Antileishmanial metabolites from Lantana balansae

ABSTRACT

Eleven compounds, 12-oxo-phytodienoic acid (1), persicogenin (2), eriodictyol 3′,4′,7-trimethyl ether (3), phytol (4), spathulenol (5), 4-hydroxycinnamic acid (6), onopordin (7), 5,8,4′-trihydroxy-7,3′-dimethoxyflavone (8), quercetin (9), jaceosidin (10), and 8-hydroxyluteolin (11), were isolated from an ethanol extract of Lantana balansae Briq., Verbenaceae, that was found to possess antileishmanial activity. The structures of the compounds were determined by NMR spectroscopy and HR mass spectrometry, and 1, 2, 3, 7, 8 and 9 were investigated for antiprotozoal activity toward promastigotes of Leishmania amazonensis and Leishmania braziliensis. Compound 1 was shown to be the most potent, with the IC₅₀ values 2.0 µM toward L. amazonensis and 0.68 µM toward L. braziliensis, although less potent than the positive control Amphotericin B. All compounds have been reported previously, but this is the first report of the isolation of a cyclopentenone fatty acid (1) and flavanones (2 and 3) from a Lantana species.

Keywords:
Flavonoids; Lantana balansae; Leishmania amazonensis; L. braziliensis; 12-oxo-phytodienoic acid

Introduction

The knowledge of the traditional uses of plants to treat different conditions has not only been helpful in the search for new biologically active compounds, but also contributed to preserve the information obtained directly from the people living in isolated rural communities. Based on such knowledge, extensive phytochemical studies of different Lantana species, particularly L. camara, have led to the identification of lantadenes (pentacyclic triterpenoids), flavonoids and phenylpropanoids as the characteristic secondary metabolites of this species. The biological and pharmacological evaluation of crude extracts, essential oils and isolated compounds have shown that they possess a broad range of biological activities, for example antiprotozoal (antiplasmodial, antimalarial, leishmanicidal), antiviral, antioxidant, antiproliferative and cytotoxic activities (Ghisalberti, 2000; Grace-Lynn et al., 2012; Sousa and Costa, 2012).

Lantana balansae Briq., Verbenaceae, is a perennial shrub with a pungent odor that grows in the mountain region of Cochabamba, Bolivia, where it is locally known as "k'ichita". An infusion of fresh leaves of L. balansae is used in the traditional medicine to treat digestive disorders and muscle spasms (personal communication with local people where the plant was collected). Previous studies of L. balansae have reported the antimicrobial activity of the methanol extract (Salvat et al., 2004) and the chemical composition of its essential oil (De Viana et al., 1973; Sena Filho et al., 2012). As part of our search for bioactive secondary metabolites from the native flora of Bolivia, an ethanol extract of L. balansae was assayed for leishmanicidal activity toward Leishmania amazonensis and Leishmania braziliensis. As the extract displayed significant activity toward both species of Leishmania it was selected for a more detailed study. Herein, we wish to report the secondary metabolites isolated from L. balansae as well as the leishmanicidal activities of six metabolites.

Materials and methods

General

1D and 2D NMR spectra were recorded at room temperature with a Bruker Avance II 400 or a Bruker Avance 500 spectrometer. The chemical shifts (δ) are reported in ppm relative to solvent signals (δ _H 7.16 and δ _C 128.0 for C₆D₆, δ _H 2.05 and δ _C 206.0 for acetone-d₆, and δ _H 2.49 and δ _C 39.5 for DMSO-d₆), while the coupling constants (J) are given in Hz. HR-ESI-MS experiments were performed with a Waters Q-TOF Micro system spectrometer, using H₃PO₄ for calibration and as internal standard. Vacuum liquid chromatography (VLC) separations were carried out on Merck silica gel 60G (Merck), while column chromatography (CC) was performed using silica gel 60 (230–400 mesh, Merck) and gel permeation on Sephadex LH-20 (GE-Healthcare). Analytical TLC plates were visualized with UV light at 254 nm and spraying with vanillin followed by heating. Preparative TLC (PTLC) was run on 20 cm × 20 cm glass-coated plates (1 mm thickness, Analtech).

Plant material

The aerial parts of Lantana balansae Briq., Verbenaceae, were collected near Independencia, Cochabamba, Bolivia at coordinates 17º11.17′ S 66º43.58′ W and an elevation of 2789 m. Voucher specimens, taxonomically identified by Lic. Modesto Zárate, are kept at "Herbario Forestal Martín Cárdenas", Cochabamba, under accession number MZ-3946.

Extraction and isolation

The air-dried and ground leaves and flowers of L. balansae (1308 g) were extracted twice at room temperature by maceration in 95% EtOH for 48 h. After filtration the combined solutions were concentrated under reduced pressure to yield 136.6 g of a dark residue. The crude organic extract was suspended in a mixture of H₂O:MeOH (9:1, v/v, 500 ml) and extracted four times with 500 ml hexane followed by the extraction with ethyl acetate (four times, 500 ml). After evaporation of the solvents, the two fractions (23.5 and 43.6 g, respectively) were fractionated. VLC chromatography (hexane:CH₂Cl₂ 1:0 to 0:1) of the hexane extract yielded ten major fractions (A–J). Fraction E (3.0 g) was subjected to VLC (heptane:EtOAc 1:0 to 8:2) which gave nine subfractions (E1–E9). E4 (582.5 mg) was purified by CC on Sephadex LH-20 (CHCl₃:MeOH 1:1) to yield 4 (39.3 mg) and 5 (43.0 mg). F (1.2 g) was fractionated by Sephadex LH-20 CC (CHCl₃:MeOH 1:1) yielding six subfractions (F1–F6). Compound 10 (3.3 mg) was obtained pure from F5 (53.0 mg). G (485.0 mg) and H (789.0 mg) were fractionated by Sephadex LH-20 CC (CHCl₃:MeOH 1:1) to yield eleven (G1–G11) and five (H1–H5) subfractions, respectively. Compound 2 (9.0 mg) was obtained from G8 and 3 (7.7 mg) from G11. Sequential purification of H3 (510.0 mg) by Sephadex LH-20 CC (CHCl₃:MeOH 1:1) and VLC (PE:EtOAc 1:0 to 7:3) yielded 1 (26.1 mg). Purification of the EtOAC extract (8.0 g) by VLC (CH₂Cl₂:Me₂CO 1:0 to 0:1) yielded seven major fractions (A–G). C (1.4 g) was subjected to CC on Sephadex LH-20 (MeOH) to give fifteen subfractions (C1–C15), from which compounds 7 (6.4 mg), 11 (4.6 mg) and 9 (4.1 mg) were obtained pure from C10, C14 and C15, respectively. C8 and C11 were purified by PTLC (CH₂Cl₂:Me₂CO 8:2) to give 8 (3.3 mg) and 6 (5.2 mg).

12-Oxo-phytodienoic acid (1)

Colorless oil; ¹H (400 MHz, C₆D₆) δ_H 2.14 (H-2, 2H, t, 7.4 Hz), 1.50 (H-3, 2H, tt, 7, 7 Hz), 1.10 (H-4, 2H, m), 1.06 (H-5, 2H, m), 1.06 (H-6, 2H, m), 1.13 (H-7, 2H, m), 1.06 (H-8, 2H, m), 2.26 (H-9, 1H, m), 6.65 (H-10, 1H, dd, 5.8, 2.6 Hz), 6.00 (H-11, 1H, dd, 5.8, 2.2 Hz), 1.85 (H-13, 1H, ddd, 7.8, 4.5, 2.2 Hz), 2.50 (H-14a, H, m), 2.34 (H-14b, 1H, m), 5.33 (H-15, 1H, dtt, 11, 7, 1 Hz), 5.24 (H-16, 1H, dtt, 11, 7, 1 Hz), 1.97 (H-17, 2H, ddq, 7, 7, 1 Hz), 0.89 (H-18, 3H, t, 7.5 Hz); ¹³C (100 MHz, C₆D₆) δ _c 179.9 (C-1), 34.1 (C-2), 24.9 (C-3), 29.2 (C-4), 29.7 (C-5), 27.5 (C-6), 29.3 (C-7), 34.5 (C-8), 46.9 (C-9), 166.1 (C-10), 133.1 (C-11), 209.8 (C-12), 51.5 (C-13), 28.5 (C-14), 125.9 (C-15), 133.8 (C-16), 20.9 (C-17), 14.4 (C-18); HR-ESI-MS m/z 293.2148 [M+H]⁺ (caldc. for C₁₈H₂₉O₃ 293.2117); [α_D²⁰] +60.7 (c 0.89, CDCl₃).

Persicogenin (2)

Colorless oil; ¹H (400 MHz, C₆D₆) δ_H 4.70 (H-2, 1H, dd, 12.8, 3.1 Hz), 2.50 (H-3a, 1H, dd, 17.1, 12.8 Hz), 2.30 (H-3b, 1H, dd, 17.1, 3.1 Hz), 6.21 (H-6, 1H, d, 2.3 Hz), 6.08 (H-8, 1H, d, 2.3 Hz), 6.62 (H-2′, 1H, d, 2.1 Hz), 6.35 (H-5′, 1H, d, 8.4 Hz), 7.03 (H-6′, 1H, dd, 8.4, 2.1 Hz), 3.09 (OMe-7, 3H, s), 3.08 (OMe-4′, 3H, s), 12.84 (OH-5, 1H, brs), 5.47 (OH-3′, 1H, s); ¹³C (100 MHz, C₆D₆) δ_c 79.0 (C-2), 43.4 (C-3), 196.2 (C-4), 165.2 (C-5), 95.3 (C-6), 168.3 (C-7), 94.5 (C-8), 163.3 (C-9), 103.8 (C-10), 132.5 (C-1′), 113.3 (C-2′), 146.5 (C-3′), 147.0 (C-4′), 110.1 (C-5′), 118.0 (C-6′), 55.1 (OMe-7), 55.3 (OMe-4′); HR-ESI-MS m/z 317.1046 [M+H]⁺ (caldc. for C₁₇H₁₇O₆ 317.1025).

Eriodictyol 3′,4′,7-trimethyl ether (3)

Colorless oil; ¹H (400 MHz, C₆D₆) δ _H 4.76 (H-2, 1H, dd, 13.1, 2.9 Hz), 2.62 (H-3a, 1H, dd, 17.1, 13.1 Hz), 2.39 (H-3b, 1H, dd, 17.1, 2.9 Hz), 6.25 (H-6, 1H, d, 2.2 Hz), 6.16 (H-8, 1H, d, 2.2 Hz), 6.69 (H-2′, 1H, d, 2 Hz), 6.52 (H-5′, 1H, d, 8.8 Hz), 6.68 (H-6′, 1H, dd, 9, 2 Hz), 3.07 (OMe-7, 3H, s), 3.36 (OMe-4′, 3H, s), 3.40 (OMe-5′, 3H, s), 12.91 (OH-5, 1H, brs); ¹³C (100 MHz, C₆D₆) δ_c 79.7 (C-2), 43.9 (C-3), 196.6 (C-4), 165.7 (C-5), 95.6 (C-6), 168.6 (C-7), 94.9 (C-8), 163.7 (C-9), 104.2 (C-10), 131.8 (C-1′), 111.0 (C-2′), 150.8 (C-3′), 150.6 (C-4′), 112.2 (C-5′), 119.4 (C-6′), 55.4 (OMe-7), 55.9 (OMe-4′), 55.9 (OMe-5′); HR-ESI-MS m/z 331.1207 [M+H]⁺ (caldc. for C₁₈H₁₉O₆ 331.1182).

Onopordin (7)

Yellow powder; ¹H (500 MHz, Acetone-d₆) δ_H 6.58 (H-3, 1H, s), 6.60 (H-6, 1H, s), 7.50 (H-2′, 1H, d, 2.2 Hz), 6.99 (H-5′, 1H, d, 8.3 Hz), 7.47 (H-6′, 1H, dd, 8.3, 2.2 Hz), 3.87 (OMe-8, 3H, s), 12.25 (OH-5, 1H, brs); ¹³C (125 MHz, acetone-d₆) δ_c 165.4 (C-2), 103.8 (C-3), 183.6 (C-4), 157.7 (C-5), 94.8 (C-6), 154.0 (C-7), 132.2 (C-8), 154.1 (C-9), 105.8 (C-10), 123.9 (C-1′), 114.2 (C-2′), 146.6 (C-3′), 150.3 (C-4′), 116.7 (C-5′), 120.2 (C-6′), 60.8 (OMe-8); HR-ESI-MS m/z 331.0685 [M+H]⁺ (caldc. for C₁₆H₁₃O₇ 317.0661).

5,8,4′-Trihydroxy-7,3′-dimethoxyflavone (8)

Yellow powder; ¹H (500 MHz, acetone-d₆) δ_H 6.71 (H-3, 1H, s), 6.64 (H-6, 1H, s), 7.65 (H-2′, 1H, d, 2.0 Hz), 7.01 (H-5′, 1H, d, 8.3 Hz), 7.62 (H-6′, 1H, dd, 8.3, 2.0 Hz), 3.87 (OMe-7, 3H, s), 3.99 (OMe-3′, 3H, s), 13.25 (OH-5, 1H, brs); ¹³C (125 MHz, acetone-d₆) δ_c 165.4 (C-2), 103.9 (C-3), 183.7 (C-4), 157.6 (C-5), 94.8 (C-6), 132.1 (C-7), 153.9 (C-8), 157.0 (C-9), 105.8 (C-10), 123.7 (C-1′), 110.5 (C-2′), 148.9 (C-3′), 151.5 (C-4′), 116.4 (C-5′), 121.4 (C-6′), 60.7 (OMe-7), 56.6 (OMe-3′); HR-ESI-MS m/z 331.0826 [M+H]⁺ (caldc. for C₁₇H₁₅O₇ 331.0818).

Quercetin (9)

Yellow powder; ¹H (500 MHz, Acetone-d₆) δ_H 6.26 (H-6, 1H, d, 2.0 Hz), 6.53 (H-8, 1H, d, 2.0 Hz), 7.83 (H-2′, 1H, d, 2.2 Hz), 6.99 (H-5′, 1H, d, 8.5 Hz), 7.69 (H-6′, 1H, dd, 8.5, 2.2 Hz), 12.17 (OH-5, 1H, brs); ¹³C (125 MHz, acetone-d₆) δ_c 164.9 (C-2), 136.7 (C-3), 176.6 (C-4), 162.3 (C-5), 99.1(C-6), 146.9 (C-7), 94.4 (C-8), 157.8 (C-9), 104.2 (C-10), 123.8 (C-1′), 115.8 (C-2′), 145.8 (C-3′), 148.3 (C-4′), 116.3 (C-5′), 121.5 (C-6′); HR-ESI-MS m/z 303.0546 [M+H]⁺ (caldc. for C₁₅H₁₁O₇ 303.0505).

Leishmanicidal detection assay

The antileishmanial assay was performed using a colorimetric method-XXT (Salamanca et al., 2008). Briefly, the activity was measured in vitro on cultures of the Leishmania parasite in the promastigote forms, of complex L. amazonensis (clon 1: Lma, MHOM/BR/76/LTB-012) and complex L. braziliensis (strand M2904 C192 RJA) that were cultivated at 26 ºC in Schneider medium (pH 6.8) supplemented with inactivated (by heating to 56 ºC for 30 min) bovine calf serum (10%). Parasites in logarithmic phase of growth, at a concentration of 1 × 10⁶ parasites/ml, were seeded in the wells of 96-well plates. Solutions of compounds to be assessed at concentration range of 0.09–100 µg/ml were added. DMSO (1%) and amphotericin B (0.5 µg/ml) were used as negative and positive controls during the evaluations. All assays were performed in triplicate and the micro well plates were incubated for 72 h at 26 ºC. After incubation, a solution of XTT (1 mg/ml) in PBS (pH 7.0 at 37 ºC) with PMS (Sigma–Aldrich, 0.06 mg/ml) was added (50 µl/well), and the plates were kept at 26 ºC for another 4 h. The optical density of each well was determined with a StatFax (Model 2100 series plate reader) at 450 nm, and the IC₅₀ values were calculated using Microsoft's Excel 2000 program.

Results and discussion

As described in the Experimental part of the crude ethanol extract after liquid–liquid partition yielded the hexane and ethyl acetate fractions, from which the eleven compounds were isolated and identified as: 12-oxo-phytodienoic acid (1) (Bohlmann et al., 1983), persicogenin (2) (Pisutthanan et al., 2006), eriodictyol 3′,4′,7-trimethyl ether (3) (Fernandez et al., 1988), phytol (4) (Hasan et al., 1991), spathulenol (5) (Vieira et al., 2013), 4-hydroxycinnamic acid (6) (Pan and Lundgren, 1995), onopordin (7) (Reynaud and Raynaud, 1984), 5,8,4′-trihydroxy-7,3′-dimethoxyflavone (8) (Whalen and Mabry, 1979), quercetin (9) (Slowing et al., 1994), jaceosidin (10) (Ulubelen et al., 1979), and 8-hydroxyluteolin (11) (Taskova et al., 2008). The presence of terpenes (4 and 5) and flavonoids (7–11) in this species is in agreement with previous studies of the genus, however, this is the first report of the isolation of a cyclopentenone fatty acid (1) and the flavanones (2 and 3) from a Lantana species. A large number of secondary metabolites have been reported from the genus Lantana, and this has been excellently reviewed by Sousa and Costa (2012).

The structures of all isolated compounds were elucidated by a combination of high resolution mass spectrometry and high field NMR spectroscopy. For all compounds COSY, NOESY, HMQC and HMBC 2D NMR spectra were recorded and analyzed. In the case that the NMR data previously was reported in the same solvent, the comparison with the published data was also used to identify the compounds.

Phytooxylipins (Blee, 1998) may possess potent phytohormone activities (Böttcher and Pollmann, 2009; Pollmann, 2009). They are derived from oxidized C₁₆ and C₁₈ fatty acid precursors that are abundant in the cellular membranes of higher plants (Gfeller et al., 2010), by the octadecanoid pathway (Mithöfer et al., 2004). Phytooxylipins play an important role in plant response to various environmental stress conditions, and recently it was shown that 12-oxo-phytodienoic acid (1) inhibits the proliferation of human breast cancer cells by targeting cyclin D1 (Altiok et al., 2008). Flavonoids are the largest group of natural phenolic compounds in higher plants, and many studies have shown that they exhibit a broad range of biological activities. Examples are anti-inflammatory, antitumor, antibacterial, antileishmanial and free radical scavenging activities (Grecco et al., 2010; Mittra et al., 2000; Samuelsson and Bohlin, 2010).

The hexane and ethyl acetate extracts of L. balansae both possessed activity toward the promastigotes of L. amazonensis and L. braziliensis, with IC₅₀ values between 1 and 10 µg/ml (see Table 1 for details). Due to limited testing capacity we decided to evaluate the antileishmanial activity of a selection of the compounds obtained from these extracts, compounds 1–3 and 7–9, and the results are presented in Table 1. The most potent compound was found to be 1. Although it is less potent compared to the positive control amphotericin B, the antileishmanial activity of 1 is nevertheless interesting with the IC₅₀ values 2.0 µM toward L. amazonensis and 0.68 µM toward L. braziliensis. The cyclopentenone moiety of 1 possessing an α,β-unsaturated carbonyl group may be important for its activity, as Michael acceptors are known to be important for a number of biological activities (Ghantous et al., 2010). The flavanones 2 and 3 were less active among the tested flavonoids, while the flavones 7 and 8 as well as flavonol 9 show antileishmanial activity although they are obviously much less potent compared to the positive control. This finding is in agreement with the previously reported comparison of the activities of naringenin, eriodictyol, and (+)-taxifolin with those of the corresponding unsaturated derivatives apigenin, luteolin, and quercetin (Tasdemir et al., 2006). The general difference between flavanones and flavones/flavonols is the lack of the C-2/C-3 double bond in the former, affecting both the molecular form as well as the conjugation. However, without detailed information about the mechanism of action it is difficult to rationalize any SAR's. Compound 8 is slightly more potent compared to 7 and 9, especially toward L. braziliensis. Of the three, 8 is the most lipophilic which may be important for its ability to reach the target. 3-Methoxyquercitin has previously been shown to be more potent toward promastigote forms of L. amazonensis than quercetin (Taleb-Contini et al., 2004), so this appears to be a trend. The in vitro and in vivo antileishmanial activity of quercetin (9) has been extensively study, and the results presented here are in agreement with previous data (da Silva et al., 2012).

Table 1
In vitro leishmanicidal activity on promastigote forms of Leishmania ssp.

	IC50 (µg/ml)a
	L. amazonensis	L. braziliensis
EtOH extract	6.0 ± 0.2	4.9 ± 1.2
Hexane fraction	6.1 ± 1.3	1.3 ± 0.3
EtOAc fraction	9.9 ± 0.5	5.3 ± 1.0
1	0.60 ± 0.1 (2.0 µM)	0.20 ± 0.01 (0.68 µM)
2	>100	90 ± 4.2 (285 µM)
3	>100	> 100
7	22 ± 4.2 (70 µM)	16 ± 0.46 (51 µM)
8	14 ± 1.81 (42 µM)	2.7 ± 0.2 (8.2 µM)
9	20 ± 0.77 (66 µM)	41 ± 2.7 (136 µM)
Controlb	0.21 ± 0.06 (0.23 µM)	0.08 ± 0.04 (0.087 µM)

• ^a
  Data are expressed as mean standard deviation of three determinations.
• ^b
  Amphotericin B was used as positive control.

Acknowledgments

The financial support of the Swedish International Development Agency (SIDA) in a frame of collaboration agreement between Lund University (Sweden) and San Simón University (Bolivia) is gratefully acknowledged. We thank to Lic. Modesto Zárate for identifiying the plant.

References

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Publication Dates

• Publication in this collection
  Mar-Apr 2016

History

• Received
  05 Mar 2015
• Accepted
  09 Nov 2015