Evaluation of the bactericidal and trypanocidal activities of triterpenes isolated from the leaves, stems, and flowers of Lychnophora pinaster

Evaluation of the bactericidal and trypanocidal activities of triterpenes isolated from the leaves, stems, and flowers of Lychnophora pinaster

Viviane G. C. Abreu^I; Jacqueline A. Takahashi^I; Lucienir P. Duarte^I; Dorila Piló-Veloso^I; Policarpo A. S. Junior^II; Rosana O. Alves^II; Alvaro J. Romanha^II; Antônio F. C. Alcântara^I,*

^IDepartamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Brazil

^IILaboratório de Parasitologia Celular e Molecular, Centro de Pesquisas René Rachou-Fiocruz, Brazil

ABSTRACT

The phytochemical investigation on the aereal parts of Lychnophora pinaster Mart., Asteraceae, was carried to isolation of triterpenes. 3-O-Acetyl-lupeol (1), 3-O-acetyl-pseudotaraxasterol (2), and 3-O-acetyl-α-amyrin (3) were isolated from hexanic extract and 4,4-dimethyl-cholesta-22,24-dien-5-ol (4), α-amyrin (5), and lupeol (6) were isolated from hexanic/dichlorometanic extract of the leaves. Compounds Δ⁷-bauerenyl acetate (7), friedelin (8), stigmasterol (9), and sitosterol (10) were isolated from the hexanic/dichlorometanic extract of the stems. The steroids 9 and 10 were also isolated from the hexanic/dichlorometanic extract of the flowers. Triterpenes 1, 3, 4, and 7 are described for the first time in the genus Lychnophora. The apolar fractions of the leaf and stem extracts and some isolated triterpenes showed low trypanocidal activity. Moreover, apolar fractions of the leaf and stem extracts and 5 showed antibacterial action against Staphylococcus aureus.

Keywords:Lychnophora pinaster triterpenes bactericidal activity trypanocidal activity

Introduction

The Asteraceae family comprises about 23000 species distributed in 1600 genera (Gao et al., 2010). The genus Lychnophora (Asteraceae) is endemic in the central region of Brazil, mainly in rupestral fields in the states of Minas Gerais, Goiás, and Bahia (Robinson, 1999). This genus comprises 68 species (Mansanares et al., 2002) and some of them are listed as species threatened with extinction (Silveira et al., 2005). Lychnophora species are popularly used to treat wounds, bruise, pain, rheumatism, and inflammation (Borsato et al., 2000). Lychnophora species also show antitumoral (Merten et al., 2006), antimicrobial (Saúde et al., 2002), anti-pyretic, analgesic (Ferraz Filha et al., 2006), antioxidant, anticonvulsivant (Taleb-Contini et al., 2008), and trypanocidal (Grael et al., 2005) properties. Their biological activities have been attributed to sesquiterpene lactones, lignans, and caffeoylquinic acid derivatives isolated from polar extracts of the leaves and roots (Santos et al., 2005). The trypanocidal activity of Lychnophora species has been attributed to flavanoids, sesquiterpene lactones, and caffeoylquinic acids isolated from their alcoholic extracts (Takeara et al., 2003).

Lychnophora pinaster Mart., Asteraceae, is popularly used as analgesic, anti-rheumatic, and trypanocidal agents (Silveira et al., 2005). This species also shows significant antinociceptive and anti-inflammatory activities (Guzzo et al., 2008). Rutin, quercetin, isochlorogenic acid, caffeic acid, isovitexin, vitexin, sesquiterpene derivatives (Alcântara et al., 2005; Leite et al., 2008), and the triterpenes lupeol, a mixture of α- and </font>&#946;<font size="2" face="Verdana, Arial, Helvetica, sans-serif">-amyrin, and friedelin (Silveira et al., 2005) were previously isolated from its aerial parts. Although several studies have lately related triterpenes to treatment of the trypanosomiases (Rosas et al., 2007; Leite et al., 2006; Cunha et al., 2006) and bactericidal activity (Al-Fatimi et al., 2010), no data are available about the trypanocidal and bactericidal activities of triterpenes isolated from Lychnophora species. The present work describes an investigation of the chemistry of leaves, stems, and flowers of L. pinaster for triterpenes, which were tested for antibacterial and trypanocidal activities.

Materials and Methods

General procedures

Uncorrected melting points were determined using Mettler equipment, model FP80 SNR H22439. The IR spectra were taken on a Perkin Elmer - Spectrum One (ATR) spectrometer. The ¹H and ¹³C NMR spectra as well as the ¹H-¹H COSY, ¹H-¹H NOESY, ¹H-¹³C HMBC, ¹H-¹³C HMQC, and ¹H-¹³C HSQC experiments were performed on a Bruker DRX400 AVANCE spectrometer, using CDCl₃ as solvent. The chemical shifts were measured in parts per million (Δ) relative to TMS, which was used as an internal standard. The coupling constants (J) were recorded in Hertz. Mass spectra (GC-EI-MS) were obtained using a Varian 2800 gas chromatograph coupled to Varian Saturn 4000 electron impact mass spectrometer. The chromatographic separations were obtained using 100% dimethylpolysiloxane HP-1 column (50 m x 0.25 mm i.d., 0.20 mm film) under the following conditions: column temperature programmed from 150 ºC (isothermal for 7 min) to 300 ºC at the rate of 40 ºC/min, maintaining final temperature for 40 min. The mobile phase was H₂ (1.0 mL/min). The temperature of the injector and detector were 250 ºC and 300 ºC, respectively. An aliquot (3.0 µL) of sample solution (10 µg/100 µL) was injected and desorption in the GC injector was carried out in the split mode (1:15). The detector voltage was 300 V, the electron impact ionization potential was 70 eV, the acquisition rate was twenty spectra/s, and the mass range was 30 to 500 (m/z). The compounds were identified through comparison of the fragmentation profile of mass spectrum of the sample with the correspondent available in the NIST (2005) standard mass fragmentation data bank.

Plant material

Lychnophora pinaster Mart., Asteraceae, was collected in April 2007 at Moeda Mountain, in Nova Lima city, Minas Gerais State (Brazil). A voucher specimen of L. pinaster was deposited in the herbarium of the Instituto de Ciências Biológicas of the Universidade Federal de Minas Gerais, under code BHCB: 24322.

Extraction and isolation of constituents

Leaves, stems, and flowers of L. pinaster were previously separated, dried at 60 ºC until constant weight was achieved (about one week), and finally powdered. The samples were named L (5698.0 g), S (4604.0 g), and F (710.0 g), respectively. All samples were submitted to EtOH extraction for a week, providing the corresponding extracts LE (625.16 g), SE (144.37 g), and FE (79.48 g) after solvent evaporation. LE, SE, and FE were submitted to chromatographic column (CC) using silica gel as the stationary phase (CCS) and elution with hexane, CH₂Cl₂, and EtOAc in increasing polarity order. Fractions of LE eluted with hexane (94.92 g) were again submitted to CCS eluted with hexane and CH₂Cl₂ in increasing polarity order, successively providing a mixture of 1 and 2 (5.14 g) and compound 3 recrystallized from MeOH (1.19 g). The LE fractions eluted with hexane/CH₂Cl₂ (1:1) (LE-2; 7.59 g) were again submitted to CCS eluted with hexane and CH₂Cl₂ in increasing polarity order. The LE-2 fractions eluted with hexane/CH₂Cl₂ (4:1) were recrystallized from MeOH, providing 4 (0.009 g). The LE-2 fractions eluted with hexane/CH₂Cl₂ (3:1) were recrystallized from MeOH, providing 5 (1.250 g). The LE-2 fractions eluted with hexane/CH₂Cl₂ (1:1) were recrystallized from MeOH, providing a mixture of 5 and 6 (1.139 g).

The SE fractions eluted with hexane/CH₂Cl₂ (9:1) provided a white solid, which was recrystallized from acetone (SE-1; 0.126 g). SE-1 was submitted to CC using alumina as stationary phase and eluted with hexane and CH₂Cl₂ in increasing polarity order. The fractions eluted with hexane provided 7 (0.051 g). The SE-1 fractions eluted with hexane/CH₂Cl₂ (7:3) provided 8 (0.035 g). The SE fractions eluted with hexane/CH₂Cl₂ (4:1) were washed with petroleum ether and recrystallized from EtOH, providing a mixture of 5 and 6 (0.041 g). The SE fractions eluted with hexane/CH₂Cl₂ (1:1) were dissolved in a mixture of MeOH and activated charcoal, and filtered to remove chlorophyll. The white solid was submitted to CCS eluted with hexane and CH₂Cl₂, in increasing polarity order. The fractions eluted with CH₂Cl₂ were recrystallized from EtOH, providing a mixture of 9 and 10 in a ratio of 2.5:1.0 (0.340 g). The FE fractions eluted with hexane/CH₂Cl₂ (1:1) (7.231 g) underwent the same procedure for chlorophyll extraction described above. The white solid was submitted to CCS eluted with hexane and CH₂Cl₂ in increasing polarity order. The fractions eluted with hexane/CH₂Cl₂ (1:1) were recrystallized from MeOH, providing a mixture of 9 and 10 in a ratio of 2.5:1.0 (1.165 g).

Antibacterial tests

Bioassays were conducted with Gram-positive (Staphylococcus aureus ATCC 29213, Bacillus cereus ATCC 11779, and Listeria monocytogenes ATCC 15313) and Gram-negative (Escherichia coli ATCC 25723, Salmonella typhimurium ATCC 14028, and Citrobacter freundi ATCC 8090) bacteria. In the agar diffusion test (Takahashi et al., 2006), microorganisms were individually inoculated in vials containing Brain Heart Infusion broth (BHI) and subsequently incubated in an oven at 37 ºC for 18 h. An aliquot of this material was transferred to a tube containing solutions of NaCl and MgSO₄.7H₂O to prepare the inoculum. Petri dishes containing culture medium (#1 Antibiotic Broth) and inoculum were prepared. Sterile 6 mm diameter filter paper discs were impregnated with 1 mL of each extract (100 µg/mL). Disks containing each sample were placed on the Petri dishes with the aid of sterile tweezers. A disk containing the positive control (disk impregnated with antibiotic chloramphenicol) or negative control (disk containing the solvent used to dissolve the sample) was placed at the center of each disk. The inhibition zones were read after 24 h of incubation.

In the minimum inhibitory concentration (MIC) test (Lana et al., 2006), microorganisms were inoculated into test tubes containing BHI broth and incubated in an oven at 37 ºC for 18 h. Then, this suspension was transferred to a tube containing sterile saline solution to reach a suspension (inoculum) compatible with the McFarland scale 5. To assay each sample, test tubes containing BHI culture medium were used. Samples dissolved in DMSO were placed in the test tubes. The inoculum was added to each test tube. The tubes were incubated in an oven at 35 ºC for 18 h. Readings were done after 18 h of incubation. MIC value was assigned to the tube containing the smallest dilution that did not present turbidity, and thus was the lowest concentration that inhibited the growth of the test microorganism.

Trypanocidal test

The in vitro assay with T. cruzi blood stream forms was carried out using blood from Swiss albino mice collected in the parasitaemia peak (7^th day) after infection with the Y strain of T. cruzi (Oliveira et al., 2006). The infected blood was diluted with normal murine blood and RPMI 1640 medium 1:2 (pH 7.2-7.4) to the concentration of 2.0 x 10⁶ trypomastigotes/mL. Solutions of each sample were prepared in DMSO and added to infected blood in duplicate to the wells of a 96 microwell plate providing a final drug concentration of 500 µg/mL (1.2-3.0 mM). To reproduce the blood bank conditions, plates were incubated at 4 ºC for 24 h. The experiments were repeated two times. Afterwards, the parasite concentration was evaluated using an optical microscope with 400x magnification. DMSO and gentian violet were used as negative and positive controls, respectively. DMSO was not added to the positive control. The trypanocidal activity was expressed as percent reduction of the parasite number (lysis) comparing the wells with drugs with those without drugs.

Results and Discussion

Identification of the isolated compounds

The mixture of 1 and 2 showed strong IR absorptions at 1733, 1244, and 1174 cm^-1, which are characteristic of ester groups. The ¹H NMR spectrum showed signals at δ_H 5.36-5.12 and 4.69-4.45 attributed to alkenylic and carbinolic hydrogen atoms, respectively. The other hydrogen signals are registered between δ_H 2.0 and 0.8, characteristic of hydrogen atoms of pentacyclic triterpenes. The ¹³C NMR showed signals at δ_C 174.0, 171.0, 81.3, and 80.9, which were assigned to two ester groups (Olea & Roque, 1990). Signals at δ_C 151.2 and 109.6 are characteristic of lupeol derivatives and signals at δ_C 139.8 and 118.9 are characteristic of pseudotaraxasterol derivatives. The ¹³C NMR data of the mixture of 1 and 2 are in agreement with the corresponding data described for 3-O-acetyl-lupeol and 3-O-acetyl-pseudotaraxasterol, respectively (Mahato & Kundu, 1994).

The IR spectrum of 3 was similar to that of 1 and 2, also indicating the structure of an acetylated triterpene. The mass spectrum showed the molecular ion peak at m/z 468, which corresponds to C₃₂H₅₂O₂. The ¹H NMR spectrum showed signals at δ_H 5.16 and 4.51, attributed to alkenyl and carbinolic hydrogen atoms, respectively. The other hydrogen signals are registered between δ_H 2.0 and 0.8. The ¹³C NMR spectrum showed signals at δ_C 171.4, 139.6, and 124.3, which are characteristic of acetylated triterpene with ursan skeleton. The HSQC contour map showed correlations between singlet signal at δ_H 2.05 with the carbon signal at δ_C 21.3, characteristic of an acetyl group. The signal at δ_H 2.05 correlates with carbon signal at δ_C 81.0 and signal at δ_H 4.51 correlates with carbon signal at δ_C 171.4, by HMBC experiments, indicating the position of the acetyl group at C-3. The ¹³C NMR data of 3 are in agreement with the corresponding data described for 3-O-acetyl-α-amyrin (Mahato & Kundu, 1994).

Compound 4 was only identified by GC/MS analysis. The GC chromatogram of 4 showed a peak (TR = 54.942 min; molecular ion peak: m/z 412) with mass spectrum similar to that of 4,4-dimethyl-cholesta-22,24-dien-5-ol (Zhuang et al., 2010), according to the NIST data bank.

The IR spectrum of 5 showed absorptions at 3276 and 1036 cm^-1, which are characteristic of hydroxyl groups. The ¹H NMR spectrum showed signals at δ_H 5.17 and 4.61, which are attributed to alkenylic and carbinolic hydrogen atoms, respectively. The other hydrogen signals are registered between δ_H 2.0 and 0.8. The ¹³C NMR spectrum showed signals at δ_C 79.1, characteristic of hydroxyl groups, and signals at δ_C 139.6 and 124.4, which are characteristic of the ursan skeleton. The ¹³C NMR data of 5 are in agreement with the corresponding data described for α-amyrin (Costa et al., 2008).

The IR spectrum of the mixture of 5 and 6 showed strong absorptions at 3278, 1189, and 1096 cm^-1, which are characteristic of hydroxyl groups. The ¹H NMR spectrum showed signals at δ_H 5.17-5.10 and δ_H 4.67-4.56, which are attributed to alkenylic and carbinolic hydrogen atoms. The other hydrogen signals are registered between δ_H 2.0 and 0.8. The ¹³C NMR spectrum showed signals at δ_C 139.6 and 124.4, which are characteristic of the ursan skeleton, and signals at δ_C 150.9 (non-hydrogenated carbon) and 109.3 (methylene carbon), which are characteristic of triterpenes with lupan skeleton. The ¹³C NMR data of the mixture of 5 and 6 are in agreement with the corresponding data described for α-amyrin and lupeol (Costa et al., 2008), respectively.

The IR spectrum of 7 showed strong absorptions at 1736, 1376, and 1217 cm^-1, which are characteristic of ester groups. The ¹H NMR spectrum showed a singlet signal at δ_H 5.46 and a doublet signal at δ_H 4.57 (J = 10.9 and 4.5 Hz), which are attributed to alkenylic and carbinolic hydrogen atoms. The other hydrogen signals are registered between δ_H 2.5 and 0.8. The ¹³C NMR spectrum showed signals at δ_C 171.0, 145.5, and 116.3, which are characteristic of acetylated triterpenes with bauerenyl skeleton. The ¹³C NMR data of 7 are in agreement with the corresponding data described for Δ⁷-bauerenyl acetate (Mahato & Kundu, 1994).

The ¹H NMR spectrum of 8 only showed signals at δH 2.4 to 0.7, which are characteristic of aliphatic hydrogen atoms. The ¹³C NMR spectrum showed a signal at δ_C 213.3, attributed to carbonylic carbon, and signals at δ_C 59.5 to 6.8. The ¹³C NMR data of 8 are in agreement with the corresponding data described for friedelin (Mahato & Kundu, 1994).

The ¹H NMR spectrum of the mixture of 9 and 10 showed multiplet signals at δ_H 5.36 and 5.03, both signals were attributed to alkenylic hydrogen atoms. The ¹³C NMR spectrum showed signals at δ_C 140.8, 138.3, 129.3, and 121.7, which are in agreement with the corresponding data described for stigmasterol. The ¹³C NMR data of 10 are similar to those of 9, except for the signals attributed to the alkenylic carbon atoms, which were only recorded two signals at δ_C 140.8 and 121.7. These ¹³C NMR data are in agreement with the corresponding data described for sitosterol (Costa et al., 2008).

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Mixture of 3-O-acetyl-lupeol (1) and 3-O-acetyl-pseudotaraxasterol (2): IR (cm^-1) v 2921, 2851, 1733, 1463, 1378, 1244, 1174, 1009, 979; ¹H NMR (400 MHz, CDCl₃) δ_H 5.36-5.12 (m), 4.69-4.45 (m); ¹³C NMR data of 1 (100 MHz, CDCl₃) δ_C 174.0 (CO₂R), 151.2 (C-20), 109.6 (C-29), 81.3 (C-3), 55.6 (C-5), 50.6 (C-9), 48.3 (C-18), 47.9 (C-19), 43.1 (C-17), 42.4 (C-14), 41.2 (C-8), 40.3 (C-22), 38.8 (C-1/C-4), 38.0 (C-13), 37.1 (C-10), 35.2 (C-16), 34.5 (C-7), 29.8 (C-21), 28.2 (C-23), 27.7 (C-2), 27.3 (C-15), 25.3 (C-12), 21.2 (C-11), 19.5 (C-30), 18.3 (C-6), 18.0 (C-28), 16.2 (C-25), 16.0 (C-26), 15.8 (C-24), 14.5 (C-27); ¹³C NMR data of 2 (100 MHz, CDCl₃) δ_C 171.0 (CO₂Me), 139.8 (C-20), 118.9 (C-21), 80.9 (C-3), 55.6 (C-5), 50.6 (C-9), 48.6 (C-18), 42.4 (C-14/C-22), 41.2 (C-8), 38.8 (C-1/C-4/C-13), 37.1 (C-10), 36.6 (C-16/C-19), 34.5 (C-7/C-17), 28.3 (C-23), 27.7 (C-2/C-12), 26.9 (C-15), 22.8 (C-29), 21.7 (C-30), 21.6 (C-11), 18.3 (C-6), 17.8 (C-28), 16.3 (C-25), 16.0 (C-26), 15.8 (C-24), 14.8 (C-27).

3-O-acetyl-α-amyrin (3): white solid; mp 238.0-240.0 ºC; IR (cm^-1) v 2923, 1733, 1449, 1387, 1378, 1365, 1243, 1022, 1003, 984, 968; ¹H NMR (400 MHz, CDCl₃) δ_H 5.16 (m, H-12), 4.51 (m, H-3), 2.05 (s, CO₂Me); ¹³C NMR (100 MHz, CDCl₃) δ_C 171.4 (CO₂Me), 139.6 (C-13), 124.3 (C-12), 81.0 (C-3), 59.0 (C-18), 55.2 (C-5), 47.7 (C-9), 42.1 (C-14), 41.5 (C-22), 40.0 (C-8), 39.6 (C-19/C-20), 38.7 (C-1/C-4), 36.9 (C-10), 33.7 (C-17), 32.9 (C-7), 31.2 (C-21), 28.7 (C-15), 28.1 (C-23/C-28), 27.2 (C-2), 26.6 (C-16), 23.3 (C-11/C-27), 21.3 (C-30), 18.3 (C-6), 17.4 (C-29), 16.9 (C-26), 15.6 (C-24/C-25); MS (70 eV) m/z (rel. int.) 468 [M⁺] (2), 454 (1), 368 (1), 270 (2), 249 (2), 218 (100), 203 (26), 189 (22), 175 (6), 147 (7), 135 (14), 95 (14), 69 (11), 55 (9).

4,4-dimethyl-cholesta-22,24-dien-5-ol (4): MS (70 eV) m/z (rel. int.) 412 [M⁺] (47), 395 (80), 136 (56), 122 (82), 93 (100), 55 (22), 43 (42).

α-amyrin (5): IR (cm^-1) v 3276, 3058, 2946, 2918, 2852, 1638, 1464, 1387, 1378, 1189, 1096, 1036, 992, 822; ¹H NMR (400 MHz, CDCl₃) δ_H 5.17 (m, H-12), 4.61 (m, H-3); ¹³C NMR (100 MHz, CDCl₃) δ_C 139.6 (C-13), 124.4 (C-12), 79.1 (C-3), 59.0 (C-18), 55.2 (C-5), 47.7 (C-9), 42.1 (C-14), 41.5 (C-22), 40.0 (C-8), 39.6 (C-19/C-20), 38.7 (C-1/C-4), 36.9 (C-10), 33.7 (C-17), 32.9 (C-7), 31.2 (C-21), 28.7 (C-15), 28.1 (C-23/C-28), 27.2 (C-2), 26.6 (C-16), 23.3 (C-11/C-27), 21.4 (C-30), 18.3 (C-6), 17.4 (C-29), 16.9 (C-26), 15.6 (C-24/C-25); MS (70 eV) m/z (rel. int.) 426 [M⁺] (12), 412 (2), 218 (100), 189 (35), 107 (70) , 81 (47).

Mixture of 5 and lupeol (6): IR (cm^-1) v 3278, 3078, 3058, 2946, 2918, 2852, 1646, 1464, 1387, 1378, 1189, 1096, 1036, 992, 880, 822; ¹H NMR (400 MHz, CDCl₃) δ_H 4.67 (s, H-29a), 4.56 (s, H-29b); ¹³C NMR data of 6 (100 MHz, CDCl₃) δ_C 150.9 (C-20), 109.3 (C-29), 79.0 (C-3), 55.2 (C-5), 50.4 (C-9), 48.3 (C-18), 48.0 (C-19), 43.0 (C-17), 42.8 (C-14), 40.8 (C-8), 40.0 (C-22), 38.8 (C-4), 38.7 (C-1), 38.1 (C-13), 37.1 (C-10), 35.6 (C-16), 34.3 (C-7), 29.7 (C-21), 28.1 (C-23), 27.4 (C-2/C-15), 25.2 (C-12), 20.9 (C-11), 19.3 (C-30), 18.3 (C-6), 18.0 (C-28), 16.1 (C-25), 15.9 (C-26), 15.6 (C-24), 14.5 (C-27).

Δ⁷-bauerenyl acetate (7): white solid; mp 267.2-275.4 ºC; IR (cm^-1) v 2960, 2848, 1736, 1473, 1461, 1376, 1217, 736, 718; ¹H NMR (400 MHz, CDCl₃) δ_H 5.46 (m, H-7), 4.57 (dd, J = 10.9 and 4.5 Hz, H-3), 2.10 (s, CO₂Me); ¹³C NMR (100 MHz, CDCl₃) δ_C 171.0 (CO₂Me), 145.5 (C-8), 116.3 (C-7), 81.2 (C-3), 54.9 (C-18), 50.6 (C-5), 48.1 (C-9), 41.3 (C-14), 38.0 (C-20), 37.8 (C-4/C-13), 37.7 (C-22), 36.6 (C-1), 35.4 (C-19), 35.1 (C-10), 32.4 (C-12), 32.1 (C-17/C-28), 31.6 (C-16), 29.2 (C-21), 28.9 (C-15), 27.5 (C-23), 25.7 (C-29), 24.2 (C-2), 24.0 (C-6), 23.7 (C-26), 22.7 (C-27), 22.6 (C-30), 16.9 (C-11), 15.8 (C-24), 13.1 (C-25).

friedelin (8): white solid; mp 257.8-259.0 ºC; IR (cm^-1) v 2954, 2930, 2848, 1736, 1472, 1462, 1378, 1171, 730, 719; ¹³C NMR (100 MHz, CDCl₃) δ_C 213.3 (C-3), 59.5 (C-10), 58.2 (C-4), 53.1 (C-8), 42.8 (C-18), 42.2 (C-5), 41.5 (C-2), 41.3 (C-6), 39.7 (C-13), 39.3 (C-22), 38.3 (C-14), 37.4 (C-9), 36.0 (C-16), 35.6 (C-11), 35.4 (C-19), 35.0 (C-29), 32.8 (C-21), 32.4 (C-15), 32.1 (C-28), 31.8 (C-30) 30.5 (C-12), 30.0 (C-17), 28.2 (C-20), 22.3 (C-1), 20.3 (C-26), 18.7 (C-27), 18.2 (C-7), 17.9 (C-25), 14.7 (C-24), 6.8 (C-23).

Mixture of stigmasterol (9) and sitosterol (10): ¹H NMR (400 MHz, CDCl₃) δ_H 5.36 (m, H-6), 5.03 (m, H-22/H-23), 3.53 (m, H-3); ¹³C NMR data of 9 (100 MHz, CDCl₃) δ_C 140.8 (C-5), 138.3 (C-22), 129.3 (C-23), 121.7 (C-6), 71.8 (C-3), 56.9 (C-14), 56.0 (C-17), 51.2 (C-24), 50.1 (C-9), 42.3 (C-13), 42.2 (C-4), 40.5 (C-20), 39.7 (C-12), 37.2 (C-1), 36.5 (C-10), 31.9 (C-7/C-8/C-25), 31.7 (C-2), 29.0 (C-16), 25.4 (C-28), 24.4 (C-15), 21.2 (C-21/C-26), 21.1 (C-11), 19.4 (C-19), 19.0 (C-27), 12.1 (C-29), 12.0 (C-18); ¹³C NMR data of 10 (100 MHz, CDCl₃) δ_C 140.8 (C-5), 121.7 (C-6), 71.6 (C-3), 56.9 (C-14), 56.0 (C-17), 50.1 (C-9), 45.2 (C-24), 42.3 (C-13), 42.2 (C-4), 39.7 (C-12), 37.2 (C-1), 36.5 (C-10), 35.5 (C-20), 33.4 (C-22), 31.9 (C-7/C-25), 31.7 (C-2), 31.4 (C-8), 29.0 (C-16), 25.5 (C-23), 22.6 (C-28), 24.4 (C-15), 21.2 (C-21), 20.9 (C-26), 21.1 (C-11), 19.4 (C-19), 18.6 (C-27), 12.1 (C-29), 12.0 (C-18).

Evaluation of the bactericidal and trypanocidal activities

The trypanocidal activities over the T. cruzi were 13±0.3% for 5 and 5±0.7% for LE, SE, and mixture of 5 and 6. So, the extracts of LE and SE and the triterpene 5 and mixture of 5 and 6 showed low effect against intact bloodstream forms of the parasite (Oliveira et al., 2006).

In the agar diffusion tests, LE and SE were not active against Bacillus cereus, Listeria monocytogenes, Escherichia coli, Salmonella typhimurium, and Citrobacter freundi. Therefore, these extracts were active against Staphylococcus aureus, a very virulent bacterium, with great ability to produce localized infections that may become fatal if untreated (Shorr, 2007). Among the tested fractions, only 5 and mixture of 5 and 6 were active against S. aureus with a minimum inhibitory concentration greater than 1024 µg/mL. Although compound 3 only differs at C-3 position in relation to 5, the former did not show bactericidal activity.

Conclusions

The present work describes the isolation of 3-O-acetyl-lupeol, 3-O-acetyl-α-amyrin, 4,4-dimethyl-cholesta-22,24-dien-5-ol, and Δ⁷-bauerenyl acetate for the first time in the genus Lychnophora. The apolar extracts and triterpenes isolated from aerial parts of L. pinaster show low trypanocidal activity. As consequence, the trypanocidal activity previously described in the literature is not related to the triterpenes isolated from aerial parts of Lychnophora pinaster. Apolar extracts of leaves and stems and the triterpene α-amyrin, which was also isolated from these extracts, show effect against S. aureus. As acetyl α-amyrin did not show activity, the free hydroxyl group at C-3 may be related to the bactericidal activity of this triterpene.

Acknowledgements

The authors thank Conselho Nacional de Desenvolvimento Científico e Tecnológico, Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, and Fundação de Amparo à Pesquisa do Estado de Minas Gerais for their financial support.

Received 13 Oct 2010

Accepted 2 Feb 2011

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Publication Dates

• Publication in this collection
  03 June 2011
• Date of issue
  Aug 2011

History

• Received
  13 Oct 2010
• Accepted
  02 Feb 2011