirine (2), rum-
(methanol-d , 270 MHz) d: 0.98 (1H, d, J = berine (3) and the other alkaloid characteri-
5.9 Hz, H-6’’’), 3.00 (2H, m, H-5’’’, H-2’’’), zed as mitrajavine (4).
3.10 (2H, m, H-4’’’, H-3’’’), 3.16 (1H, m, H- Compound 4, was obtained as yellow
4’’), 3.26 (1H, m, H-5’’), 3.69 (1H, m, H-3’’), syrup and, crystallized in EtOH/H O. Its IR 4.51 (1H, brs, H-1’’’), 5.09 (1H, d, J = 7.1 Hz, H-1’’), 5.32 (1H, d, J = 7.2 Hz, H-2’’), 6.88 1710 cm-1, associated to –COOR group and (2H, d, J = 8.6, H-3’,H-5’), 6.19 (1H, brs, broad band at 3400 cm-1 –NH. The high reso- H-6), 6.41 (1H, brs, H-8), 7.99 (2H, d, J = 8.6 Hz, H-2’, H-6’), 12.56 (1H, s, OH-5).13C NMR (methanol-d , 67 MHz) ä: 157.2 (C-2), 134.5 (C-3), 178.0 (C-4), 161.5 (C-5), 98.6 (C-6), the identification of an ABX aromatic system 164.6 (C-7), 93.6 (C-8), 158.9 (C-9), 104.0 for three aromatic protons which resonated (C-10), 121.5 (C-1’), 103.4 (C-1’’), 114.8 at d 7.15 (d, J= 8.6 Hz), 6.86 (d, J= 2.2 Hz), (C-2’), 131.5 (C-3’), 160.1 (C-4’), 131.1 and 6.75 (dd, J = 8.6, 2.2 Hz). The resonance (C-5’), 114.8 (C-6’), 75.8 (C-2’’), 76.8 (C-3’’), of the indolic NH proton was found at d 7.87 70.7 (C-4’’), 75.8 (C-5’’), 67.0 (C-6’’), 107.1 ppm as well the olefinic proton 17 which re- (C-1’’’), 71.0 (C-2’’’), 70.8 (C-3’’’), 72.6 (C-4’’’), sonated at d 7.54 ppm. Two signals singlet of 68.4 (C-5’’’), 16.7 (C-6’’’).
Scientific Journal of the Experimental Faculty of Sciences, at the Universidad del Zulia Volume 16 Nº 2, April-June 2008 Leishmanicidal activity of alkaloids from Hamelia patens control DMSO
Time (hours)
Figure 3. Effect of palmirine (2) on growth and proliferation of Leishmania mexicana promastigotes.
IC50 = 56µg/ml
[Palmirine] mg/ml
Figure 4. Dosis-answer curve for palmirine (2) against L. mexicana promastigotes.
Figure 5. Effect of rumberine (3) on the growth and proliferation of Leishmania mexicana promastigotes.
Scientific Journal of the Experimental Faculty of Sciences, at the Universidad del Zulia Volume 16 Nº 2, April-June 2008 A.I. Suárez et. al. / Ciencia Vol. 16, Nº 2(2008) 148 - 155 Rumberine

[Rumberine] pg/ml
Figure 6. Dosis-answer curve for rumberine (3) against L. mexicana promastigotes.
methoxy groups showed resonance at d 3.78 physical and spectral data of the compounds and 3.74 ppm. Protons clearly belonging to we isolated were in good agreement with tho- pyrane and quinolizidine rings presents in indole alkaloids, which are frequently isola- here the unequivocal assignment of the 13C ted in the Rubiaceae, were also observed as multiplets between 2.5 and 4.5 ppm. Finally providing additional information with res- one doublet integrating for three protons pect to previously published data (table 1).
was found at 1.37 ppm, this signal sugges- Compound 5 was shown by FAB-MS to
ted one methyl group coupled to one proton.
have the molecular formula C H O . The UV All this analyzed data were consistent with a yohimbine type structure. The 13C spectrum 207, 268, and 365 nm. These are typical sig- confirmed by HMQC was consistent with the nals of the flavonol skeleton. The 1H NMR proposed structure. 21 signals resonances (CD OD) spectrum of compound 5 suggested
indicated the presence of eleven sp2 carbons a 3-glycosylated flavonoid which displayed including one ester carbonyl (168.1 ppm); the characteristic signals of the kaempferol the signal at 131.2 ppm was unambiguously nucleus: two doublets at d 6.19 and 6.39 ppm (J = 1.8 Hz), assigned to the H-6 and H-8 pro- tons, respectively, of the A-ring; and a pair of were confirmed by the resonances at d 51.2 doublets integrating for two protons each one indicated a typical A B aromatic systems at d substituted olefin were identified at 107.9 6.87 and 8.06 ppm (J = 8.8 Hz) in the C ring.
The glycosidic nature was confirmed by the two anomeric protons at d 4.51 (brs, 1H) and 5.09 (d, J = 7.1 Hz, 1H). The glucose b-linked paring our data with yohimbane alkaloids in to 3-OH, was evident from the large constant the literature, we found a good correlation coupling of H-1 while L-rhamnose is á-linked with the reported data for mitrajavine (4) (7,
to glucose d 4.51 (brs, 1H). The 13C spectrum 8), this alkaloid was previously found in Mi- was compared with literature spectra and all the signals indicated a flavone substituted at Isopteropodine (C H N O ) 1, rumberi-
the 3 position. Finally we found all our data ne (C H N O ) 2, and palmirine (C H N O )
matched with the known kaempferol-rutino- 3, had been fully characterized (4). Both the
Scientific Journal of the Experimental Faculty of Sciences, at the Universidad del Zulia Volume 16 Nº 2, April-June 2008 Leishmanicidal activity of alkaloids from Hamelia patens !C NMR data (CDCl!) for H. patens alkaloids Compound 6, was isolated from the
HMQC to H-3 and H-4. Finally a doublet of EtOAc extract as yellow amorphous solid.
doublet signal at 2.82 (dd, J= 4.2, 16.3 Hz) The FAB MS exhibited a molecular ion peak belonging to the protons H-4 confirmed the at 290, and the composition of the molecu- lar formula was determined as C H O . The spectrum were characterized by a DEPT ex- presented by the resonances at 7.02 ppm (d, periment, which shows that 6 was a flavo-
J= 1.8 Hz); 6.86 ppm (d, J= 1.8, 8.6 Hz) and, noid which have seven quaternary carbons, 6.76 ppm (d, J= 8.6 Hz). At ä 5.90 (d, J=2.3 seven methines, and one methylene. The ab- Hz) and 6.01 (d, J= 2.3 Hz) were found two structure. Comparison of the 13C NMR data signals integrating for one proton were ob- of 6 with those of reported isoflavan (10, 11)
served at 4.18 and 4.85 ppm , assigned by Scientific Journal of the Experimental Faculty of Sciences, at the Universidad del Zulia Volume 16 Nº 2, April-June 2008 A.I. Suárez et. al. / Ciencia Vol. 16, Nº 2(2008) 148 - 155 References
ment and a literature survey established the
structure of compound 6 as (-) epicatechine.
1. MORTON J.F. Atlas of medicinal plants of
middle America. Bahamas to Yucatan. Ch.
Pure indole alkaloids 1-4 were tested in
C. Thomas publisher, Springfield (USA), 1981.
vitro for their ability to inhibit the growth of 2. AQUINO R., CIAVATTA L.M., SIMONE F. PIZZA Leishmania mexicana. Comparison of the C.A. Phytochemistry 29: 2358-2360, 1990.
leishmanicidal activity showed that com-
pounds 2 and 3 have the highest values,
with IC of 56 µM and 61 M respectively. (fi- Medica 57: 199, 1991.
gures 3-6 ). These values are slightly higher RAMON J.L., PASCUAL C., RUMBERO A. Tet-
drugs used for the treatment of leishmania- rahedron Lett 34: 3197-3200, 1979.
(12), which blockage at sterol biosynthesis Cubana Plant. Med. 11: 1-4, 2006.
at the level of squalene epoxidase (13), and 6. RAMIREZ J.L., GUEVARA P. Mol Biochem
Parasitol 22: 177-183, 1987.
ducing alteration in the lipid bilayer (14) . We Planta Medica 13: 241-246,
can not give any structure-activity rela-tionships with our results, but it is interes- ting to point out that mitrajavine 4, did not
show any activity in our assays; however, P., PHILLIPSON J.D., LEE C.M. Planta
Medica 245-254, 1967.
were reported with high leishmanicidal acti- 10. MABRY T.J., MARKHAM K.R., THOMAS M.B.
vity against Leishmania major promastigo- The systematic identification of fla-
tes (15). Nor did compound 1 show any mea-
vonoids Springer-Verlag, New York, Heilde-
ningful difference, in comparison with the 11. EL-SOHLY H.N, JOSHI A., LI X.C., ROSS S.A. Although all compounds 1-4 have been
Phytochemistry 52: 141-1451, 1999.
reported (4, 8), to our knowledge, this is the 12. AGRAWAL P.K . 13C NMR of flavonoids El-
first report of antileishmanial activities for these alkaloids. The results obtained with the oxindole alkaloids 1-3 indicate that the
R.S. Cell. Biol. International 21: 337-339,
activity could be related to the presence of a substituent with oxygen in the 5 position of 14. RYDER N. S., MIETH H. Curr Top Med Mycol
the indole ring. Further studies with deriva- tives of these compounds will be necessary 15. HERNÁNDEZ M. A., DAGGER F., NICOLAS P., to determine the structure-activity rela- HERNÁNDEZ A., BENEDETTI E. L. Eur. J.
tionship, but they could be considered tem- Cell. Biol. 59: 412-424, 1992.
plates for future antileishmanial drugs.
J.W. Planta Medica 66: 531-536, 2000.
17. MARKUS V., GUIDO P. J. Nat. Prod. 62:
Scientific Journal of the Experimental Faculty of Sciences, at the Universidad del Zulia Volume 16 Nº 2, April-June 2008


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