Quercitrin: An Antileishmanial Flavonoid Glycoside from Kalanchoe pinnata

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

Quercitrin (quercetin 3-O-α-L-rhamnopyranoside), one of the constituents of the biologically active aqueous extract obtained from Kalanchoe pinnata, is demonstrated to be a potent antileishmanial compound (IC₅₀ ≈ 1 μg/mL) with a low toxicity profile. This is the first time that antileishmanial activity is demonstrated for a flavonoid glycoside.

Continuous studies on the antileishmanial properties of Kalanchoe pinnata (KP) have already led to significant results. We have previously shown that oral treatment of Leishmania amazonensis-infected animals with an aqueous leaf extract of KP significantly reduced the lesion size and parasite load to levels comparable to i. p. administration of glucantime [1]. More recently, we have reported an important remission of human cutaneous leishmaniasis upon oral KP treatment without toxic effects [2].

Here, we describe the isolation of quercitrin from a KP aqueous extract (KPAE) and its activity on intracellular Leishmania amazonensis amastigotes.[*]

With the aim of finding antileishmanial compounds that could be responsible for KPAE activity, the extract was partitioned with dichloromethane in acid (F1) and alkaline medium (F2) and then with ethyl acetate (F3). The three organic fractions exhibited high antileishmanial activity, but only F3 showed no cytotoxicity like the KPAE (Fig. [1]). Activities of F1 and F2 were not selective, affecting both Leishmania’s amastigote and its host cell. Due to this fact, only F3 was chosen for purification.

Antiamastigote activity-guided fractionation of F3 led to the isolation of quercitrin (quercetin 3-O-α-L-rhamnopyranoside) that inhibited, at 100 μg/mL (223 μM), 93.9 % of amastigote growth. This flavonoid was more active and much less cytotoxic than its biosynthetic aglycone precursor quercetin (IC₅₀ > 100 μg/mL, data not shown) for which antileishmanial activity has been recently described [3]. In the lactate dehydrogenase release cyctotoxicity assay (100 μg/mL), quercitrin exhibited a 17.3 % release compared to 57.5 % in the case of quercetin that turned out to be more toxic for macrophages (quercetin’s cytotoxicity expressed as IC₅₀ seems to be near to 100 μg/mL).

The antileishmanial activity of quercitrin was tested at different concentrations and compared to the commercial drug Pentostam® (IC₅₀ ≈ 20 μg/mL). As shown in Fig. [2], quercitrin exhibits significant antileishmanial activity at 0.01 μg/mL (0.022 μM) with an IC₅₀ value of approximately 1 μg/mL (2 μM) and no significant toxicity below 10 μg/mL (data not shown). The IC₅₀ value for quercitrin’s cytotoxicity is higher than 100 μg/mL as 17.3 % of LDH release was observed at this concentration. It is interesting to note that this concentration corresponds to 100 times the IC₅₀ value for antileishmanial activity.

Quercitrin showed higher antileishmanial activity (93.9 % of inhibition) than F3 (67.4 %) at the same concentration (Fig. [1]) thereby confirming its contribution to the activity of the mother fraction.

Despite the activity of quercitrin on the amastigote form of L. amazonensis, no activity was observed on the promastigote form. On the contrary, quercitrin increased the growth of promastigotes at 72 hours (Fig. [3]).

The mechanism by which quercitrin acts is still unknown. However, as was found for pentavalent antimonials, quercitrin acts only on the Leishmania amastigote form. Although a wide spectrum of biological activities have already been demonstrated for flavonoids, few studies have been devoted to the antileishmanial activity of this class of natural polyphenols. Luteolin, a common flavonoid in the human diet, was already described as a promising antileishmanial drug. Free quercetin itself, as seen above, possesses antileishmanial activity, but is highly toxic [3]. Proanthocyanidins exert antileishmanial activity as well as modulatory effects on nitric oxide and tumor necrosis factor-alpha release in RAW 264.7 cells [4].

Our present study demonstrates that the aqueous leaf extract of Kalanchoe pinnata possesses antileishmanial activity. Quercitrin, one of the flavonoids present in this extract, is shown to be a very potent and safe antileishmanial compound in vitro. To our knowledge, this is the first report on the antileishmanial activity of a flavonoid glycoside. The in vivo activity of quercitrin is currently under investigation in our laboratories.

Materials and Methods

Reversed-phase chromatography was performed on RP-2 (70 - 230 mesh, Merck) or RP-18 silanized silica (40 - 63 μM, Merck) and size exclusion chromatography on G-15 - 120 Sephadex (40 - 120 μ, Sigma). Eluates were monitored by thin-layer chromatography (TLC) on silica gel 60 F₂₅₄ (Merck) using butanol/acetic acid/water (BAW 8 : 1:1). After visualization under UV, ceric sulfate solution was used. ¹H- and ¹³C-NMR spectra were recorded on a Bruker DRX-300 spectrometer (¹H: 300 MHz; ¹³C: 75 MHz). MS analysis was performed on a Biflex III (Bruker Daltonics, Billerica, MA, USA) in the reflectron mode, IS1 : 19 kV, IS2 : 16.5 kV, reflector: 20 kV. α-Cyano-4-hydroxycinnamic acid (HCCA, Aldrich, Milwaukee, WI) was used as matrix.

Chemicals: Sodium stibogluconate (Pentostam), Glaxo Wellcome Research and Development (GW387208A0044X60). This research has complied with all relevant federal guidelines and institutional policies related to the use of animal models for research purpose.

Plant material: K. pinnata was collected at the UFRJ campus out of blowing season (Ilha do Fundão, Rio de Janeiro). A voucher specimen (292.697) is deposited at the herbarium of the Rio de Janeiro’s Botanical Garden (Brazil).

Extraction and isolation: Fresh leaves (6.76 kg) were triturated and extracted with distilled water at 20 % (w/v) for 30 minutes at 50 °C. The extract was concentrated at the same temperature to 1/5 of its initial volume and partitioned with dichloromethane at pH 2 and afterwards at pH 11, affording F1 (423.1 mg) and F2 (113.0 mg), respectively. The residual aqueous phase was neutralized and partitioned using ethyl acetate affording F3 (1.110 g). An aliquot of F3 (1.067 g) was re-suspended in distilled water (5 mL) and chromatographed on an RP-2 column (29.0 × 2.2 cm; H₂O/MeOH gradient) affording 3 fractions. The only flavonoid fraction F3 - 2 eluted with 3 : 7 MeOH/ H₂O (elution volume: 300 mL; 496.7 mg) was purified on an RP-18 column (32.0 × 2.5 cm; H₂O/MeOH gradient), yielding two flavonoid fractions. The first flavonoid fraction (eluted with 1 : 9 EtOH/ H₂O - elution volume: 230 mL; 30.7 mg), enriched in one compound, was chromatographed on a Sephadex G-15 column (31.0 × 0.8 cm; H₂O) affording quercitrin as a yellow powder: 10.0 mg; Rf = 0.83; ¹H- and ¹³C-NMR (CD₃OD) as reported before [5]; [M + Na]⁺ was determined as 471.097. For MS analysis the sample was diluted in H₂O/MeOH (50/50) (1 μg/mL). 0.5 μL of sample solution, followed by 0.5 μL of matrix were spotted on the target. Mixing was accomplished by pipetting up and down, and the solution was allowed to dry at room temperature. MALDI spectra were internally calibrated using matrix peaks.

Biological assay: Parasite Leishmania amazonensis (MHOM/BR/75/Josefa strain) promastigotes transfected with green fluorescent protein (L. amazonensis-GFP promastigotes) were used. They were routinely isolated from mouse lesions and maintained as promastigotes in Dulbecco’s modified Eagle’s medium containing 10 % heat-inactivated fetal calf serum and 150 μg of geneticin/mL at 26 °C.

Antileishmanial activity. For antiamastigote activity, mouse peritoneal macrophages were plated in triplicates in 24-well culture microplates at 2 × 10⁵ cells/well in 0.4 mL complete medium. After 1 h at 37 °C and 5 % CO₂, the non-adherent cells were removed, and the cells were infected with 10⁷ promastigotes/well for 4 h at 34 °C. Free parasites were washed away with pre-warmed saline, and the infected cells were cultured for further 72 h in the presence or absence of 0.5 mL plant material. After collecting the culture supernatants, the cells were transferred using 200 μL of distilled water to black microplates, and the fluorescence was read in a plate reader fluorimeter (Fluoroskan; LabSystems) at 435-nm excitation and 538-nm emission.

For antipromastigote activity, promastigotes were suspended in duplicate at 2 × 10⁴ in 100 μL medium containing serum and antibiotics in 96-well culture microplates. The parasite suspension was mixed with 100 μL of double-concentrated plant compounds and incubated at 26 °C for 72 h. The number of promastigotes/sample was determined under an optical microscope.

Release of lactate dehydrogenase (LDH) by macrophages. The release of LDH was determined using 50 μL of culture supernatant collected at the end of the antileishmanial assay. The LDH content, which represents an indirect indication of cytotoxicity, was determined colorimetrically using a commercial kit (Doles Reagentes e Equipamentos para Laboratórios LTDA). The specific release was calculated as percentage of controls (non-treated macrophages as negative control and Triton detergent-treated macrophages as positive control).

Acknowledgements

M. F. Muzitano thanks CAPES (Brazil) for a fellowship.

References

• 1 Da Silva S AG, Costa S S, Mendonça S CF, Silva E M, Moraes V LG, Rossi-Bergmann B. Therapeutic effect of oral Kalanchoe pinnata leaf extract in murine leishmaniasis.  Acta Trop. 1995;  60 201-5
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  CrossrefPubMedSearch in Google Scholar
• 3 Mittra B, Saha A, Chowdhury A R, Pal C, Mandal S, Mukhopadhyay S. et al . Luteolin, an abundant dietary component is a potent anti-leishmanial agent that acts by inducing topoisomerase II-mediated kinetoplast DNA cleavage leading to apoptosis.  Mol Med. 2000;  6 527-41
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• 4 Kolodziej H, Kayser O, Kiderlen A F, Ito H, Hatano T, Yoshida T. et al . Proanthocyanidins and related compounds: anti-leishmanial activity and modulatory effects on nitric oxide and tumor necrosis factor-alpha-release in the murine macrophage-like cell line RAW 264.7  Biol Pharm Bull. 2001;  24 1016-21
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  CrossrefPubMedSearch in Google Scholar

Sônia S. Costa

Núcleo de Pesquisas de Produtos Naturais

Universidade Federal do Rio de Janeiro

21941-590 Rio de Janeiro, RJ

Brazil

Phone: +55-21-2562 6512

Fax: +55-21-2562 6512

Email: sscosta@nppn.ufrj.br

References

• 1 Da Silva S AG, Costa S S, Mendonça S CF, Silva E M, Moraes V LG, Rossi-Bergmann B. Therapeutic effect of oral Kalanchoe pinnata leaf extract in murine leishmaniasis.  Acta Trop. 1995;  60 201-5
  CrossrefPubMedSearch in Google Scholar
• 2 Torres-Santos E C, Da Silva S AG, Costa S S, Santos A PPT, Almeida A P, Rossi-Bergmann B. Toxicological analysis and effectiveness of oral Kalanchoe pinnata on a human case of cutaneous leishmaniasis.  Phytother Res. 2003;  17 801-3
  CrossrefPubMedSearch in Google Scholar
• 3 Mittra B, Saha A, Chowdhury A R, Pal C, Mandal S, Mukhopadhyay S. et al . Luteolin, an abundant dietary component is a potent anti-leishmanial agent that acts by inducing topoisomerase II-mediated kinetoplast DNA cleavage leading to apoptosis.  Mol Med. 2000;  6 527-41
  PubMedSearch in Google Scholar
• 4 Kolodziej H, Kayser O, Kiderlen A F, Ito H, Hatano T, Yoshida T. et al . Proanthocyanidins and related compounds: anti-leishmanial activity and modulatory effects on nitric oxide and tumor necrosis factor-alpha-release in the murine macrophage-like cell line RAW 264.7  Biol Pharm Bull. 2001;  24 1016-21
  CrossrefPubMedSearch in Google Scholar
• 5 Slowing K, Sollhuber M, Carretero E, Villar A. Flavonoid glycosides from Eugenia jambos .  Phytochemistry. 1994;  37 255-8
  CrossrefPubMedSearch in Google Scholar

Sônia S. Costa

Núcleo de Pesquisas de Produtos Naturais

Universidade Federal do Rio de Janeiro

21941-590 Rio de Janeiro, RJ

Brazil

Phone: +55-21-2562 6512

Fax: +55-21-2562 6512

Email: sscosta@nppn.ufrj.br