Journal Pre-proof An enquiry into antileishmanial activity and quantitative analysis of polyhydroxylated steroidal saponins from Solanum paniculatum L. leaves Alexander B. Valerino-D´ıaz (Conceptualization) (Investigation) (Writing - original draft), Ana C. Zanatta (Methodology) (Validation) (Writing - review and editing) (Visualization), Daylin Gamiotea-Turro (Investigation) (Writing - review and editing), Ana Carolina Bolela ˜ Bovo Candido (Investigation), Lizandra Guidi Magalhaes (Methodology) (Validation) (Writing - review and editing), Wagner Vilegas (Writing - review and editing), Lourdes Campaner dos Santos (Supervision) (Writing - review and editing)
PII:
S0731-7085(20)31521-1
DOI:
https://doi.org/10.1016/j.jpba.2020.113635
Reference:
PBA 113635
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received Date:
20 April 2020
Revised Date:
28 August 2020
Accepted Date:
8 September 2020
Please cite this article as: Valerino-D´ıaz AB, Zanatta AC, Gamiotea-Turro D, Candido ACBB, ˜ LG, Vilegas W, Santos LCd, An enquiry into antileishmanial activity and Magalhaes quantitative analysis of polyhydroxylated steroidal saponins from Solanum paniculatum L. leaves, Journal of Pharmaceutical and Biomedical Analysis (2020), doi: https://doi.org/10.1016/j.jpba.2020.113635
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An enquiry into antileishmanial activity and quantitative analysis of polyhydroxylated steroidal saponins from Solanum paniculatum L.
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leaves.
Alexander B. Valerino-Díaza, Ana C. Zanattaa, Daylin Gamiotea-Turroa, Ana Carolina Bolela
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Bovo Candidob, Lizandra Guidi Magalhãesb, Wagner Vilegasc and Lourdes Campaner dos
a
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Santosa*.
UNESP - São Paulo State University, Institute of Chemistry. Rua Prof. Francisco Degni, 55,
14800-060, Araraquara, São Paulo, Brazil; E-mail:
[email protected],
b
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[email protected],
[email protected] Research Group on Natural Products, Center for Research in Sciences and Technology,
University of Franca, Av. Dr. Armando Salles Oliveira, 201, 14404-600 Franca, São Paulo, Brazil; E-mail:
[email protected];
[email protected] Jo
c
UNESP - São Paulo State University, Institute of Biosciences. Praça Infante Dom Henrique, s/n,
11330-900, São Vicente, São Paulo, Brazil; E-mail:
[email protected] 1
*Corresponding author: a* Rua Prof. Francisco Degni, 55 Bairro: Quitandinha, 14800-060, Araraquara, São Paulo, Brazil. Telefone: +55 16 3301-9657. Fax: +55 16 3322-2308. E-mail:
[email protected] (Lourdes Campaner dos Santos)
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Highlights
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Graphical abstract
Determination of C-22 stereocenter configuration for two steroidal saponins through
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NMR techniques.
Quantification of eight steroidal saponins using an UHPLC-ESI-MS
High antileishmanial activity of steroidal saponins isolated form S. paniculatum leaves, against promastigotes form of L. (L.) amazonensis.
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Abstract Solanum paniculatum L. is species whose fruits are widely consumed in Brazil as a tonic
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beverage with higher content of steroidal saponins. In this work, we developed an analytical method for the quantification of the eight saponins present in the 70% ethanol extract from the
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leaves using ultra-high-performance liquid chromatography coupled to mass spectrometry
(UHPLC-MS). Besides, the eight spirostanic saponins were screened for in vitro antileishmanial
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activity against promastigote and amastigote forms of Leishmania (L.) amazonensis. Substances
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1, 2 and 3 were found to be the most active compounds, with inhibitory concentration (IC50) values of 8.51 ± 4.38, 10.75 ± 6.85 and 10.45 ± 4.21 μM, respectively, against promastigote
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forms and effective concentration (EC50) values of >25, 17.73 ± 0.99 and 19.57 ± 0.84 μM, respectively, against amastigote forms. The cytotoxic test with compounds 1-3 evidenced low
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toxicity in murine macrophage cells, with values above 50 μM at concentration lower than 25 µM. These findings show that saponins 1-3 should be evaluated in further studies for the treatment of cutaneous leishmaniasis.
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Keywords: UHPLC-ESI-MS; spirostanic saponins; Solanum paniculatum L.; quantitative analysis; Leishmania amazonensis; antileishmanial activity.
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1 Introduction Saponins are a large class of secondary metabolites glycosides widely distributed in the plant kingdom [1]. Most of these metabolites have detergent properties, due to the presence of a lipid-
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soluble aglycone and hydrophilic chain consisting of one or more glycoside units, which can reduce the surface tension of aqueous solutions [2]. Literature reports that saponins may protect
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plants from the attack of insects and herbivores [3].
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Saponins have also been employed for pharmaceutical purposes [2], since they display a wide range of activities, like antiviral, antitumor, antiparasitic, antibacterial, anti-inflammatory,
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molluscicidal, hemolytic [2], anticancer [4], antioxidant [5], immunomodulator [6 ̶ 8] and on plasma cholesterol in human blood [9]. In addition, saponins can be utilized as the primary
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metabolites for initiating the semi-synthesis of steroidal drugs [10]. Solanum paniculatum L. (Solanaceae) is a species found in South America (Paraguay and
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Argentina) and broadly distributed in Brazil [11]. It is known in Brazil as jurubeba, jurupeba, jubeba, juripeba, jubeba, juvena, juina or juna [12, 13]. The fruits are employed in many medicinal and culinary applications, as well as in beverage production [14, 15]. The leaves are employed for the treatment of gastric disorders, or as diuretic or tonic, against anemia, arthritis,
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bronchitis, cough, and liver disorders [16], being also used as an important component in several phytotherapic formulations [14]. Previous works, led to the isolation and structural determination of new spirostanic saponins from the 70% ethanol extract of S. paniculatum leaves [14, 17]. Due to the difficulty for detecting and quantifying steroidal saponins by liquid chromatography coupled to diode array detectors (LC-DAD), the present study sought to develop an ultra-
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performance liquid chromatography couple mass spectrometry (UHPLC-MS) method for the quantification of spirostanic saponins in 70% ethanol extract of S. paniculatum L. leaves. The data obtained in this study may potentially be important for the quality assessment of S. paniculatum phytomedicines. Leishmaniasis is a parasitic disease caused by Leishmania parasites. The disease affects roughly 12 million people and 350 million people at risk of contracting the disease in 98 countries
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worldwide [18]. The clinical features depends on the parasite’s characteristics and on the
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effectiveness of the immune response, and according to the clinical manifestations, the
leishmaniasis can be divided into cutaneous (localized and disseminated), muco-cutaneous, and
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visceral or kala-azar [18]. In the spectrum of Leishmania parasites, the L. (L.) amazonensis
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belongs to the L. mexicana complex and is associate to diffuse cutaneous leishmaniasis (DCL) [18, 19]. This cutaneous form is characterized by suppression of the natural early response, as
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reported by inhibition of macrophage production of pro-inflammatory molecules [20]. The treatment of leishmaniasis has been historically undertaken by the administration of
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pentavalent antimonials (as first choice), amphotericin B (AmpB) (polyenic antibiotic), pentamidine (dybenzamidine) and paromomycin (broad-spectrum amino glycoside antibiotic). All of these drugs are found to be expensive, and provoke huge side effects on patients. Among the side effects, include myalgia, arthralgia, and high toxicity to the liver, heart, kidneys, spleen,
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hypotension, local pain after injection, and in some cases parasites resistance [21]. Over the past few years, scientists have paid considerable attention to the search for compounds,
extracts or fractions, obtained from natural sources, which have essentially specific properties that make them suitable to be used for the treatment of leishmaniasis infection [22]. Literature
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reports the inhibitory effect of saponin aqueous fraction from Zinziber officinalis Roscoe (ginger) against promastigote forms of L. (L.) amazonensis [21]. In fact, phytopreparations based on Solanum species are widely used in several parts of the world for the treatment of cutaneous leishmaniasis and it was demonstrated the in vitro anti-leishmanial effect of solamargine and solasonine from S. lycocarpum fruits towards promastigotes of L. (L.)
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amazonensis [23]. Thus, in this work we have also evaluated the effect of the spirostanic saponins isolated from S.
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paniculatum leaves on promastigotes forms of L. (L.) amazonensis and the most active
compounds were also tested against the amastigote forms. Finally, our study also assessed the
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cytotoxic effect of the saponins in murine macrophage cells.
2 Materials and Methods
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2.1 Plant material
Solanum paniculatum L. leaves were collected on February 20th, 2015 from the orchard of
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medicinal plants in São Paulo State University (UNESP), at the Faculty of Pharmaceutical Sciences of Araraquara, São Paulo State, Brazil, located on GPS 21°48’52.44’’S and 48°12’ 07.13 W as described by Valerino-Díaz et al. [14]. The data collected were added to the SisGen platform (National System of Management of Genetic Heritage and Associated
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Traditional Knowledge) as genetic patrimony with registration number AB8B70B.
2.2 Extraction and isolation The preparation of 70% ethanol extract and isolation of the pure compounds 1-8 were already described in the Materials and Methods section of Valerino-Díaz et al. [14].
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2.3 Sample preparation The extract were submitted to solid phase extraction (C18 cartridges, Chromabond®, 45 µm, 500 mg, 6 mL) prior to UHPLC analysis to eliminate chlorophylls and low-polarity compounds. The
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cartridges were previously activated with MeOH (4.5 mL) and equilibrated with H2O (4.5 mL). The ethanol 70% ethanol extract (10 mg) of S. paniculatum leaves was previously solubilized in
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1.5 mL of MeOH/H2O solution (9:1, v/v) loaded into the cartridge and eluted with 1.5 mL of MeOH/H2O (9:1, v/v). The dried sample was suspended in MeOH to a concentration of 10
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µg.mL-1. The saponins standards were redissolved in MeOH to obtain the stock solution at the
(0.22 μm, 25 mm).
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2.4 ESI-mass spectrometry conditions
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concentration of 1000 µg.mL-1. All sample solutions were filtered through a Millex® PTFE filter
Direct flow infusion analysis were performed using Thermo Scientific LTQ XL linear ion trap
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(IT) analyzer equipped with an electrospray ionization (ESI) source (Thermo, San Jose, CA, USA). The MS conditions employed were as follows: capillary voltage: 5.98 kV, capillary temperature: 350 °C; cone voltage: -45 V and syringe pump flow rate: 10.0 µL.min-1. Full scan analysis were recorded in m/z range from 200 to 2000. Multiple-stage fragmentations (ESI-MSn)
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were performed using the collision-induced dissociation (CID) method against helium for ion activation.
2.5 UHPLC analysis
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UHPLC-ESI-IT-MS analysis were performed using Thermo Scientific® ultra-performance liquid chromatography equipment, which consisted of an automatic injector (Accela AS), a Thermo Scientific LTQ XL linear ion trap (IT) analyzer equipped with an electrospray ionization (ESI) source (Thermo, San Jose, CA, USA) quaternary pump (Accela 600). Optimizations of the chromatographic conditions of the extract were performed using ACQUITY UPLC BEH column
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(C18 5.0 x 2.1 mm, 1.7 μm) at room temperature. The optimization method adopted for the extract consisted of linear gradient program (5-100% v/v) in eluent B (MeOH) executed over a
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period of 20 min. The injection volume used for each sample was 10 μL and the flow rate was 0.300 mL.min-1. After maintaining the column with 100% B for 5 min, the column was
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reconditioned to the initial condition for analysis to be performed in sequence. The fragmentation
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experiments were performed by collision-induced dissociation (CID) with Selected Ion Monitoring (SIM) detection for MS/MS acquisition in negative ion mode with collision energy
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of 30% and activation time of 30 ms. The MS conditions employed were as follows: source voltage: 5.98 kV, source current: 5.09 uA, capillary temperature: 350 °C; capillary voltage: -45
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V. The mass scan range was monitored from 200 to 2000. The mass spectra were processed in Xcalibur software (version 2.0).
2.6 Saponin quantification parameters
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The quantitative analysis of saponins in the 70% ethanol extract was carried out based on the following procedure: preparation of solutions, corresponding to standard saponins (1-8) isolated (> 95% purity) from the 70% ethanol extract of S. paniculatum leaves [14]. The UHPLC-ESI-ITMS quantification analysis was conducted by the construction of an analytical curve based on the concentration variation of an external standard (saponins). The solutions of the standards were
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used in the construction of eight analytical curves; this involved the preparation of dilutions from a stock solution of each 1000 μg.mL-1 standard, using methanol as diluent. The results were plotted in a graph where the areas of the peaks from the selected corresponding [M−H]- ion of each substance (saponin) obtained were related to the concentration level (i.e. peak areas versus concentration). Each concentration level was analyzed in triplicate. The average areas of the
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chromatographic peaks were interpolated as a function of concentration using linear regression equation and correlation coefficients. The limit of detection (LOD) was calculated taking as
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criteria a S/N ratio of 3, while the limit of quantification (LOQ) was determined based on a S/N
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ratio of 10.
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2.7 Cells and parasites
J774 (murine macrophages) were cultivated in RPMI 1640 medium (Gibco) without phenol red
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supplemented with 10% FBS (Gibco), 10 U.mL-1 penicillin and 10 μg.mL-1 streptomycin (Gibco), and 10% sodium bicarbonate, pH 7.4 at 37 °C with 5% CO2 in 25 cm2 culture flask.
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Promastigote forms of L. (L.) amazonensis (IFLA/BR/67/PH8) were maintained in culture medium RPMI 1640 (Gibco, Grand Island, USA) without phenol red and supplemented with 10% fetal bovine serum (FBS) (Gibco), 10 U.mL-1 penicillin and 10 μg.mL-1 streptomycin (Gibco), and 10% sodium bicarbonate (pH 7.4). The parasite culture was maintained in
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biological oxygen demand (B.O.D.) (Quimis, Diadema, BR) incubator at 24 °C in a 25 cm2 culture flask. Promastigote forms of L. (L.) amazonensis in the stationary growth phase (5-day culture) were used in all the experiments.
2.8 Cytotoxicity assay
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Cytotoxic activity J774A.1 macrophages was assessed by 3-(4,5-dimethylthiazol-2yl)-2,5diphenyl-2H-tetrazoliumbromide (MTT) (Sigma–Aldrich) assay as described by Guimarães et al., 2013 with modifications [24]. In brief, J774A.1 macrophages cultivated in RPMI 1640 medium were seeded (2 × 105 cells.well-1) in 96-well culture plates (TPP); and incubated at 37 °C in the presence of 5% CO2. After 24 h, compounds 1, 2 and 3 were added in the plates (3.12 to 50 μM AmpB (0.047-0.78 µM) and incubated for 48 h. After the period of incubation, 10 µL
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MTT solution (5 mg.mL-1) (Sigma-Aldrich) was added in each well and the plates were
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incubated for 4 h at 37 °C. Subsequently, the supernatant was removed and the formazan crystal was solubilized with 100 µL of 100% DMSO (Sytnh). Absorbance was measured at 570 nm with
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a microplate reader (Libra S12–Biochrom). Results were expressed as the average percentage of
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non-viable cells in relation to the negative control (0.1% DMSO). Cytotoxic Concentration of
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50% of the cells) were determined by the non-linear regression curve.
2.9 Antileishmanial activity of saponins against promastigote forms of L. (L.). amazonensis
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The determination of chemical activity of saponins against promastigote forms of L. (L.) amazonensis was performed as described by Corral et al. [25] and De Souza Araújo et al. [26]. Promastigotes forms of L. (L.) amazonensis (3 × 106 cells.mL-1) cultivated in RPMI 1640 medium supplemented were placed in 96-well culture plates (TPP – Trasadingen, Sweden) with
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different concentrations of compounds (3.12-50.00 μM) and amphothericin B (AmpB) (SigmaAldrich, St Louis, USA) with concentrations 0.011-0.19 µM, previously dissolved in dimethylsulfoxide (DMSO, Synth - São Paulo, BR), to a total volume of 200 μL.well-1. The plates were incubated with the compounds at 24 °C in B.O.D. (Quimis) incubator for 24 h. Thereafter, 2 mM of resazurin solution (20 µL) (Sigma-Aldrich) in phosphate-buffered saline
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were added in all the wells. The plates were incubated for further 4 h under the same conditions. Absorbance measurements were subsequently carried out under 570 and 600 nm in a Sunrise microplate reader (Libra S12, Biochrom - Holliston, EUA). As a negative control, RPMI 1640 medium (Gibco) containing 0.1% DMSO (Synth) was used and as a positive control, parasites grown with AmpB at the concentrations described above were used. The concentration that
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inhibited 50% of the cell growth (IC50) was determined by regression analysis of the data.
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2.10 Antileishmanial activity of saponins against intracellular amastigote forms of L. (L.) amazonensis
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The determination of chemical activity of saponins against amastigote forms of L. (L.)
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amazonensis was performed as described by Casa et al. 2018 with modifications [27]. In brief, macrophages (J774A.1) cultivated in RPMI 1640 medium supplemented, were suspended and
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adjusted to a concentration of 2 x 105 cells.well-1 and seeded in a 24-well round-coverslip culture plate incubated for 24 h at 37 °C in the presence of 5% CO2. Adherent macrophages were
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infected with metacyclic promastigotes (stationary growth phase, 5-day culture) at a concentration of 1 x 106 cells.well-1 (10:1 ratio), during 4 h at 34 °C. Parasites not internalized in the macrophages were removed, and the infected culture was incubated with different concentrations of AmpB (0.005 to 0.095 μM) and compounds 1, 2 and 3 (1.56 to 25 μM) for 48 h
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at 37 °C and 5% CO2. After incubation, the coverslips were washed, fixed with methanol and stained with Giemsa solution (Synth). The slides were analyzed in an optical microscopy (Nikon New York, USA) by counting 200 macrophages and was determined the number of amastigote within each infected cell. The EC50 was calculated in comparison to the negative control (RPMI
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1640 medium containing 0.1% DMSO). The 50% effective concentration values (EC50) were determined for the percentage of amastigote reduction using non-linear regression curves.
2.11 Statistical analysis
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All experiments were performed in triplicate, each being repeated three times. The IC50, EC50 and CC50 values were determined by the non-linear regression curve using the GraphPad Prism
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software version 5.0 for Windows (GraphPad software, San Diego, USA). The results were
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expressed as mean ± standard deviation of nine determinations (n = 9).
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3 Results and Discussion
Recently, the authors of the present work reported the successful isolation and characterization of
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eight spirostanic saponins from S. paniculatum leaves, six of them being novel compounds which had hitherto not been described in the literature (1, 4-8) [14]. The authors were, however,
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unable to provide a complete description of all the stereocenters of compounds 2 and 3 in the aforementioned work. Thus, the present work sought to report the stereochemistry of C-22, which helped provide a complete description of compounds 2 and 3.
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3.1 Compound 2
The patterns observed in 1H and 13C NMR for compound 2 are very close to each other when compared with compound 1 [14]. Nonetheless, the aforementioned paper failed to describe the configuration of the stereocenter C-22 for compound 2. The determination of the stereocenter C22 was performed using the criteria proposed by Matsushita et al. [28]. These authors observed
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the existent association between 1H NMR chemical shifts of the hydrogen and the methyl group H3-21 and H-16 along with the 13C NMR chemical shifts of C-20 in 23-hydroxyspirostanols. Specifically, with regard to 22-α-O-spirostanol derivatives, reports in the literature show that H321 and H-16 absorb frequency between δH 1.17-1.26 and δH 4.49-4.56 respectively. When it comes to the chemical shift of 13C, 22-α-O-spirostanol derivatives absorb frequency between δC 35.0-36.2. On the other hand, in the case of 22-β-O-spirostanol derivatives, H3-21 and H-16
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absorb frequency between δH 1.52-1.54 and δH 5.18-5.20, respectively. With regard to the
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chemical shift of 13C in 22-α-O-spirostanol derivatives, C-20 is found to absorb frequency between δC 43.0-43.1 respectively [28]. A plausible reason that can help elucidate these
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phenomena is that, in C-22-β-configuration, both H3-21 and H-16 lie closer to hydroxyl group at
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C-23 position. Compound 2 showed chemical shifts of H-21 at δH 1.09, H-16 δH 4.45 and δC 40.3 [14]; this helped associate the 22-α-O-configuration (C-22 S) with sapogenol. Based on the data
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presented, one can establish the configuration of the C-22 stereocenter in compound 2; this was identified as the new 6-O-α-L-rhamnopyranosyl-(1→3)-β-D-quinovopyranosyl-(22S,23R,25R)-
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3β,6α,23-trihydroxy-5α-spirostane.
3.2 Compound 3
Compound 3 was isolated and characterized in line with the descriptions provided in the work of
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Valerino-Diaz et al. [14]. The present work however, did not report the description of C-22 stereocenter. Based on the same criteria employed for the determination of the C-22 stereocenter configuration for compound 2, compound 3 showed chemical shifts of H-21 at δH 1.11, H-16 δH 4.47 and δC 39.6 [14]; this helped determine the 22-α-O-configuration (C-22S) [28]. Thus, the
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structure of compound 3 was identified as the new 6-O-β-D-xylopyranosyl-(1→3)-β-Dquinovopyranosyl-(22S,23R,25S)-3β,6α,23-trihydroxy-5α-spirostane.
3.3 MS/MS analysis of saponins Mass spectrum analysis conducted in negative mode helped to evaluate the fragmentation pattern
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for each of the spirostanic saponins studied. A similar fragmentation pattern was observed from molecular ions with m/z = 725 (compounds 3, 4, 6, 8) and m/z = 739 (compounds 1, 2, 5, 7)
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respectively. Table 1 presents the fragmentation patterns of the eight spirostanic saponins. The study carried out on the fragmentation of the precursor ions enabled us to determine the linkage
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of the glycosides in the aglycone by the typical cleavages of sugar units, considering the neutral
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losses of 146 (rhamnose) and 146 (quinovose) for compounds 1, 2, 5, 7; and 132 (xylose) and
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146 (quinovose) for compounds 2, 3, 6, 8, respectively.
3.4 Quantitative analysis of saponins
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In this study, the quantification of saponins in the 70% ethanol extract of S. paniculatum leaves helped to determine the contents of each one present in the extract. The UHPLC-ESI-MS technique was applied in this work for the quantification of eight steroidal saponins in 70% ethanol extract of S. paniculatum leaves by standard calibration curve. The standard saponins
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were confirmed by UHPLC-ESI-MS in negative mode using Selected Ion Monitoring (SIM) analysis. An analysis of the parameters evaluated in the study helped to obtain relevant information regarding the efficiency of the method adopted and to assess crucial uncertainties associated with the results. The compounds quantified in this work have been identified as follows: 6-O-α-L-rhamnopyranosyl-(1→3)-β-D-quinovopyranosyl-(22S,23R,25S)-3β,6α,23-
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trihydroxy-5α-spirostane (1); 6-O-α-L-rhamnopyranosyl-(1→3)-β-D-quinovopyranosyl(22S,23R,25R)-3β,6α,23-trihydroxy-5α-spirostane (2); 6-O-β-D-xylopyranosyl-(1→ 3)-β-Dquinovopyranosyl-(22S,23R,25S)-3β,6α,23-trihydroxy-5α-spirostane (3); 6-O-β-D-xylopyranosyl(1→ 3)-β-D-quinovopyranosyl-(22S,23R,25R)-3β,6α,23-trihydroxy-5α-spirostane (4); 3-O-α-Lrhamnopyranosyl-(1→ 3)-β-D-quinovopyranosyl-(22S,23S,25R)-3β,6α,23-trihydroxy-5α-
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spirostane (5); 3-O-β-D-xylopyranosyl-(1→ 3)-β-D-quinovopyranosyl-(22S,23S,25R)-3β,6α,23trihydroxy-5α-spirostane (6); 6-O-α-L-rhamnopyranosyl-(1→3)-β-D-quinovopyranosyl-
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(22S,25S)-1β.3β,6α-trihydroxy-5α-spirostane (7) and 6-O-β-D-xylopiranosyl-(1→ 3)-β-D-
spirostanic saponins are depicted in Figure 1.
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quinovopyranosyl-(22S,25S)-3β,4β,6α-trihydroxy-5α-spirostane (8). The structures of the
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The 70% ethanol extract chromatogram of S. paniculatum leaves, as well as the Selected Ion Monitoring (SIM) chromatograms corresponding to the ions m/z = 725 (compounds 3, 4, 6, 8)
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and m/z = 739 (compounds 1, 2, 5, 7) are represented in Figures 2A, 2B, and 2C. With regard to the method applied in this study, selectivity was verified by comparing the retention times (Rt) of
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the standard saponins (1-8) with those of the peaks obtained from selected ion monitoring chromatograms corresponding to m/z 725 and 739 ions, respectively.
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3.5 Linearity and sensibility
For the analysis of linearity and sensibility, calibration curves were constructed for each standard saponin prepared at different concentrations according to the amount of compounds present in the extract: compounds 1, 3 and 7 (0.1563 to 2.500 μg.mL-1); 2, 4 and 8 (0.0781 to 1.2500 μg.mL-1) and 5 and 6 (0.0391 to 0.6250 μg.mL-1). To assess the linearity and sensibility of the method, the calibration curves were constructed in a range of five concentrations, which were
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injected in triplicate in the column. All the results corresponding to the calibration curves analysis are depicted in Table 2. The linearity range evaluated for each standard presented correlation coefficients (R2) higher than 0.99; the values are within the stipulated range required by the Brazilian National Health Surveillance Agency (ANVISA) in terms of the active principles present in vegetal raw materials
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[29]. These results indicate good proportionality between the concentration of the standard and the area of the chromatographic peak in the concentration range analyzed.
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3.6 Limits of detection and quantification
The limit of detection (LOD) is defined as the lowest concentration of analyte that can be
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detected, but not necessarily quantified, under experimental conditions established by the
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instrumental technique used. The quantification limit (LOQ) is the lowest analyte concentration, which can be quantified in the sample with acceptable accuracy and precision, under the
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experimental conditions adopted [30]. The LOD and LOQ values obtained for each of the
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compounds evaluated are reported in Table 2.
3.7 Contents of steroidal saponins
The contents of the isolated and identified substances (saponins) in the 70% ethanol extract of S.
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paniculatum leaves, were determined by the interpolation of their areas in the calibration curve of each standard, with the corresponding areas determined in the Selected Ion Monitoring (SIM) chromatogram of the 70% ethanol extract for each compound (1-8). The data are shown in Table 3. The highest concentration values obtained by this method among the eight saponins investigated corresponded to compounds 1 and 3, which recorded values of 71.2 0.2 and 103.5
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0.2 mg.g-1 respectively, while the lowest concentration values corresponded to compounds 5 and 6, which recorded concentration values of 17.5 0.1 and 14.8 0.04 mg.g-1 of leaf dry weight, respectively.
3.8 Cytotoxic and antileishmanial activities
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One of the main problems associated with the use of the drugs against leishmaniasis is the toxicity [21]. Initially, the cytotoxic was determined using J774A.1 cells due to this cell line was
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used to obtain intracellular amastigote form. The results obtained in this study demonstrated that J774A.1 macrophages incubated with compounds 1, 2, and 3 at concentration 50 µM for 48 h
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showed a cell viability of about 70% and CC50 values higher than 50 µM. On the other hand,
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cells incubated with AmpB at concentration 0.78 µM showed a cell viability of 44.47 ± 1.87% and CC50 value was 0.57 ± 0.35 µM (Table 4). For this reason, the concentrations used against
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amastigote forms, were lower than 50 µM for compounds 1, 2 and 3 and lower than 0.005 µM for AmpB, which exhibited concentrations cell viability higher than 75%.
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Table 5 and 6 show the results of the antileishmanial activity of steroidal saponins (1-8) against promastigote and amastigote forms of L. (L.) amazonensis, respectively. According to the Japanese Global Health Innovative Technology (GHIT) fund, compounds isolated from plants or natural sources that present IC50 values 25, 17.73 ± 0.99 and 19.57 ± 0.84 µM,
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respectively. The positive control, AmpB, presented EC50 value of 0.022 ± 0.016 µM against
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amastigote forms L. (L.) amazonensis (Table 6). This outcome may be attributed to the fact that the active compounds relative to the promastigote forms of Leishmania ssp are incapable of
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reaching or affecting the intracellular amastigote forms by virtue of the inability of the
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compounds to cross the host cell membranes in the amastigote forms state [36].
4 Conclusions
The present study reported the development of a UHPLC-MS Selected Ion Monitoring (SIM)
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quantitative method, which was used to determine the contents of spirostanic saponins in S. paniculatum leaves. The analytical method employed presented good linearity, sensibility, selectivity and reproducibility. This work unfolds the first UHPLC-ESI-IT-MS analytical procedure developed for the quantification of spirostanic saponins in S. paniculatum L.
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The results obtained in the study demonstrated for the first time the antileishmanial activity of steroidal saponins, isolated from S. paniculatum leaves, against promastigotes forms of L. amazonensis in vitro. The compounds were also found to have low cytotoxicity. Further studies on the active spirostanic saponins isolated from S. paniculatum leaves are needed in order to develop suitable, potential candidate drugs for the treatment of cutaneous leishmaniasis infections. The findings of the study show that S. paniculatum leaves are a rich source of
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behind its popularity as medicinal plant among the Brazilian populace.
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steroidal saponins. The results reinforce the relevance of the species and help clarify the reasons
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Author statement
Alexander B. Valerino-Díaz: Conceptualization, Investigation, Writing - Original draft. Ana C. Zanatta: Methodology, Validation, Writing - Review and Editing, Visualization. Daylin
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Gamiotea-Turro: Investigation, Writing - Review and Editing. Ana Carolina Bolela Bovo Candido: Investigation. Lizandra Guidi Magalhães: Methodology, Validation, Writing Review and Editing. Wagner Vilegas: Writing - Review and Editing. Lourdes Campaner dos
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Santos: Supervision, Writing - Review and Editing.
Funding
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This research was funded by the Pro-Rector's Office (PROPG) at UNESP - São Paulo State University, (2015/04899-3), the Coordination for the Improvement of Higher Education Personnel (CAPES, Grant No. 187715-1), and the São Paulo State Research Foundation (FAPESP, Grant No. 2009/52237-9). Declaration of interests
Notes
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The authors declare no competing financial interests.
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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Acknowledgments
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The authors are grateful to Brian Newman for the exhausted revision of the manuscript.
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References [1]
C. Y. Cheok, H. A. K. Salman, R. Salaiman, Extraction and quantification of saponins: A review. Food Res. Int. 59 (2014) 16–40. https//doi.org/10.1016/j.foodres.2014.01.057. S. G. Sparg, M. E. Ligth, J. van Staden, Biological activities and distribution of plants saponins. J. Ethnopharmacol. 94 (2004) 219–243.
[3]
ro
https://doi.org/10.1016/j.jep.2004.05.016.
of
[2]
J. M. Augustin, V. Kuzina, S. B. Anderson, S. Back, Molecular activities,
-p
biosynthesis and evolution of triterpenoid saponins. Phytochemistry. 72 (2011) 435–
T. –C. Chen, J. –F. Lu, J. –S. Wang, L. –J. Lin, H. –I. Kuo, B. –H. Chen,
lP
[4]
re
457. https//doi.org/10.1016/j.phytochem.2011.01.015.
Antiproliferation effect and apoptosis mechanism of prostate cancer cells PC-3 by
ur na
flavonoids and saponins prepared for Gynostemma pentaphyllum. J. Agr. Food Chem. 59 (2011) 11318–11329. https://doi.org/10.1021/jf2018758. [5]
K. W. Chan, N. M. H. Khong, S. Iqbal, M. Ismail, Isolation and antioxidative properties of phenolic-saponins rich fraction from deffated rice bran. J. Cereal Sci. 57
Jo
(2013) 480–485. https://doi.org/10.1016/j.jcs.2013.02.002.
[6]
A. Estrada, G. S. Katselis, B. Laarvel, B. Barl, Isolation and evaluation of immunological adjuvant activities of saponins from Polygala senega L. Comp. Immunol. Microbiol. Infect. Dis. 23 (2000) 27–43. https://doi.org/10.1016/S01479571(99)00020-X
21
[7]
H. Sun, L. Chen, K. Wang, J. Zhou, Structure – function relationship of saponins from roots of Platycodon glandiflorum for hemolytic and adjuvant activity. Int. Immunopharmacol. 11 (2011) 2047–2056. https://doi.org/10.1016/j.intimp.2011.08.018.
[8]
S. G. Verza, F. Silveira, S. Cibulsky, S. Kaiser, S. Ferreira, G. Gossman, Immunoadjuvant activity, toxicity assays, and determination by UPLC/Q-TOF/MS of
of
triterpenic saponins from Chenopodium quinoa seeds. J. Agr. Food Chem. 60 (2012) 3113–3118. https://doi.org/10.1021/jf205010c.
M. Marrelli, F. Comforti, F. Araniti, G. A. Statti, Effects of Saponins on Lipid
ro
[9]
Metabolism: A Review of potential health benefits in the treatment of obesity. Molecules.
H. Sheng, H. Sung, Synthesis, biology and clinical significance of pentacyclic
re
[10]
-p
21 (10) (2016) 1404–1424. https://doi.org/10.3390/molecules21101404.
triterpenes: a multi-target approach to prevention and treatment of metabolic and vascular
[11]
lP
diseases. Nat Prod Rep. 28 (2011) 543–593. https://doi.org/10.1039/c0np00059k. J. Sakah Kaunda, Y-J. Zhang, The Genus Solanum: An Ethnophamacological,
ur na
phytochemical and biological properties review. Nat. Products and Bioprospect. 9 (2019) 77–137. https://doi.org/10.1007/s13659-019-0201-6. [12]
R. Coimbra, Notas de Fitoterapia. Catálogo dos Dados Principais sobre Plantas utilizadas em Medicina e Farmácia, 1958, 2.ed. Ed. Silva Araujo-Roussel. Rio de
Jo
Janeiro, Brasil, pp. 241.
[13]
M. Corrêa. Dicionário das Plantas Úteis do Brasil. Ministério da Agricultura. Instituto Brasileiro de Desenvolvimento Florestal, 1984, vol III, pp. 395.
[14]
A. B. Valerino-Diaz, D. Gamiotea-Turro, A. C. Zanatta, W. Vilegas, C. H. G. Martins, T. S. Silva, L. Rastrelli, L. C. Santos, New polyhydroxylated steroidal saponins from
22
Solanum paniculatum L. leaf alcohol tincture with antibacterial activity against oral pathogens. J. Agr Food Chem. 66 (2018) 8703–8713. https://doi.org/10.1021/acs.jafc.8b01262. [15]
M. A. Miranda, R. F. J. Tiossia, M. R. Silva, K. C. Rodrigues, C. C. Kuenha, L. G. R. Oliveira, In vitro leishmanicidal and cytotoxic activities of the glycoalkaloids from
of
Solanum lycocarpum (Solanaceae) fruits. Chem & Biodivers. 10 (2013) 642–648. https://doi.org/10.1002/cbdv.201200063.
K. Nurit-Silva, R. Costa Siva, I. J. L. D. Basílio, M. F. Agra, Leaf epidermal characters of
ro
[16]
Brazilian species of Solanum section Torva as taxonomic evidence. Can. J. Microbiol. 58
G. M. Vieira júnior, C. Quintino da Rocha, TdS. Rodrigues, C. Hakiko Hiruma-Lima,
re
[17]
-p
(2012) 806–814. https://doi.org/10.1139/b2012-046.
Vilegas, W, New steroidal saponins and antiulcer activity from Solanum paniculatum L.
[18]
lP
Food Chem. 86 (2015) 160–167. https://doi.org/10.1016/j.foodchem.2014.08.005. World Health Organization (WHO): Global Health Observatory Data 2018.
ur na
Leishmaniasis. Situation and trends. Available at: https://www.who.int/gho/neglected_diseases/leishmaniasis/en/#:~:text=Out%20of%2020 0%20countries%20and,endemic%20for%20leishmaniasis%20in%202018. (Accessed on August 15th, 2020).
F. T. Silveira, R. Lainson, C. E. P. Corbett, Clinical and immunopathological spectrum
Jo
[19]
of American cutaneous Leishmaniasis with special reference to the disease in Amazonian Brazil - A review. Mem. Inst. Oswaldo Cruz. 99 (2004) 239–251. https://doi.org/10.1590/S0074-02762004000300001.
23
[20]
R. Crupi, E. Gugliandolo, R. Siracusa, D. Impellizzeri, M. Cordaro, R. Di Paola, D. Britti, S. Cuzzocrea, N-acetyl-L-cysteine reduces Leishmania amazonensis-induced inflammation in BALB/c mice. Vet. Res. 16 (2020) 1–13. https://doi.org/10.1186/s12917-020-2234-9.
[21]
M. C. Duarte, G. S. V. Tavares, D. G. Valadares, D. P. Lage, T. G. Ribeiro, L. M. R.
of
Lage, M. R. Rodrigues, A. A. G. Faraco, M. Soto, E. S. da Silva, et al, Antileishmanial activity and mechanism of action from a purified fraction of Zinziber
28. https://doi.org/10.1016/j.exppara.2016.03.026.
C. W. Wright, J. D Phillipson, Natural products and the development of selective
-p
[22]
ro
officinalis Roschoe against Leishmania amazonensis. Exp. Parasitol. 166 (2016) 21–
re
antiprotozoal drugs. Phytother Res, 4 (2000) 127–139.
[23]
lP
https://doi.org/10.1002/ptr.2650040402.
D. R. A. Mans, T. Beerens, I Magali, R. C. Soekhoe, G. J. Schoone, K. Oedairadjsingh, J.
ur na
A. Hasrat, E. van den Bogaart, H. D. F. H. Schallig, In vitro evaluation of traditionally used Surinamese medicinal plants for their potential anti-Leishmanial efficacy. J Ethnopharmacol, 180 (2016) 70–77. https://doi.org/10.1016/j.jep.2016.01.012. T. T. Guimarães, M do C. F. R. Pinto, J. S. Lanza, M. N. Melo, R. L. do Monte-Neto, I. M. M. de Melo, E. B. T. Diogo, V. F. Ferreira, C. A. Camara, W. O. Valença, et. al.
Jo
[24]
Potent naphthoquinones against antimony-sensitive and -resistant Leishmania parasites: Synthesis of novel α- and nor- α-lapachonebased 1,2,3-triazoles by copper-catalyzed azide-alkyne cycloaddition. Eur. J. Med. Chem. 63 (2013) 523-530. https://doi.org/10.1016/j.ejmech.2013.02.038.
24
[25]
M. J. Corral, E. González, M. Cuquerella, J. M Alunda, Improvement of 96-well microplate assay of estimation of cell growth and inibition of Leishmania with Alamar Blue. J. Microbiol. Methods. 94 (2013) 111–116. https://doi.org/10.1016/j.mimet.2013.05.012.
[26]
P. S. Souza Araújo, S. S. Carvalho de Oliveira, C. M. d´Avila-Levy, A. L. Souza dos
of
Santos, M. H. Branquinha. Susceptibility of prosmatigotes and intracelular amastigotes from distinct Leishmania species to the calpain inhitor MDL 28170.
[27]
ro
Parasitol. Res. 117 (2018) 2085–2094. https://doi.org/10.1007/s00436-018-5894-7. D. M. Casa, D. B. Scariot, N. M. Khalil, C. V. Nakamura, R. M. Mainardes, Bovine
-p
serum albumin nanoparticles containing amphotericin B were effective in treating
re
murine cutaneous Leishmaniasis and reduced the drug toxicity. Exp. Parasitol, 192
[28]
lP
(2018) 12-18. https://doi.org/10.1016/j.exppara.2018.07.003.
S. Matsushita, Y. Yanai, A. Fusyuku, T. Ikeda, M. Ono, T. Nohara, Distinction of
ur na
Absolute Configuration at C-22 of C-23-hydroxylspirostane and C-23hydroxyspirosolane glycosides. Chem Pharm Bull. 5 (2007) 1079–1081. 000000000010.1248/cpb.55.1079. Brasil Anvisa. Agência Nacional de Vigilância Sanitária. Resolução da Diretoria
Jo
[29]
Colegiada - RDC Nº 166, 25/07/2017. Guia para validação de métodos analíticos, Diário Oficial da União: Brasília, 2017.
[30]
M. C Marcucci. Validação de princípios ativos de plantas medicinais e fitoterápicos. Farmacognosia, Coletânea Científica. 2011, pp. 271–293.
25
[31]
K. Katsuno, , J. N., Duncan, K., Hooft van Huijsduijnan, R., Kanako, T., Kita, K., Mowbray, C. E. Burrows, D. Schmatz, P. Warner, B. T. Slingsby, Hit and criteria in drug discovery for infectious diseases of the developing world. Nat. Rev. Drug Discov. 14 (2015) 751–758. https://doi.org/10.1038/nrd4683.
[32]
M. E. Rogers, M. L. Chance, P. A. Bates, The role of promastigote secretory gel in the
of
origin and transmission of the infective stage of Leishmania Mexicana by the sandfly Lutzomyia longipalpis. Parasitol, 124 (2002) 495–507.
[33]
ro
https://doi.org/10.1017/s0031182002001439.
H. Fadel, I. Sifaoui, A. López-Arencibia, M. Reyes-Batlle, S. Hajaji, O. Chiboub, I. A.
-p
Jiménez, I. L. Bazzocchi, J. Lorenzo-Morales, S. Benayache, J. E. Piñero, Assessment of
re
the antiprotozoal activity of Pulicaria inuloides extracts, an Algerian medicinal plant: Leishmanicidal bioguided fractionation. Parasitol Res. 117 (2018) 531–537.
[34]
lP
https://doi.org/10.1007/s00436-017-5731-4.
S. N. Marango, C. Khayeka-Wandabwa, J. A. Makwali, B. N. Jumba, J. K. Choge, E. O.
ur na
Adino, C. O. Anjili, Experimental therapeutic assays of Tephrosia vogelii against Leishmania major infection in murine model: in vitro and in vivo. BMC Res Notes, 10 (2017) 1–12. https://doi.org/10.1186/s13104-017-3022-x. P. S. Lage, M. A. Chávez-Fumagalli, J. T. Mesquita, L. M. Mata, S. O. Fernandes, V. N. Cardoso, M. Soto, C. A. Tavares, J. P. Leite, A. G. Tempone, E. A. Coelho,
Jo
[35]
Antileishmanial activity and evaluation of the mechanism of action of strychnobiflavone flavonoid isolated from Strychnos pseudoquina against Leishmania infantum. Parasitol Res. 114 (2015) 4625–4635. https://doi.org/10.1007/s00436-015-4708-4.
26
[36]
G. de Muylder, K. K. H. Ang, S. Chen, M. R. Arkin, J. C. Engel, J. H. McKerrow. A screen against Leishmania intracellular amastigotes: comparison to a promastigotes screen and identification of a host cell-specific hit. Plos Neglect. Trop. D. 5 (2011),
Jo
ur na
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re
-p
ro
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e1253. https://doi.org/10.1371/journal.pntd.0001253.
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Figure captions
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Figure 1. Spirostanic saponins (1-8) isolated from S. paniculatum leaves [14].
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Figure 2. (A). UHPLC-ESI-MS analysis of steroidal saponins in 70% ethanol extract of S. paniculatum leaves. (B) UHPLC-ESI-MS Selected Ion Monitoring (SIM) analysis of steroidal saponins with m/z = 725 ions (Negative mode). (C) UHPLC-ESI-MS Selected Ion Monitoring
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(SIM) analysis of steroidal saponins with m/z = 739 ions (Negative mode).
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Table 1. Identification of spirostanic saponins from 70% ethanol extract of S. paniculatum leaves by ESI-MSn. Rt (min)
[M-H]-
MS2
MS3
1
11.13
739
593
447
2
10.72
739
593
447
3
11.24
725
593
447
4
10.78
725
593
447
5
11.67
739
593
447
6
11.68
725
593
447
7
11.84
739
593
447
8
11.90
725
593
447
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Compound
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Table 2. Statistical analysis of regression equation, correlation coefficients and linearity range in the quantification of steroidal saponins by UHPLC/ESI/MS.
1 2 3 4 5 6 7 8
R2 0.9999 0.9994 0.9999 0.9989 0.9975 0.9972 0.9985 0.9992
Linear range (µg.mL-1) 0.098 – 2.5 0.028 – 1.25 0.087– 2.5 0.008 –1.25 0.048 – 0.625 0.054 – 0.625 0.21– 2.5 0.04– 1.25
LOD (µg.mL-1)
LOQ (µg.mL-1)
0.03 0.009 0.03 0.003 0.02 0.02 0.07 0.01
0.09 0.03 0.09 0.008 0.05 0.05 0.21 0.04
Y: peak area count; X: concentration of standard (µg.mL-1)
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Regression equationa Y=214537X+2921 Y=92856X+12324 Y=180448X+20120 Y=141575X+3491 Y=145255X+6854 Y=174515X+2527 Y=163353X+36486 Y=177970X + 7794
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Table 3. Contents of steroidal saponins in 70% ethanol extract of S. paniculatum leaves. µg.mL-1 RSDa
mg.g-1 RSD
1 2 3 4 5 6 7 8
0.71 0.02 0.26 0.01 1.04 0.02 0.26 0.003 0.17 0.01 0.15 0.004 0.43 0.02 0.32 0.004 3.34 0.091
71.2 0.2 25.8 0.1 103.5 0.2 25.7 0.03 17.5 0.1 14.8 0.04 43.0 0.2 32.1 0.04 333.6 0.09
Total Content RSD: Relative Standard Deviation.
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Compound
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Table 4. In vitro cytotoxicity evaluation in J774A.1 macrophages after 48 h of incubation. % of viability ± SDb 25 12.5
6.25
3.12
CC50 (µM)a
Compounds
50
1 2 3
75.27±2.01 69.10±4.67 71.69±2.50 0.78
78.94±0.73 76.82±2.32 76.34±0.93 0.39
81.47±0.27 87.49±1.15 78.72±0.83 0.19
97.27±1.53 99.03±0.88 89.74±5.87 96.54±3.19 91.34±2.29 97.81±2.00 0.095 0.047
>50 >50 >50
Amphotericin B
44.47±1.87
60.18±1.89
68.52±2.71
82.28±4.32 99.45±1.79
>0.78
a
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CC50 cytotoxic concentration values represent the concentration of compound required to kill 50% of the J774A.1 macrophage cells tested; b SD: Standard Deviation.
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Table 5. Leishmanicidal activity of steroidal saponins against promastigote forms of L. amazonensis and determination of IC50 values within 24 h of incubation. Compounds
IC50 (µM)a
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8.51±4.38 10.75±6.85 10.45±4.21 14.31±3.57 22.53±1.29 >50 37.11±1.62 21.58±1.04
68.43±2.13
54.31±2.41
44.81±3.17
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Amphotericin B
19.27±2.23 11.36±1.81 0.079±0.039
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Inhibition concentration 50 (IC50) Data were expressed as mean ± standard deviation (SD) of nine determinations (n = 9).
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b
3.12
87.80±0.65 81.68±4.51 64.95±11.66 30.18±4.22 21.84±3.89 95.63±6.17 88.13±2.97 44.14±3.62 40.98±5.19 0.62±0.88 80.84±6,65 80.27±1.72 65.33±4.53 37.37±2.26 2.10±2.01 86.67±3.03 78.84±2.24 35.51±2.65 20.96±9,65 3.65±1.17 72.64±7.40 53.96±1.36 31.73±3.22 18.95±6.87 2.43±0.72 43.50±8.03 30.34±5.036 27.10±1.32 25.28±9.21 8.56±0.31 53.23±5.53 43.14±9.14 31.00±5.80 8.10±9.38 0.00±0,00 71.12±1.86 51.38±3.89 37.59±8.20 25.57±1.05 6.84±0.89 0.19 0.095 0.047 0.023 0.011
1 2 3 4 5 6 7 8
a
50
% of growth inhibition ± SDb 25 12.5 6.25
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Table 6. Leishmanicidal activity of steroidal saponins against amastigote forms of L. amazonensis and determination of EC50 values within 48 h of incubation. % amastigote reduction ± SDa 12.5
6.25
3.12
1.56
EC50 (µM)a
1 2 3
44.05±0.91 56.91±1.71 50.88±0.97 0.095
29.02±2.40 41.55±1.82 43.29±1.31 0.047
12.12±0.37 28.45±2.17 32.05±0.87 0.023
0.00±0.00 18.79±0.86 20.79±1.21 0.011
0.00±0.00 0.00±0.00 0.00±0.00 0.005
>25 17.73±0.99 19.57±0.84
Amphotericin B
59.21±0.83
53.27±0.91
50.38±1.17
47.96±0.58 42.15±1.18 0.022±0.016
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Data were expressed as mean ± standard deviation (SD) of nine determinations (n = 9).
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Compounds
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