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Leishmanicidal activity in vitro of Musa paradisiaca L. and Spondias mombin L. fractions
Veterinary Parasitology 187 (2012) 79–84
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Leishmanicidal activity in vitro of Musa paradisiaca L. and Spondias mombin L. fractions Marina Parissi Accioly a , Claudia Maria Leal Bevilaqua a,∗ , Fernanda C.M. Rondon a , Selene Maia de Morais a , Lyeghyna K.A. Machado a , Camila A. Almeida a , Heitor Franco de Andrade Jr. b , Roselaine P.A. Cardoso b a b
Programa de Pós-graduac¸ão em Ciências Veterinárias/Universidade Estadual do Ceará, Brazil Laboratório de Protozoologia/Universidade de São Paulo, Brazil
a r t i c l e
i n f o
Article history: Received 27 June 2011 Received in revised form 12 October 2011 Accepted 21 December 2011 Keywords: Visceral leishmaniasis Secondary metabolites Banana tree Cajazeira
a b s t r a c t Visceral leishmaniasis (VL) is a zoonotic disease characterized by infection of mononuclear phagocytes by Leishmania chagasi. The primary vector is Lutzomyia longipalpis and the dog is the main domestic reservoir. The control and current treatment of dogs using synthetic drugs have not shown effectiveness in reducing the incidence of disease in man. In attempt to find new compounds with leishmanicidal action, plant secondary metabolites have been studied in search of treatments of VL. This study aimed to evaluate the leishmanicidal activity of Musa paradisiaca (banana tree) and Spondias mombin (cajazeira) chemical constituents on promastigotes and amastigotes of L. chagasi. Phytochemical analysis by column chromatography was performed on ethanol extracts of two plants and fractions were isolated. Thin layer chromatography was used to compare the fractions and for isolation the substances to be used in vitro tests. The in vitro tests on promastigotes of L. chagasi used the MTT colorimetric method and the method of ELISA in situ was used against amastigotes besides the cytotoxicity in RAW 264.7 cells. Of the eight fractions tested, Sm1 and Sm2 from S. mombin had no action against promastigotes, but had good activity against amastigotes. The fractions Mp1 e Mp4 of M. paradisiaca were very cytotoxic to RAW 264.7 cells. The best result was obtained with the fraction Sm3 from S. mombin with IC50 of 11.26 g/ml against promastigotes and amastigotes of 0.27 g/ml. The fraction Sm3 characterized as tannic acid showed the best results against both forms of Leishmania being a good candidate for evaluation in in vivo tests. © 2012 Published by Elsevier B.V.
1. Introduction Leishmaniasis is a zoonotic disease with worldwide distribution and is considered by the World Health Organization (WHO) one of the six most important tropical diseases. Visceral leishmaniasis (VL) is caused by the subgenus Leishmania leishmania, Leishmania donovani complex
∗ Corresponding author at: Av. Dede Brasil, 1700, 60740-913 Fortaleza, Ceará, Brazil. Tel.: +55 85 31019853; fax: +55 85 31019840. E-mail address: [email protected] (C.M.L. Bevilaqua). 0304-4017/$ – see front matter © 2012 Published by Elsevier B.V. doi:10.1016/j.vetpar.2011.12.029
(Gontijo and Melo, 2004; Rath et al., 2003). It is considered endemic in the Mediterranean and extends to Latin America from Mexico to Argentina (Lainson and Rangel, 2005). Although several animal species may become infected (Luppi et al., 2008), the main domestic reservoir of this disease is the dog (Diniz et al., 2008) and Lutzomyia longipalpis is the primary sand fly vector (Wood et al., 2003). As recommended by the WHO, the control of LV is highlighted by the serological survey of dogs and euthanasia in cases in which they are positive, diagnosis and treatment of human cases and residual insecticide application based on pyrethroid (Melo, 2004). Treatment of dogs with drugs
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used in humans such as N-methyl glucamine, sodium stibogluconate, amphotericin B and others may decrease the symptoms, but are not able to eliminate all of the parasites. Therefore, the use of drugs in dogs is very limited and results in the dog remaining infected, maintaining the transmission cycle (Mishra et al., 2009). In Brazil, ministerial decree No. 1426 of July, 11th of 2008, prohibits the treatment of canine visceral leishmaniasis with products for human use or those not registered in the Ministério da Agricultura, Pecuária e Abastecimento. Therefore, there is a need to develop new therapies aimed at the control and treatment in the quest for a better quality of life and control of diseases of public health importance (Araújo, 2006). The use of medicinal plants and studies on the chemistry and pharmacology of natural products have grown considerably in the second half of the 20th century (Albuquerque et al., 2007). Many compounds derived from natural sources have pharmacological activities and may be used for drug development (Lawrence, 2003). Several compounds isolated from plants such as terpenoids, steroids, flavonoids, alkaloids, phenolic compounds and naphthoquinones have been studied to evaluate their effects on promastigotes and amastigotes of different species of leishmania. Some of these substances can be found in the leaves of cajazeira (Spondias mombin L.), banana (Musa paradisiaca L.) trees and other plants common in the Brazilian Northeast which present easy access to the population, and have being reported as having biochemical and pharmacological activities in vitro (Kolodziej and Kinderlen, 2005). Compounds from the banana trees leaves, such as the tannins have shown antimicrobial properties (Olivo et al., 2007) and the leaves from the cajazeiras have show besides the antimicrobial properties, healing action (Njoku and Akumefala, 2007). The objective of this study was to evaluate the leishmanicidal effect in vitro of the fractions obtained from crude extracts of leaves of cajazeira and banana tree on promastigotes and amastigotes of Leishmania chagasi. 2. Material and methods 2.1. Collection of plants Samples of the aerial parts of S. mombin and M. paradisiaca were collected and identified in the Herbarium Prisco Bezerra at the Federal University of Ceara, under numbers 44,919 and 44,920, respectively. 2.2. Obtaining the fractions and chemical analysis The fractions of S. mombin and M. paradisiaca used in biological tests were obtained from plant leaves by solvent extraction and column chromatography of crude extracts: Plant leaves were triturated, immersed into a methanol:water (4:1) mixture and left at room temperature for one week. After this period the leaves were filtered and the resultant solutions were placed in a rotary evaporator to eliminate the solvent to obtain the crude extracts. The crude extracts were submitted to chromatographic column, using silica gel as stationary phase. Chromatographic column is generally used as a
purification technique; it isolates desired compounds from a mixture. These extracts were mixed with silica gel (1:1) and placed on the top of a glass column filled with silica gel 60 and then eluted with the solvents hexane, chloroform, ethyl acetate and methanol in mixtures of increasing polarity. The solvents were passed through the column by gravity. 10 mL fractions of solvents were collected sequentially in labeled tubes and the composition of each fraction was analyzed by thin layer chromatography plates, using iodine and/or alcoholic FeCl3 solution for revelation. The main fractions were rechromatographed or submitted to crystallization for further purification. The fractions selected for leishmanicidal tests were named according to the solvents mixture used for elution. For M. paradisiaca: Mp1 - hexane: chloroform (50:50), Mp2 - ethyl acetate:methanol (90:10), Mp3: ethyl acetate:methanol (50:50), Mp4: methanol 100%. For S. mombin: Sm1 - chloroform:ethyl acetate (80:20), Sm2 - ethyl acetate:chloroform (90:10), Sm3 - ethyl acetate:methanol (80:20), Sm4 - methanol 100%. The selected fractions were analyzed by phytochemical tests and infrared and nuclear magnetic resonance spectroscopy (Silverstein & Webster, 1997) to characterize the types of secondary metabolites. The qualitative phytochemical tests of phenols, tannins, catechins, leucoanthocyanidins, flavonoids, steroids, terpenes, saponins and alkaloids were carried out in accordance with Matos (2009) and Harborne (1998). 2.3. In vitro tests 2.3.1. Test on promastigote The strains of L. chagasi MHOM46/LC/HZ1 were grown in M199 plus 10% fetal calf serum, 5% sterile human urine, and gentamicin (40 mg/ml) in a BOD chamber at 23.6 ◦ C (Tempone et al., 2005a). The promastigotes were counted in a neubauer chamber and the concentration used was 1 × 105 promastigotes/well. The fractions at concentrations of 100, 50, 25, 12.5 and 6.25 g/ml were, added to the plates with logarithmic phase promastigotes and left overnight. The viability of parasites was evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT) assay (Tempone et al., 2005b). Subsequently SDS 10% and 4 N HCl were added and read at 570 nm using a microplate reader. The experiment included pentamidine (40 g/ml) as a positive control and the medium without treatment as a negative control. 2.3.2. Test on amastigotes RAW 264.7 cells grown in Dulbecco’s medium plus 10% fetal calf serum and 40 mg/ml gentamicin were seeded in 96-well plates incubated at 36.6 ◦ C in 5% CO2 for 4 days to form the carpet cellular or confluent cells (Rondon et al., 2011). After the formation of these confluent cells, was added in the ratio 10:1 promastigote parasites/cell and were incubated again for 24 h. Before starting the tests, the infected cells were stained and parasites observed under a microscope. When we observed more than 50% of infected cells in a microscope field, the parasite load was considered appropriate to perform the tests with drugs in different concentrations. The fractions were added and
873, 758 873, 772 828, 765, 690 680 1096 1085 1105, 1026 1074 1214 1199 1261 1214 – 1323 1341 – – – 1442 1447 1445 1742 1718 1718 1732 – – – –
1625 1613 1625, 1583 1610
1063 1041 1076 1150, 1075 – 1242 1234 1236 – – – – 1378 – – 1382 1465 1448 1418 1526, 1429 1647 1670, 1647 1674, 1610 1682, 1647 – – – –
Alkyl C H stretch Alcohol/phenol O H Stretch
The phytochemical analysis of crude aqueous methanol extract of S. mombin revealed the presence of hydrolysable tannins (formation of dark blue precipitate in reaction with a hydro-alcoholic acid solution of FeCl3 ) and saponins (formation of stable spoon by shaking with water). In the crude methanol extract of M. paradisiaca, the phytochemical analysis showed condensed tannins (formation of dark green precipitate in reaction with hydro-alcoholic acid solution of FeCl3 ) and steroids (formation of a green color complex in presence of Lieberman-Burchard reagent). Table 1 shows infrared spectroscopic data of fractions obtained by chromatographic treatment of plants methanol extract. All S. mombin fractions showed similar infrared spectrum demonstrating the presence of phenolic hydroxyl (3408 cm−1 to 3424 cm−1 ), ester carbonyl groups (1742 cm−1 to 1718 cm−1 ), two aromatic skeleton bands (1625–1583 cm−1 and 1442 cm−1 to 1447 cm−1 ) and C O bands in the ester groups probably due to linkage with sugars (1377–1341 cm−1 ), C O from phenol groups (1261–1199 cm− 1), C O of sugar moiety (1096–1026 cm−1 ). For M. paradisiaca fractions Mp1 differ from the others since it gave a positive Lieberman-Burchard
Table 1 Infrared absorptions (cm−1 ) of M. paradisiaca and S. mombim fractions.
3. Results
C O ester
Alkenyl C C stretch
2.3.4. Statistical analysis The IC50 values (drug concentration able to inhibit 50% of parasites) with a confidence interval of 95% were calculated using a curve of non-linear regression using the statistical software GraphPad Prism 5.0. The entire experiment was performed in triplicate.
2930 2932 – –
C H (CH2 ) C H ()
C H (CH3 )
2.3.3. Cytotoxicity RAW 264.7 cells grown in Dulbecco’s medium plus 10% fetal calf serum and gentamicin were incubated in 96-well plates for 4 days at 36.6 ◦ C in 5% CO2 . The cells not adsorbed were removed, and 100 g mL−1 of plant fractions were added and incubated overnight OPD was added, and the reaction was stopped using 4 N HCl and read at 492 nm in a microplate reader.
M. paradisiaca 3407 Mp1 3366 Mp2 3362 Mp3 3407 Mp4 S. mombim Sm1 3408 Sm-2 3385 Sm3 3424 Sm4 3236
C O ester
C O phenol
C O alcohol/ether
Aromatic C H bending
incubated overnight. Thereafter, the testing was performed in situ by ELISA using the modified methodology of Piazza et al. (1994). Saponin (0.01% Sigma) diluted in 1× PBS supplemented with 1% bovine serum albumin (Sigma) was added to microplates for 30 min at 37 ◦ C. Blocking was done with 1× PBS plus 5% skim milk for 30 min at 37 ◦ C. Plates were washed 3 times, and the serum of rabbits immunized with promastigotes of L. chagasi diluted 1:500 in 1× PBS plus 3% skim milk, 0.05% Tween 20, and 10% fetal calf serum was added after drying and maintained overnight at 37 ◦ C. Conjugated anti-rabbit IgG (Sigma) was diluted 1:10,000 in PBS plus 3% skim milk and 0.05% Tween-20 was added, and after further washing orthophenylenediamine chromogen (OPD) was added. The reaction was stopped using 4 N HCl and read at 492 nm in microplate reader. The antileishmanial activity against intracellular amastigotes was determined with infected macrophages (Tempone et al., 2004). The experiment included amphotericin B (40 g/ml) as a positive control and medium with cells and promastigotes as a negative control.
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– 680 684 807, 699
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Table 2 IC50 values of the fractions from the leaves of banana tree (M. paradisiaca) and cajazeira (S. mombin). Fractions
IC50 promastigotes (g/ml)
IC95%
IC50 amastigotes (g/ml)
IC95%
Cytotoxicity (100 g/ml) % mortality
M. paradisiaca Mp1 Mp2 Mp3 Mp4
915.2c 1.83ac 21.60bc 1.70ac
3.41–24,441 0.34–9.79 11.12–41.95 0.02–12.91
15.07b 14.18b 95.31bc 16.54b
3.71–61.30 2.60–77.15 15.00–605.5 6.36–42.98
63.2g 36.3d 44.18f 68.7h
S. mombin Sm1 Sm2 Sm3 Sm4 Pentamidine Amphotericin B
246.10c 253.60ac 11.26abc 58.10bc 0.29–52.99abc –
3.92–15,458 8.37–7683 8.5–148.76 28.25–119.50 0.01–497.30 –
17.07abc 0.61a 0.27a 42.16b – 4.08–219.20abc
2.43–119.60 0.13–2.95 0.02–3.28 29.54–60.17 – 0.58–286.8
0.0a 31.3c 22.9b 40.2e – 1.7a
Equal letters in the same column indicate no statistical difference (p > 0.05).
test for steroid. The absorption bands in the infrared spectrum (3407, 2930, 1647, 1465, 1378 and 1063 cm−1 ) correspond to a sterol with a double band and an O H group. Mp2, Mp3 and Mp4 showed characteristics of condensed tannins as demonstrated by reaction with FeCl3 with infrared bands displayed in Table 1 (phenol O H, alkenyl C C of aromatic ring, C O phenol, C O alcohol/ether, aromatic C H). Four fractions from the crude extracts of the leaves of M. paradisiaca and four from S. mombin were obtained by silica gel column chromatography eluted with different polarity solvent mixtures: IC50 values on promastigotes, amastigotes and cytotoxicity are shown in Table 2. Among the fractions tested against promastigotes, the most effective were Mp2 and Mp4 with IC50 of 1.70 and 1.83 g/mL, respectively. No fraction differed statistically from the positive control pentamidine. The fractions Sm2 and Sm3 of S. mombin showed good leishmanicidal activity in vitro against L. chagasi amastigotes obtaining IC50 values of 0.61 g/ml and 0.27 g/ml, respectively and showed no (Sm2) and low (Sm3) cytotoxicity to RAW 264.7 cells. No fraction differed from the positive control amphotericin B. The fraction that showed better in vitro activity against both forms of the parasite and no cytotoxicity to RAW 264.7 cells, was Sm3 eluted with acetate: methanol (80:20) from S. mombin chromatographic column, with IC50 of 11.26 g/ml and 0.27 g/ml of promastigotes and amastigotes, respectively. 4. Discussion The phytochemical analysis of crude extract of S. mombin was similar to that described by Ayoka et al. (2006), which confirmed the presence of tannins, anthraquinones, flavonoids, leucociandins and saponins in aqueous and methanol extracts. Infrared spectroscopy data shown in Table 1 are characteristic of gallotannins or ellagitannins reported for this species (Corthout et al., 1990, 1991). Compounds Sm1 and Sm2 are galloylquinic acids due to absorptions in 1 HNMR spectra from quinic acid moiety (three oxymethine protons at ı 4.21, 4.35 and 5.03, two sets of methylene protons at ı 1.95–2.20, 2.00–2.09, and 2.15), and galloyl protons in the range of 6.77–7.56. References
and further reading may be available for this article. To view references and further reading you must purchase this artiSm3 and Sm4 present characteristics of galloyl-glucose by analysis of 1 HNMR spectra which show the presence of a glucose unity due to a broad signal between 4.38 and 4.28 ppm (3H), a large multiplete at 3.8 ppm (4H) and a multiplete centered in 7.19 ppm due to aromatic protons of galloyl moieties (Buziashvili et al., 1973). Oliveira (2007) reported in the crude extract of M. paradisiaca the presence of tannins, phenolic compounds, anthocyanins, alkaloids and phytosterols as beta-sitosterol and stigmasterol, also detected in this work. Mp-1 showed phytochemical and spectral characteristics of a steroid and the other fractions as condensed tannins. Bioassay-guided fractionation of the bark of Khaya senegalensis led to the isolation of two dimeric proanthocyanidins, catechin-(4a,6)-catechin (1) and catechin(4a,8)-catechin) (2), with immunostimulating activities. Anti-Leishmania screening of these compounds was conducted against L. donovani, Leishmania major, Leishmania infantum promastigotes and retested against L. donovani amastigotes persisting in RAW macrophages as host cells. Isolated compounds (1) and (2) were not active against promastigotes (EC50 25.0 g/ml), but exhibited significant effects when tested against amastigotes indicating an indirect immunostimulating principle (EC50 = 3.85 and 3.98 g/mL, respectively) with no cytotoxicity (Kayser and Abreu, 2001). Gallotannins were found in the seeds of Mangifera indica and showed antimicrobial effects, probably because of the property of hydrolysable tannins to form complexes by binding to metal ions (Engels et al., 2009). The same activity of gallotannins was reported by Tian et al. (2009a,b) with the plant Galla chinensis. There are also reports of polyphenols with antibacterial action, but the principle antibactericidal were not characterized (Kabuki et al., 2001). However, Engels et al. (2009) showed in subsequent searches with hydrolysable tannins that antimicrobial activity may be due to the presence of gallotannins, especially since the HPLC extract profiles were very similar to those reported by Kabuki et al. (2001). Other biological effects attributed to the tannins are their antiviral and antitumor activities (Yang et al., 2000). The
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antimicrobial activity of hydrolysable tannins has been attributed to several mechanisms of action, in particular its ability to interact with proteins and inhibit enzymatic activity (Konish et al., 1993). Another mechanism may be related to the ability of complexation with metal ions (Chung et al., 1998). This ability was correlated with the protection of plants against predators, such as animals, insects and microorganisms (Hatano et al., 1999). However, further studies of isolation of large amounts of hydrolysable tannins are being made to obtain a better understanding of structure-function relationship of phenolic compounds and examine its mechanism of action (Engels et al., 2009). Kolodziej et al. (2001) reported that several tannins have demonstrated activity against amastigotes of L. donovani and that differences in the leishmanicidal activity between promastigotes and amastigotes may be due to differences in biochemical or metabolic characteristics of the two stages of the parasite. Furthermore, the direct effect on amastigotes may be indicative of an activation of the functions of macrophages. The fraction Mp1 from M. paradisiaca showed absorptions in the spectra of carbon-13 and hydrogen corresponding to a mixture of the phytosterols -sitosterol and stigmasterol. There are no studies about leishmanicidal action of these two phytosteroids, however, they demonstrate antimicrobial activity, anti-inflammatory (Nishioka et al., 2002), anticarcinogenic (Awad and Fink, 2000) and cholesterol lowering (Law, 2000). Bouic and Lamprecht (1999) reported -sitosterol increasing the activity of Tlymphocytes and natural killer cells. Nishioka et al. (2002) reported that the antimicrobial and anti-inflammatory actions of Mallotus peltatus were caused by a combination of several substances, among them the -sitosterol. The same anti-inflammatory effect of -sitosterol and stigamasterol isolated from the leaves of Croton pullei were covered by Rocha et al. (2008), although the mechanisms responsible for the effect are not yet fully understood. Another biological activity demonstrated in M. paradisiaca was antiulcerogenic and the two compounds responsible for this activity were two acilglicosides of -sitosterol (Kovganko and Kashkan, 1994). We conclude that some fractions used showed good leishmanicidal activity in vitro, suggesting that the use of herbal and natural products as potential agents for the treatment of tropical diseases caused by protozoa. The gallotannins are probably the secondary metabolites responsible for this activity and promising compounds for further studies to be conducted in vivo. Acknowledgments This work received financial support from CNPq (grant 464390/0). Dr. Claudia M.L. Bevilaqua and Selene Maia de Morais have a grant from CNPq. Authors would like to thank Dr. Nilce Viana Gramosa (CENAUREN/UFC) for obtaining the spectra of 1H and 13C NMR. References Albuquerque, U.P., Medeiros, P.M., Almeida, A.L.S., Monteiro, J.M., Neto, E.M.F.L., Melo, J.G., Santos, J.P., 2007. Medicinal plants of the caatinga
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Leishmanicidal activity in vitro of Musa paradisiaca L. and Spondias mombin L. fractions Marina Parissi Accioly a , Claudia Maria Leal Bevilaqua a,∗ , Fernanda C.M. Rondon a , Selene Maia de Morais a , Lyeghyna K.A. Machado a , Camila A. Almeida a , Heitor Franco de Andrade Jr. b , Roselaine P.A. Cardoso b a b
Programa de Pós-graduac¸ão em Ciências Veterinárias/Universidade Estadual do Ceará, Brazil Laboratório de Protozoologia/Universidade de São Paulo, Brazil
a r t i c l e
i n f o
Article history: Received 27 June 2011 Received in revised form 12 October 2011 Accepted 21 December 2011 Keywords: Visceral leishmaniasis Secondary metabolites Banana tree Cajazeira
a b s t r a c t Visceral leishmaniasis (VL) is a zoonotic disease characterized by infection of mononuclear phagocytes by Leishmania chagasi. The primary vector is Lutzomyia longipalpis and the dog is the main domestic reservoir. The control and current treatment of dogs using synthetic drugs have not shown effectiveness in reducing the incidence of disease in man. In attempt to find new compounds with leishmanicidal action, plant secondary metabolites have been studied in search of treatments of VL. This study aimed to evaluate the leishmanicidal activity of Musa paradisiaca (banana tree) and Spondias mombin (cajazeira) chemical constituents on promastigotes and amastigotes of L. chagasi. Phytochemical analysis by column chromatography was performed on ethanol extracts of two plants and fractions were isolated. Thin layer chromatography was used to compare the fractions and for isolation the substances to be used in vitro tests. The in vitro tests on promastigotes of L. chagasi used the MTT colorimetric method and the method of ELISA in situ was used against amastigotes besides the cytotoxicity in RAW 264.7 cells. Of the eight fractions tested, Sm1 and Sm2 from S. mombin had no action against promastigotes, but had good activity against amastigotes. The fractions Mp1 e Mp4 of M. paradisiaca were very cytotoxic to RAW 264.7 cells. The best result was obtained with the fraction Sm3 from S. mombin with IC50 of 11.26 g/ml against promastigotes and amastigotes of 0.27 g/ml. The fraction Sm3 characterized as tannic acid showed the best results against both forms of Leishmania being a good candidate for evaluation in in vivo tests. © 2012 Published by Elsevier B.V.
1. Introduction Leishmaniasis is a zoonotic disease with worldwide distribution and is considered by the World Health Organization (WHO) one of the six most important tropical diseases. Visceral leishmaniasis (VL) is caused by the subgenus Leishmania leishmania, Leishmania donovani complex
∗ Corresponding author at: Av. Dede Brasil, 1700, 60740-913 Fortaleza, Ceará, Brazil. Tel.: +55 85 31019853; fax: +55 85 31019840. E-mail address: [email protected] (C.M.L. Bevilaqua). 0304-4017/$ – see front matter © 2012 Published by Elsevier B.V. doi:10.1016/j.vetpar.2011.12.029
(Gontijo and Melo, 2004; Rath et al., 2003). It is considered endemic in the Mediterranean and extends to Latin America from Mexico to Argentina (Lainson and Rangel, 2005). Although several animal species may become infected (Luppi et al., 2008), the main domestic reservoir of this disease is the dog (Diniz et al., 2008) and Lutzomyia longipalpis is the primary sand fly vector (Wood et al., 2003). As recommended by the WHO, the control of LV is highlighted by the serological survey of dogs and euthanasia in cases in which they are positive, diagnosis and treatment of human cases and residual insecticide application based on pyrethroid (Melo, 2004). Treatment of dogs with drugs
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used in humans such as N-methyl glucamine, sodium stibogluconate, amphotericin B and others may decrease the symptoms, but are not able to eliminate all of the parasites. Therefore, the use of drugs in dogs is very limited and results in the dog remaining infected, maintaining the transmission cycle (Mishra et al., 2009). In Brazil, ministerial decree No. 1426 of July, 11th of 2008, prohibits the treatment of canine visceral leishmaniasis with products for human use or those not registered in the Ministério da Agricultura, Pecuária e Abastecimento. Therefore, there is a need to develop new therapies aimed at the control and treatment in the quest for a better quality of life and control of diseases of public health importance (Araújo, 2006). The use of medicinal plants and studies on the chemistry and pharmacology of natural products have grown considerably in the second half of the 20th century (Albuquerque et al., 2007). Many compounds derived from natural sources have pharmacological activities and may be used for drug development (Lawrence, 2003). Several compounds isolated from plants such as terpenoids, steroids, flavonoids, alkaloids, phenolic compounds and naphthoquinones have been studied to evaluate their effects on promastigotes and amastigotes of different species of leishmania. Some of these substances can be found in the leaves of cajazeira (Spondias mombin L.), banana (Musa paradisiaca L.) trees and other plants common in the Brazilian Northeast which present easy access to the population, and have being reported as having biochemical and pharmacological activities in vitro (Kolodziej and Kinderlen, 2005). Compounds from the banana trees leaves, such as the tannins have shown antimicrobial properties (Olivo et al., 2007) and the leaves from the cajazeiras have show besides the antimicrobial properties, healing action (Njoku and Akumefala, 2007). The objective of this study was to evaluate the leishmanicidal effect in vitro of the fractions obtained from crude extracts of leaves of cajazeira and banana tree on promastigotes and amastigotes of Leishmania chagasi. 2. Material and methods 2.1. Collection of plants Samples of the aerial parts of S. mombin and M. paradisiaca were collected and identified in the Herbarium Prisco Bezerra at the Federal University of Ceara, under numbers 44,919 and 44,920, respectively. 2.2. Obtaining the fractions and chemical analysis The fractions of S. mombin and M. paradisiaca used in biological tests were obtained from plant leaves by solvent extraction and column chromatography of crude extracts: Plant leaves were triturated, immersed into a methanol:water (4:1) mixture and left at room temperature for one week. After this period the leaves were filtered and the resultant solutions were placed in a rotary evaporator to eliminate the solvent to obtain the crude extracts. The crude extracts were submitted to chromatographic column, using silica gel as stationary phase. Chromatographic column is generally used as a
purification technique; it isolates desired compounds from a mixture. These extracts were mixed with silica gel (1:1) and placed on the top of a glass column filled with silica gel 60 and then eluted with the solvents hexane, chloroform, ethyl acetate and methanol in mixtures of increasing polarity. The solvents were passed through the column by gravity. 10 mL fractions of solvents were collected sequentially in labeled tubes and the composition of each fraction was analyzed by thin layer chromatography plates, using iodine and/or alcoholic FeCl3 solution for revelation. The main fractions were rechromatographed or submitted to crystallization for further purification. The fractions selected for leishmanicidal tests were named according to the solvents mixture used for elution. For M. paradisiaca: Mp1 - hexane: chloroform (50:50), Mp2 - ethyl acetate:methanol (90:10), Mp3: ethyl acetate:methanol (50:50), Mp4: methanol 100%. For S. mombin: Sm1 - chloroform:ethyl acetate (80:20), Sm2 - ethyl acetate:chloroform (90:10), Sm3 - ethyl acetate:methanol (80:20), Sm4 - methanol 100%. The selected fractions were analyzed by phytochemical tests and infrared and nuclear magnetic resonance spectroscopy (Silverstein & Webster, 1997) to characterize the types of secondary metabolites. The qualitative phytochemical tests of phenols, tannins, catechins, leucoanthocyanidins, flavonoids, steroids, terpenes, saponins and alkaloids were carried out in accordance with Matos (2009) and Harborne (1998). 2.3. In vitro tests 2.3.1. Test on promastigote The strains of L. chagasi MHOM46/LC/HZ1 were grown in M199 plus 10% fetal calf serum, 5% sterile human urine, and gentamicin (40 mg/ml) in a BOD chamber at 23.6 ◦ C (Tempone et al., 2005a). The promastigotes were counted in a neubauer chamber and the concentration used was 1 × 105 promastigotes/well. The fractions at concentrations of 100, 50, 25, 12.5 and 6.25 g/ml were, added to the plates with logarithmic phase promastigotes and left overnight. The viability of parasites was evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT) assay (Tempone et al., 2005b). Subsequently SDS 10% and 4 N HCl were added and read at 570 nm using a microplate reader. The experiment included pentamidine (40 g/ml) as a positive control and the medium without treatment as a negative control. 2.3.2. Test on amastigotes RAW 264.7 cells grown in Dulbecco’s medium plus 10% fetal calf serum and 40 mg/ml gentamicin were seeded in 96-well plates incubated at 36.6 ◦ C in 5% CO2 for 4 days to form the carpet cellular or confluent cells (Rondon et al., 2011). After the formation of these confluent cells, was added in the ratio 10:1 promastigote parasites/cell and were incubated again for 24 h. Before starting the tests, the infected cells were stained and parasites observed under a microscope. When we observed more than 50% of infected cells in a microscope field, the parasite load was considered appropriate to perform the tests with drugs in different concentrations. The fractions were added and
873, 758 873, 772 828, 765, 690 680 1096 1085 1105, 1026 1074 1214 1199 1261 1214 – 1323 1341 – – – 1442 1447 1445 1742 1718 1718 1732 – – – –
1625 1613 1625, 1583 1610
1063 1041 1076 1150, 1075 – 1242 1234 1236 – – – – 1378 – – 1382 1465 1448 1418 1526, 1429 1647 1670, 1647 1674, 1610 1682, 1647 – – – –
Alkyl C H stretch Alcohol/phenol O H Stretch
The phytochemical analysis of crude aqueous methanol extract of S. mombin revealed the presence of hydrolysable tannins (formation of dark blue precipitate in reaction with a hydro-alcoholic acid solution of FeCl3 ) and saponins (formation of stable spoon by shaking with water). In the crude methanol extract of M. paradisiaca, the phytochemical analysis showed condensed tannins (formation of dark green precipitate in reaction with hydro-alcoholic acid solution of FeCl3 ) and steroids (formation of a green color complex in presence of Lieberman-Burchard reagent). Table 1 shows infrared spectroscopic data of fractions obtained by chromatographic treatment of plants methanol extract. All S. mombin fractions showed similar infrared spectrum demonstrating the presence of phenolic hydroxyl (3408 cm−1 to 3424 cm−1 ), ester carbonyl groups (1742 cm−1 to 1718 cm−1 ), two aromatic skeleton bands (1625–1583 cm−1 and 1442 cm−1 to 1447 cm−1 ) and C O bands in the ester groups probably due to linkage with sugars (1377–1341 cm−1 ), C O from phenol groups (1261–1199 cm− 1), C O of sugar moiety (1096–1026 cm−1 ). For M. paradisiaca fractions Mp1 differ from the others since it gave a positive Lieberman-Burchard
Table 1 Infrared absorptions (cm−1 ) of M. paradisiaca and S. mombim fractions.
3. Results
C O ester
Alkenyl C C stretch
2.3.4. Statistical analysis The IC50 values (drug concentration able to inhibit 50% of parasites) with a confidence interval of 95% were calculated using a curve of non-linear regression using the statistical software GraphPad Prism 5.0. The entire experiment was performed in triplicate.
2930 2932 – –
C H (CH2 ) C H ()
C H (CH3 )
2.3.3. Cytotoxicity RAW 264.7 cells grown in Dulbecco’s medium plus 10% fetal calf serum and gentamicin were incubated in 96-well plates for 4 days at 36.6 ◦ C in 5% CO2 . The cells not adsorbed were removed, and 100 g mL−1 of plant fractions were added and incubated overnight OPD was added, and the reaction was stopped using 4 N HCl and read at 492 nm in a microplate reader.
M. paradisiaca 3407 Mp1 3366 Mp2 3362 Mp3 3407 Mp4 S. mombim Sm1 3408 Sm-2 3385 Sm3 3424 Sm4 3236
C O ester
C O phenol
C O alcohol/ether
Aromatic C H bending
incubated overnight. Thereafter, the testing was performed in situ by ELISA using the modified methodology of Piazza et al. (1994). Saponin (0.01% Sigma) diluted in 1× PBS supplemented with 1% bovine serum albumin (Sigma) was added to microplates for 30 min at 37 ◦ C. Blocking was done with 1× PBS plus 5% skim milk for 30 min at 37 ◦ C. Plates were washed 3 times, and the serum of rabbits immunized with promastigotes of L. chagasi diluted 1:500 in 1× PBS plus 3% skim milk, 0.05% Tween 20, and 10% fetal calf serum was added after drying and maintained overnight at 37 ◦ C. Conjugated anti-rabbit IgG (Sigma) was diluted 1:10,000 in PBS plus 3% skim milk and 0.05% Tween-20 was added, and after further washing orthophenylenediamine chromogen (OPD) was added. The reaction was stopped using 4 N HCl and read at 492 nm in microplate reader. The antileishmanial activity against intracellular amastigotes was determined with infected macrophages (Tempone et al., 2004). The experiment included amphotericin B (40 g/ml) as a positive control and medium with cells and promastigotes as a negative control.
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– 680 684 807, 699
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Table 2 IC50 values of the fractions from the leaves of banana tree (M. paradisiaca) and cajazeira (S. mombin). Fractions
IC50 promastigotes (g/ml)
IC95%
IC50 amastigotes (g/ml)
IC95%
Cytotoxicity (100 g/ml) % mortality
M. paradisiaca Mp1 Mp2 Mp3 Mp4
915.2c 1.83ac 21.60bc 1.70ac
3.41–24,441 0.34–9.79 11.12–41.95 0.02–12.91
15.07b 14.18b 95.31bc 16.54b
3.71–61.30 2.60–77.15 15.00–605.5 6.36–42.98
63.2g 36.3d 44.18f 68.7h
S. mombin Sm1 Sm2 Sm3 Sm4 Pentamidine Amphotericin B
246.10c 253.60ac 11.26abc 58.10bc 0.29–52.99abc –
3.92–15,458 8.37–7683 8.5–148.76 28.25–119.50 0.01–497.30 –
17.07abc 0.61a 0.27a 42.16b – 4.08–219.20abc
2.43–119.60 0.13–2.95 0.02–3.28 29.54–60.17 – 0.58–286.8
0.0a 31.3c 22.9b 40.2e – 1.7a
Equal letters in the same column indicate no statistical difference (p > 0.05).
test for steroid. The absorption bands in the infrared spectrum (3407, 2930, 1647, 1465, 1378 and 1063 cm−1 ) correspond to a sterol with a double band and an O H group. Mp2, Mp3 and Mp4 showed characteristics of condensed tannins as demonstrated by reaction with FeCl3 with infrared bands displayed in Table 1 (phenol O H, alkenyl C C of aromatic ring, C O phenol, C O alcohol/ether, aromatic C H). Four fractions from the crude extracts of the leaves of M. paradisiaca and four from S. mombin were obtained by silica gel column chromatography eluted with different polarity solvent mixtures: IC50 values on promastigotes, amastigotes and cytotoxicity are shown in Table 2. Among the fractions tested against promastigotes, the most effective were Mp2 and Mp4 with IC50 of 1.70 and 1.83 g/mL, respectively. No fraction differed statistically from the positive control pentamidine. The fractions Sm2 and Sm3 of S. mombin showed good leishmanicidal activity in vitro against L. chagasi amastigotes obtaining IC50 values of 0.61 g/ml and 0.27 g/ml, respectively and showed no (Sm2) and low (Sm3) cytotoxicity to RAW 264.7 cells. No fraction differed from the positive control amphotericin B. The fraction that showed better in vitro activity against both forms of the parasite and no cytotoxicity to RAW 264.7 cells, was Sm3 eluted with acetate: methanol (80:20) from S. mombin chromatographic column, with IC50 of 11.26 g/ml and 0.27 g/ml of promastigotes and amastigotes, respectively. 4. Discussion The phytochemical analysis of crude extract of S. mombin was similar to that described by Ayoka et al. (2006), which confirmed the presence of tannins, anthraquinones, flavonoids, leucociandins and saponins in aqueous and methanol extracts. Infrared spectroscopy data shown in Table 1 are characteristic of gallotannins or ellagitannins reported for this species (Corthout et al., 1990, 1991). Compounds Sm1 and Sm2 are galloylquinic acids due to absorptions in 1 HNMR spectra from quinic acid moiety (three oxymethine protons at ı 4.21, 4.35 and 5.03, two sets of methylene protons at ı 1.95–2.20, 2.00–2.09, and 2.15), and galloyl protons in the range of 6.77–7.56. References
and further reading may be available for this article. To view references and further reading you must purchase this artiSm3 and Sm4 present characteristics of galloyl-glucose by analysis of 1 HNMR spectra which show the presence of a glucose unity due to a broad signal between 4.38 and 4.28 ppm (3H), a large multiplete at 3.8 ppm (4H) and a multiplete centered in 7.19 ppm due to aromatic protons of galloyl moieties (Buziashvili et al., 1973). Oliveira (2007) reported in the crude extract of M. paradisiaca the presence of tannins, phenolic compounds, anthocyanins, alkaloids and phytosterols as beta-sitosterol and stigmasterol, also detected in this work. Mp-1 showed phytochemical and spectral characteristics of a steroid and the other fractions as condensed tannins. Bioassay-guided fractionation of the bark of Khaya senegalensis led to the isolation of two dimeric proanthocyanidins, catechin-(4a,6)-catechin (1) and catechin(4a,8)-catechin) (2), with immunostimulating activities. Anti-Leishmania screening of these compounds was conducted against L. donovani, Leishmania major, Leishmania infantum promastigotes and retested against L. donovani amastigotes persisting in RAW macrophages as host cells. Isolated compounds (1) and (2) were not active against promastigotes (EC50 25.0 g/ml), but exhibited significant effects when tested against amastigotes indicating an indirect immunostimulating principle (EC50 = 3.85 and 3.98 g/mL, respectively) with no cytotoxicity (Kayser and Abreu, 2001). Gallotannins were found in the seeds of Mangifera indica and showed antimicrobial effects, probably because of the property of hydrolysable tannins to form complexes by binding to metal ions (Engels et al., 2009). The same activity of gallotannins was reported by Tian et al. (2009a,b) with the plant Galla chinensis. There are also reports of polyphenols with antibacterial action, but the principle antibactericidal were not characterized (Kabuki et al., 2001). However, Engels et al. (2009) showed in subsequent searches with hydrolysable tannins that antimicrobial activity may be due to the presence of gallotannins, especially since the HPLC extract profiles were very similar to those reported by Kabuki et al. (2001). Other biological effects attributed to the tannins are their antiviral and antitumor activities (Yang et al., 2000). The
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antimicrobial activity of hydrolysable tannins has been attributed to several mechanisms of action, in particular its ability to interact with proteins and inhibit enzymatic activity (Konish et al., 1993). Another mechanism may be related to the ability of complexation with metal ions (Chung et al., 1998). This ability was correlated with the protection of plants against predators, such as animals, insects and microorganisms (Hatano et al., 1999). However, further studies of isolation of large amounts of hydrolysable tannins are being made to obtain a better understanding of structure-function relationship of phenolic compounds and examine its mechanism of action (Engels et al., 2009). Kolodziej et al. (2001) reported that several tannins have demonstrated activity against amastigotes of L. donovani and that differences in the leishmanicidal activity between promastigotes and amastigotes may be due to differences in biochemical or metabolic characteristics of the two stages of the parasite. Furthermore, the direct effect on amastigotes may be indicative of an activation of the functions of macrophages. The fraction Mp1 from M. paradisiaca showed absorptions in the spectra of carbon-13 and hydrogen corresponding to a mixture of the phytosterols -sitosterol and stigmasterol. There are no studies about leishmanicidal action of these two phytosteroids, however, they demonstrate antimicrobial activity, anti-inflammatory (Nishioka et al., 2002), anticarcinogenic (Awad and Fink, 2000) and cholesterol lowering (Law, 2000). Bouic and Lamprecht (1999) reported -sitosterol increasing the activity of Tlymphocytes and natural killer cells. Nishioka et al. (2002) reported that the antimicrobial and anti-inflammatory actions of Mallotus peltatus were caused by a combination of several substances, among them the -sitosterol. The same anti-inflammatory effect of -sitosterol and stigamasterol isolated from the leaves of Croton pullei were covered by Rocha et al. (2008), although the mechanisms responsible for the effect are not yet fully understood. Another biological activity demonstrated in M. paradisiaca was antiulcerogenic and the two compounds responsible for this activity were two acilglicosides of -sitosterol (Kovganko and Kashkan, 1994). We conclude that some fractions used showed good leishmanicidal activity in vitro, suggesting that the use of herbal and natural products as potential agents for the treatment of tropical diseases caused by protozoa. The gallotannins are probably the secondary metabolites responsible for this activity and promising compounds for further studies to be conducted in vivo. Acknowledgments This work received financial support from CNPq (grant 464390/0). Dr. Claudia M.L. Bevilaqua and Selene Maia de Morais have a grant from CNPq. Authors would like to thank Dr. Nilce Viana Gramosa (CENAUREN/UFC) for obtaining the spectra of 1H and 13C NMR. References Albuquerque, U.P., Medeiros, P.M., Almeida, A.L.S., Monteiro, J.M., Neto, E.M.F.L., Melo, J.G., Santos, J.P., 2007. Medicinal plants of the caatinga
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