Chemical Prospection of Qualea grandiflora Mart. Fruit and Stem Extracts and Their in vitro and in vivo Antiplasmodial Activity

Gonçalves Neto, Geraldo S.; Costa, Romário P. da; Zanini, Camila L.; Aguiar, Anna Caroline C.; Souza, Juliana O. de; Souza, Guilherme E. de; Severino, Richele P.; Cass, Quezia B.; Cruz, Fábio C.; Oliva, Glaucius; Guido, Rafael V. C.; Sousa, Lorena R. F. de

doi:10.21577/0103-5053.20200034

Abstract

Qualea grandiflora Mart. (“pau-terra”) is a medicinal plant symbolic from Cerrado biome. Qualea species reportedly have antiparasitic properties, but their active compounds have yet to be identified. In this study, we investigated the antiplasmodial activity of Q. grandiflora fruit and stem extracts against Plasmodium falciparum. The polar extracts of both fruits and stems showed promising in vitro antiplasmodial activity against sensitive and resistant P. falciparum strains (half-maximal inhibitory concentration (IC₅₀) = 1.2 and 4.2 ng mL^-1, respectively), low cytotoxicity against human hepatic cells (IC₅₀^HepG2 ≥ 400 ng mL^-1), and a noteworthy selectivity index (SI ≥ 322 and ≥ 96, respectively). The fruit ethanolic extract revealed significant oral efficacy at 100 mg kg^-1 in a mouse model of P. berghei malaria (100% reduction in parasitemia on day 5 post-infection). To investigate the chemical composition of the extracts, we developed a method involving liquid chromatography-high resolution quadrupole-time of flight mass spectrometer. The chemical profiling of active extracts indicated 32 compounds, including gallotannins, ellagic acid derivatives and flavonoids. The common component in the active polar extracts was found to be ellagic acid (EA), which may contribute favorably to the in vitro and in vivo inhibitory activity observed in this study.

Keywords:
Qualea grandiflora ; Plasmodium falciparum ; ellagic acid

Introduction

Over 200 million cases of malaria, an infectious tropical disease caused by Plasmodium spp., were reported worldwide in 2017,¹1 http://www.who.int/malaria/publications/world-malaria-report-2017/en/, accessed in February 2020.
http://www.who.int/malaria/publications/... leading to an estimated death toll of 435,000 people. Despite numerous prevention and control measures, malaria remains one of the world’s main public health problems.

Various species of Plasmodium, e.g., P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi, are known to cause malaria infection in humans. Plasmodium protozoans, transmitted through the bites of Anopheles spp. mosquitoes have a complex life cycle, and despite the existence of antimalarial drugs such as quinolones, antifolates and artemisinin derivatives, all the known types of treatment have reportedly encountered parasite resistance, including the gold-standard treatment for malaria and artemisinin-based combination therapies (ACTs).²2 Aguiar, C. A.; de Sousa, L. R. F.; Garcia, C. R. S.; Oliva, G.; Guido, R. V. C.; Curr. Med. Chem. 2019, 26, 4380.

3 Lin, J. T.; Juliano, J. J.; Wongsrichanalai, C.; Curr. Infect. Dis. Rep. 2010, 12, 165.

4 Miotto, O.; Almagro-Garcia, J.; Manske, M.; Macinnis, B.; Campino, S.; Rockett, K. A.; Amaratunga, C.; Lim, P.; Suon, S.; Sreng, S.; Anderson, J. M.; Duong, S.; Nguon, C.; Chuor, C. M.; Saunders, D.; Se, Y.; Lon, C.; Fukuda, M. M.; Amenga-Etego, L.; Hodgson, A. V.; Asoala, V.; Imwong, M.; Takala-Harrison, S.; Nosten, F.; Su, X. Z.; Ringwald, P.; Ariey, F.; Dolecek, C.; Hien, T. T.; Boni, M. F.; Thai, C. Q.; Amambua-Ngwa, A.; Conway, D. J.; Djimdé, A. A.; Doumbo, O. K.; Zongo, I.; Ouedraogo, J. B.; Alcock, D.; Drury, E.; Auburn, S.; Koch, O.; Sanders, M.; Hubbart, C.; Maslen, G.; Ruano-Rubio, V.; Jyothi, D.; Miles, A.; O’Brien, J.; Gamble, C.; Oyola, S. O.; Rayner, J. C.; Newbold, C. I.; Berriman, M.; Spencer, C. C.; McVean, G.; Day, N. P.; White, N. J.; Bethell, D.; Dondorp, A. M.; Plowe, C. V.; Fairhurst, R. M.; Kwiatkowski, D. P.; Nat. Genet. 2013, 45, 648.

5 Parija, S. C.; Hiasindh, A. A.; Trop. Parasitol. 2016, 6, 30.

6 Tu, Y.; Nat. Med. 2011, 17, 1217.^-⁷7 Achan, J.; Talisuna, O. A.; Erhart, A.; Yeka, A.; Tibenderana, J. K.; Baliraine, F. N.; Rosenthal, P. J.; D’Alessandro, U.; Malar. J. 2011, 10, 144.

Natural products are a rich source of antimalarial compounds.⁶6 Tu, Y.; Nat. Med. 2011, 17, 1217.^,⁸8 Pan, W. H.; Xu, X. Y.; Shi, N.; Tsang, S. W.; Zhang, H. J.; Int. J. Mol. Sci. 2018, 19, 1382.Qualea grandiflora Mart., commonly known in Brazil as “pau-terra” or “pau-terra-do-cerrado,” belongs to the family Vochysiaceae and is one of the most common trees of the Brazilian cerrado biome. In folk medicine, the stem bark and leaves are used to treat inflammatory diseases and gastrointestinal disorders.⁹9 Ayres, M. C. C. A.; Escórcio, S. P.; da Costa, D. A.; Chaves, M. H.; Vieira Jr., G. M.; Cavalheiro, A. J.; Quim. Nova 2008, 31, 1481.

10 Hiruma-Lima, L. C.; Santos, H.; Kushima, C. H.; Pellizzon, G. G.; Silveira, P. C. P.; Vasconcelos, W.; Vilegas, A. R. M.; Brito, C. A. S.; J. Ethnopharmacol. 2006, 104, 207.^-¹¹11 Neto, F. C.; Pilon, A. C.; Silva, D. H.; da Silva, V. B.; Castro-Gamboa, I.; Phytochem. Rev. 2011, 10, 413. Moreover, Q. grandiflora stem and leaf extracts have shown several pharmacological properties, including antiulcerogenic, antibacterial, analgesic and anticonvulsant activity.⁹9 Ayres, M. C. C. A.; Escórcio, S. P.; da Costa, D. A.; Chaves, M. H.; Vieira Jr., G. M.; Cavalheiro, A. J.; Quim. Nova 2008, 31, 1481.^,¹²12 Gaspi, F. O. G.; Foglio, M. A.; Carvalho, J. E.; Moreno, R. A.; J. Ethnopharmacol. 2006, 107, 19.

Previous studies of Qualea species (Q. multiflora, Q. paraensis, Q. grandiflora) have described the antiparasitic properties of the plants’ extracts.¹³13 Muñoz, V.; Sauvain, M.; Bourdy, G.; Gallapa, J.; Bergeron, S.; Rojas, I.; Bravo, J. A.; Balderrama, L.; Ortiz, B.; Gimenez, A.; Deharo, E.; Ethnopharmacology 2000, 69, 127. Recently, fractions of ethyl acetate extract of leaves have shown antiparasitic activity against Trypanosoma cruzi, T. brucei gambiense and P. falciparum.¹⁴14 Cordeiro, T. M.; Borghetti, F.; Charneau, S.; Pharmacogn. Mag. 2017, 52, 668. However, to the best of our knowledge, there are no reports of the antiplasmodial properties of Qualea grandiflora Mart. fruit and stem extracts.

In this work, we investigated the in vitro and in vivo antiplasmodial activity of Q. grandiflora stem and fruit extracts. We then identified the active components of the extracts using ultra-high-performance liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (LC-HRMS). Our findings indicate that Qualea grandiflora Mart. fruit and stem extracts are alternative sources of natural products with in vitro and in vivo antiplasmodial properties.

Experimental

General experimental procedures

The solvents used were purchased from J.T. Baker (Phillipsburg, USA), all solvents were of analytical and HPLC grades. The LC-HRMS system was composed of UHPLC, 1260 Infinity II system (Agilent, Barueri, SP, Brazil), and it was equipped with a high-resolution mass spectrometer (HRMS) containing a quadrupole time-of-flight mass analyzer (QTOF, Impact HD) with an electrospray ionization (ESI) source (Bruker Daltonics, Bremen, Germany).

Plant material

The fruits and stems from Qualea grandiflora were collected in October 2015 in the cerrado at Federal University of São Carlos (UFSCar), São Carlos, SP, Brazil. The species (voucher No. 8854) were identified by Maristela Imatomi and deposited at the Herbarium of the Botany Laboratory (HUFSCar) at the same University.

Extraction and sample preparation

Air-dried (40 °C) stems (2.0 kg) were extracted with ethanol. Fruits (1.8 kg) were directly extracted with ethanol as well. The concentrated ethanolic extracts from fruits (QgEfr) and stems (QgEs), 15 g of each, were dissolved in ethanol and distilled water (1:3) for liquid-liquid partition. The extracts were partitioned, and the polar extracts obtained were used for antimalarial assays as well for chemical fingerprinting.

The ethyl acetate (EtOAc) extract from fruits (QgEAfr) and stems (QgEAs), and hydroalcoholic extracts from stems (QgHs) were concentrated for the LC analysis. The samples were prepared using 1 mg mL^-1 of extracts (QgEAfr, QgEAs and QgHs) with Milli-Q water (Massachusetts, USA) and methanol (8:2 v/v), and 1 µL of each solution were injected into the LC-HRMS system.

LC-HRMS

For the analysis, a CortecsTM C18+ (2.7 µm particle size; 10 × 0.21 cm) (Waters, Milford, MA, USA) analytical column was used with a mobile phase composed of water (A), acetonitrile (B), and 0.1% v/v of formic acid was added to both solvents. A linear gradient of 5 to 100% B in 20 min was used at a flow rate of 0.2 mL min^-1, with temperature of 40 °C.

The ionization experiments were carried out at positive [M + H]⁺ and negative [M - H]^- modes. The parameters used for mass spectrometry ionization source were as following: nebulizer, 3.0 bar; dry gas flow, 8.0 L min^-1; dry heater temperature, 180 °C; capillary voltage, 4500 V; end plate offset, 500 V; collision cell energy, 5 eV; and full-MS scan range, m/z 100-1000. The acquisition was obtained in auto MS/MS mode (number of precursors: 3) in experiments with collision energy of 20, 25, 30, 35 and 40 eV for all m/z analyzed range. The Data Analysis 4.0 software and Bruker Smart Formula (Bruker Daltonics, Bremen, Germany) were used as tools for compounds identification.

In vitro assay in Plasmodium falciparum

A culture of Plasmodium falciparum, 3D7 strain, was maintained in complete Roswell Park Memorial Institute (RPMI) medium, supplemented with Albumax II^®. This culture was synchronized using the sorbitol method,¹⁵15 Lambros, C.; Vanderberg, J. P.; J. Parasitol. 1979, 65, 418. and the resulting ring-stage culture was adjusted to 2% hematocrit and 0.5% parasitemia using fresh medium and human O+ red blood cells (RBCs).

In a 96-well plate, 20 µL of two-fold serial dilutions of each compound were prepared, to a concentration 10 times higher than the final desired concentration. Each compound was tested in duplicate, and compounds were tested in two independent experiments for result confirmation. A volume of 180 µL of the prepared culture was then distributed in each well, and positive (untreated parasitized RBCs) and negative (non-parasitized RBCs) growth controls were included in each plate. The plate was then incubated at 37 °C and 5% CO₂, for 72 h. After the incubation, the plate fluorescence was read in a spectrophotometer SpectraMax^® Gemini^TM microplate readers (Molecular Devices, California, USA), using the SYBR Green I method¹⁶16 Smilkstein, M.; Sriwilaijaroen, N.; Kelly, J. X.; Wilairat, P.; Riscoe, M.; Antimicrob. Agents Chemother. 2004, 48, 1803. and the intensity data were analyzed in OriginPro software,¹⁷17 OriginPro, version 8; OriginLab Corp., Massachussets, USA, 2008. resulting in a sigmoidal dose response curve from which the IC₅₀ values were obtained.

In vitro cytotoxicity evaluation

A culture of hepatoma cells (HepG2) was maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum until it reached confluency. For cytotoxicity evaluation, the confluent culture flask was trypsinized for deadhesion and the suspended cells were counted, followed by distribution on a 96-well plate to a final concentration of 5 × 10⁶ cells per well. The plate was then incubated for 24 h for cell adhesion. In a separate 96-well plate, 20 µL of serial dilutions of the compounds were prepared and transferred to the cell culture plate. After another 24 h of incubation, the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine cell viability in each well. Briefly, 20 µL of a 5 mg mL^-1 solution of MTT was added to each well, followed by a 3-h incubation period. The formazan crystals formed were solubilized in 100 µL of dimethyl sulfoxide (DMSO), and the plate absorbance was read in the SpectraMax^® Plus 384 microplate readers (Molecular Devices, California, USA). Intensity data were normalized and plotted using the OriginPro software,¹⁷17 OriginPro, version 8; OriginLab Corp., Massachussets, USA, 2008. and the resulting curves were analyzed to determine the half-maximal inhibitory concentration (IC₅₀^HepG2) for each compound.

In vivo assay in Plasmodium berghei

The suppressive test was performed as described.¹⁸18 Peters, W.; Exp. Parasitol. 1965, 17, 97. The P. berghei NK65 strain was obtained as a donation from New York University and maintained through weekly blood passages. For the experiments, the use of animals is in agreement with ethics and animals care which was approved by the Ethics Committee for Animal Use of Universidade Federal do Estado de São Paulo (UNIFESP) under CEUA No. 6630080816. The mice were inoculated i.p. (intraperitoneal) with 1 × 10⁵5 Parija, S. C.; Hiasindh, A. A.; Trop. Parasitol. 2016, 6, 30. infected erythrocytes, kept together for about 24 h, then randomly distributed into groups of five per cage. The mice were treated daily for three consecutive days with compounds freshly diluted in distilled water and administered orally at 100 mg kg^-1; the control groups received either the drug vehicle or the antimalarial chloroquine (CQ) administered at 20 mg kg^-1. On days 5 and 7 after the parasite inoculation, blood was taken from the tail of each mouse and used to prepare thin smears that were methanol-fixed, Giemsa-stained, and examined microscopically (1000×) to determine parasitemia. The inhibition of parasite growth was determined in relation to parasitemia in the untreated mice, considered to be 100% parasite growth. Compounds reducing the parasitemia by > 40% were considered active, between 30 and 40% partially active, and by less than 30% were considered inactive. The experiments were performed twice.

Results and Discussion

The in vitro assays indicated that the six Q. grandiflora extracts were active against both sensitive (3d7) and resistant (k1) P. falciparum strains (IC₅₀ values between 1.2 and 7 ng mL^-1) (Table 1). The inhibitory activity evaluated showed good correlation between the sensitive and resistant strains, indicating that the active compounds in the fractions showed no cross-resistance underlying the resistance mechanism of the k1 strain. Moreover, all the extracts produced very low toxic effects on human hepatic cells in assay conditions (IC₅₀^HepG2 ≥ 400 ng mL^-1), showing significant selectivity indices (SI ≥ 58) (Table 1). In view of this finding, we assessed the in vivo activity of the most potent and selective extract, the fruit ethanolic extract (QgEfr, IC₅₀ = 1.2 ng mL^-1 and SI ≥ 322), in Plasmodium berghei-infected mice.

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Table 1
In vitro antiplasmodial and cytotoxic activity of Q. grandiflora extracts against P. falciparum and HepG2

Five infected mice were treated orally with 100 mg kg^-1 of the ethanolic extract for three consecutive days after infection (Figure 1). Parasitemia was evaluated on days 5 and 7 post-infection. The antimalarial drug CQ was used as positive control (20 mg kg^-1). QgEfr extract reduced parasitemia by 100% on day 5 post-infection, similar to the CQ control, and by 83% on day 7 (Figure 1).

Figure 1
In vivo inhibitory activity evaluation of Qualea grandiflora fruit ethanolic extract (QgEfr, 100 mg kg⁻¹) and chloroquine (CQ, 20 mg kg⁻¹) against P. berghei. Summary of percentage of survival parasite after treatment with QgEfr extract and chloroquine (control). QgEfr extract reduced parasitemia by 100% on day 5 post-infection, similar to the CQ control. On day 7 QgEFr reduced parasitemia by (83 ± 12)% and CQ control maintained 100% of reduction.

To assess the chemical profile of the active polar extracts of Q. grandiflora fruits and stems, we analyzed the samples by LC-HRMS and optimized chromatographic conditions. The MS/MS data in negative ion mode [M - H]^- provided product ion spectra. To identify the compounds, we investigated each spectrum and compared it to free compound screening libraries online.¹⁹19 Aquino, A. J.; Alves, T. C.; Oliveira, R. V.; Ferreira, A. G.; Cass, Q. B.; Ind. Crops Prod. 2019, 132, 59; https://massbank.eu/, accessed in February 2020; http://mona.fiehnlab.ucdavis.edu/, accessed in February 2020; https://metlin.scripps.edu, accessed in February 2020.
https://massbank.eu/... In addition, we used data published in the literature²⁰20 Gu, D.; Yang, Y.; Bakri, M.; Chen, Q.; Xin, X.; Aisa, H. A.; Phytochem. Anal. 2013, 24, 661.

21 Singh, A.; Bajpai, V.; Kumar, S.; Sharma, K. R.; Kumar, B.; Nat. Prod. Commun. 2016, 11, 239.

22 Nasser, A. L. M.; Carli, C. B. A.; Rodrigues, C. M.; Maia, D. C. G.; Carlos, I. Z.; Eberlin, M. N.; Hiruma-Lima, C.; Vilegas, W.; Z.Naturforsch. 2008, 63, 794.

23 Zhou, Z. H.; Liu, M. Z.; Wang, M. H.; Qu, W.; Sun, J. B.; Liang, J. Y.; Wu, F. H.; Nat. Prod. Res. 2015, 29, 798.

24 Wu, S. H.; Chen, Y. W.; Yang, L. Y.; Li, S. L.; Li, Z. Y.; Fitoterapia 2008, 79, 474.

25 Bai, N.; He, K.; Roller, M.; Zheng, B.; Chen, X.; Shao, Z.; Peng, T.; Zheng, Q.; J. Agric. Food Chem. 2008, 56, 11668.

26 Dutra, R. P.; Abreu, B. V. B.; Cunha, M. S.; Batista, M. C. A.; Torres, L. M. B.; Nascimento, F. R. F.; Ribeiro, M. N. S.; Guerra, R. N. M.; J. Agric. Food Chem. 2014, 62, 2549.

27 Slimestad, R.; Biochem. Syst. Ecol. 2003, 31, 1247.^-²⁸28 Zhang, Z.; He, L.; Lu, L.; Liu, Y.; Dong, G.; Miao, J.; Luo, P.; J. Pharm. Biomed. Anal. 2015, 109, 62. to compare the exact mass and MS/MS fragmentations patterns, enabling us to identify 32 compounds from several chemical classes, including gallotannins, ellagic acid derivatives and flavonoids (Table 2).

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Table 2
Secondary metabolites identified from QgEAfr, QgEAs and QgHs of Q. grandiflora. The exact mass of compounds was assigned based on MS experimental data

Characterization

Ellagic acid (1A,2F and 3Hi) and 14 ellagic acid derivatives (2A,3A,6A,7A,13A,3F,7F,8F,9F,11F,1Hi,2Hi,4Hi and 5Hi) were identified from Q. grandiflora fruit and stem extracts. Ellagic acid was identified in the ethyl acetate (EtOAc) extracts of stems (QgEAs) (1A) and fruits (QgEAfr) (2F), as well as in hydroalcoholic extract of stems (QgHs) (3Hi). The peaks pertaining to compounds 1A,2F and 3Hi indicated the [M - H]^- ions with m/z values of 300.9991, 300.9997 and 300.9965 (C₁₄H₆O₈), respectively, with fragment ions characteristic of ellagic acid, such as those at m/z 283.9954, 273.0041, 257.0083, 245.0084, 229.0137, 201.0190, 185.0244, 173.0242, 157.0290, 145.0295, and 129.0345 (Table 2).²⁰20 Gu, D.; Yang, Y.; Bakri, M.; Chen, Q.; Xin, X.; Aisa, H. A.; Phytochem. Anal. 2013, 24, 661.^,²¹21 Singh, A.; Bajpai, V.; Kumar, S.; Sharma, K. R.; Kumar, B.; Nat. Prod. Commun. 2016, 11, 239.

The ellagic acid derivatives were identified based on the abundant ion fragments of MS² spectra with m/z values varying from 299.9883 to 299.9919. Compounds 2A (QgEAs), 1Hi (QgHs) and 11F (QgEAfr) displayed similar [M - H]^- ions at m/z 461.0731, 461.0679 and 461.0733 (C₂₁H₁₈O₁₂), respectively. Furthermore, we observed the loss of 146.0588 Da related to a hexose and of 15.0231 Da pertaining to a CH₃ group (Figure 2). The rhamnose moiety was suggested for the hexose since the compound 3’-O-methyl-4-O-(rhamnopyranosyl) ellagic acid has reportedly¹¹11 Neto, F. C.; Pilon, A. C.; Silva, D. H.; da Silva, V. B.; Castro-Gamboa, I.; Phytochem. Rev. 2011, 10, 413.^,²¹21 Singh, A.; Bajpai, V.; Kumar, S.; Sharma, K. R.; Kumar, B.; Nat. Prod. Commun. 2016, 11, 239.^,²²22 Nasser, A. L. M.; Carli, C. B. A.; Rodrigues, C. M.; Maia, D. C. G.; Carlos, I. Z.; Eberlin, M. N.; Hiruma-Lima, C.; Vilegas, W.; Z.Naturforsch. 2008, 63, 794. been identified in Q. grandiflora stems.

Figure 2
(a) Proposed fragmentation pattern of methyl-O-rhamnopyranosyl ellagic acid (2A (QgEAs), 1Hi (QgHs) and 11F (QgEAfr)); (b) proposed fragmentation pattern of methyl-O-pentopyranoside ellagic acid (3A (QgEAs), 2Hi (QgHs) and 9F (QgEAfr)).

The ellagic acid derivatives 3A (QgEAs), 2Hi (QgHs) and 9F (QgEAfr) exhibited [M - H]¯ ions at m/z 447.0574, 447.0522 and 447.0573 (C₂₀H₁₆O₁₂), respectively, and the fragmentation ions 132.0435 Da (removal of a pentose moiety) and 15.0229 Da (loss of a CH₃ group), suggesting they were methyl-pentopyranoside ellagic acids (Figure 2). Compounds 6A (QgEAs) and 4Hi (QgHs) with [M - H]^- ions at m/z 433.0414 and 433.0376, respectively, showed the loss of pentose in both compounds (133.0505 Da).

The peak (retention time (t_R) = 7.02 min) related to 13A (QgEAs) produced the deprotonated ion m/z 629.0787 (C₂₈H₂₂O₁₇), and the MS² spectra showed the elimination of a hexose moiety [M - h - 162]^-, a galloyl [M - h - 152]^-, and a methyl unit [M - h - 15]^-, allowing for the assignment of a methyl-galloyl-glucopyranosyl ellagic acid.²²22 Nasser, A. L. M.; Carli, C. B. A.; Rodrigues, C. M.; Maia, D. C. G.; Carlos, I. Z.; Eberlin, M. N.; Hiruma-Lima, C.; Vilegas, W.; Z.Naturforsch. 2008, 63, 794.^,²³23 Zhou, Z. H.; Liu, M. Z.; Wang, M. H.; Qu, W.; Sun, J. B.; Liang, J. Y.; Wu, F. H.; Nat. Prod. Res. 2015, 29, 798. Compound 5Hi (QgHs) displayed a [M - H]^- ion at m/z 477.0631 (C₂₁H₁₈O₁₃) and the loss of 162.0509 and of 15.0239 Da suggesting a glucose and a CH₃ moiety, respectively, which correspond to methyl ellagic acid-O-glucopyranoside.²⁴24 Wu, S. H.; Chen, Y. W.; Yang, L. Y.; Li, S. L.; Li, Z. Y.; Fitoterapia 2008, 79, 474.

Based on the [M - H]^- ions at m/z 329.0311 (C₁₆H₁₀O₈) and 343.0470 (C₁₇H₁₂O₈), followed by the loss of CH₃ groups [M - h - 15]^- in MS² spectra, we identified compounds 3F and 7F as di-O-methyl ellagic acid (3F) and tri-O-methyl ellagic acid (7F), respectively.²¹21 Singh, A.; Bajpai, V.; Kumar, S.; Sharma, K. R.; Kumar, B.; Nat. Prod. Commun. 2016, 11, 239.^,²⁵25 Bai, N.; He, K.; Roller, M.; Zheng, B.; Chen, X.; Shao, Z.; Peng, T.; Zheng, Q.; J. Agric. Food Chem. 2008, 56, 11668. Compound 8F produced m/z 469.0066 (C₂₁H₁₀O₁₃) and the fragment ions at m/z 425.0158, 301.0003 and 169.0150. The diagnostic ion at m/z 169.0150 corresponded to the galloyl unit and the one at 301.0003 to ellagic acid. Thus, we identified the compound as valoneic acid dilactone.²⁶26 Dutra, R. P.; Abreu, B. V. B.; Cunha, M. S.; Batista, M. C. A.; Torres, L. M. B.; Nascimento, F. R. F.; Ribeiro, M. N. S.; Guerra, R. N. M.; J. Agric. Food Chem. 2014, 62, 2549.

We also identified gallotannins. Six of gallotannin derivatives were found in the stem extracts, QgEAs (4A,5A,8A,10A,11A and 12A), together with gallic acid (9A), and six gallotannins were identified in the fruit extract, QgEAfr (1F,5F,6F,12F,13F and 14F). Gallic acid (t_R = 1.28 min) (9A) (QgEAs) was identified based on its [M - H]^- ion at m/z 169.0138 (C₇H₆O₅), which provided the product ion at m/z 124.0161 through the loss of CO₂ and hydrogen, corresponding to 44.9977 Da.²¹21 Singh, A.; Bajpai, V.; Kumar, S.; Sharma, K. R.; Kumar, B.; Nat. Prod. Commun. 2016, 11, 239.

The gallotannin derivatives 4A,8A,11A and 12A (QgEAs) with ions at m/z 453.1030 (C₂₀H₂₂O₁₂); m/z 331.0664 (C₁₃H₁₆O₁₀); m/z 483.0767 (C₂₀H₂₀O₁₄) and m/z 635.0868 (C₂₇H₂₄O₁₈), respectively, produced product ions between m/z 169.0139 and 169.0147, which were attributed to the presence of a galloyl moiety. For the compound 12A, successive losses of the galloyl group in MS² spectra and decarboxylation [M - H - 44]^- produced the characteristic fragments at m/z 465.0654, 313.0554, 300.9976, 275.0202, 211.0242, 169.0139, 125.0240 (Table 2, Figure 3). These data indicated the presence of a sugar core in the structure of compounds 4A,8A,11A and 12A. Those compounds produced the typical loss of hexose units [M - h - 162]^-. In compound 4A, the loss of 284.0883 Da pointed to hexose bound to the methyl catechol moiety. According to the fragmentation pathway of compounds 4A,8A,11A and 12A, we identified a methoxy-hydroxyphenol-O-galloyl-hexoside , a galloyl-hexose, a digalloyl-hexose and a trigalloyl-hexose, respectively.²⁰20 Gu, D.; Yang, Y.; Bakri, M.; Chen, Q.; Xin, X.; Aisa, H. A.; Phytochem. Anal. 2013, 24, 661.^,²¹21 Singh, A.; Bajpai, V.; Kumar, S.; Sharma, K. R.; Kumar, B.; Nat. Prod. Commun. 2016, 11, 239. Galloyl-hexoses 8A and 6F were identified in the EtOAc extract of stems and fruits, respectively.

Figure 3
Fragmentation pattern of trigalloyl-hexose 12A (QgEAs).

The gallotannins 10A (QgEAs) and 1F (QgEAfr) exhibited similar [M - H]^- ions at m/z 783.0664 and 783.0690 (C₃₄H₂₄O₂₂), respectively, and based on their mass loss observed in the MS² spectrum (1F) at 302.0055, 180.0634 and 25.9789 Da, we identified them as pedunculagin.²¹21 Singh, A.; Bajpai, V.; Kumar, S.; Sharma, K. R.; Kumar, B.; Nat. Prod. Commun. 2016, 11, 239. The peak (t_R = 5.63 min) of compound 5A (QgEAs) was identified as ethyl gallate since it exhibited [M - H]^- ions at m/z 197.0455 (C₉H₁₀O₅), with a loss of C₃H₅O₂ corresponding to 73.0288 Da.²⁰20 Gu, D.; Yang, Y.; Bakri, M.; Chen, Q.; Xin, X.; Aisa, H. A.; Phytochem. Anal. 2013, 24, 661.^,²¹21 Singh, A.; Bajpai, V.; Kumar, S.; Sharma, K. R.; Kumar, B.; Nat. Prod. Commun. 2016, 11, 239.

Compounds 5F,12F,13F and 14F, from fruit extracts (QgEAfr), provided similar product fragments in MS² spectra, with a mass loss [M - 162]^- of hexose moiety. Moreover, 12F showed a [M - H]^- ion at m/z 633.0742 (C₂₇H₂₂O₁₈), and an evident fragment at m/z 169.0113, thereby indicating a galloyl group, which led to the assignment of galloyl-hexahydroxydiphenoyl (HHDP)-hexose.²³23 Zhou, Z. H.; Liu, M. Z.; Wang, M. H.; Qu, W.; Sun, J. B.; Liang, J. Y.; Wu, F. H.; Nat. Prod. Res. 2015, 29, 798. Compounds 5F and 14F exhibited [M - H]¯ ions at m/z 481.0630 (C₂₀H₁₈O₁₄) and 785.0841 (C₃₄H₂₆O₂₂), respectively, with prominent ions at m/z 300.9999 and 300.9976, confirming the presence of the HHDP group in both compounds. The gallotannin 5F was characterized as HHDP-hexose. Compound 14F was identified as di-O-galloyl-HHDP-hexose isomer based on its fragment ions of m/z 615.0644, 483.0780, 419.0746, 300.9976, 275.0215, 249.0368 and on the loss of H₂O and CO₂ by HHDP, which generated ions at m/z 300.9976, 275.0215, and 249.0368.²⁰20 Gu, D.; Yang, Y.; Bakri, M.; Chen, Q.; Xin, X.; Aisa, H. A.; Phytochem. Anal. 2013, 24, 661.

Compound 13F showed an [M - H]^- ion at m/z 935.0784 (C₄₁H₂₈O₂₆), with fragment ions at m/z 783.0674, 633.0720, 301.0004, 275.0191, and 249.0386, most of them similar to the fragment ions of compound 14F. The low abundance of ions at m/z 783.0674 was attributed to the loss of a galloyl group (152.0110 Da) from the [M - H]^- ion at m/z 935.0784, which is characteristic of di-O-galloyl-lactonised valoneoyl-hexose.²⁰20 Gu, D.; Yang, Y.; Bakri, M.; Chen, Q.; Xin, X.; Aisa, H. A.; Phytochem. Anal. 2013, 24, 661.

The flavonoids in QgEAfr (4F and 10F) were identified as isorhamnetin acetyl-hexoside and apigenin-O-hexoside, respectively. In the negative ion mode [M - H]^-, the product ion spectra of both flavonoids showed loss of a hexose unit (162.0526 Da). The flavonol exhibited the [M - H]^- ion at m/z 519.0325 (C₂₄H₂₄O₁₃) with fragment ions at m/z 461.0731 and 315.0153, and the loss of 57.9594 Da in the acetyl moiety.²⁷27 Slimestad, R.; Biochem. Syst. Ecol. 2003, 31, 1247. The apigenin-O-hexoside (10F) was identified by its molecular ion [M - H]^- at m/z 431.1007, with fragment ion m/z 269.0455 corresponding to aglycone apigenin [M - h - 162]^-, and the fragment ion m/z 125.0235 corresponding to a cleavage in ring C.²⁸28 Zhang, Z.; He, L.; Lu, L.; Liu, Y.; Dong, G.; Miao, J.; Luo, P.; J. Pharm. Biomed. Anal. 2015, 109, 62. Glycosylated flavonols and triterpenes have been previously identified in Q. grandiflora leaf extracts.⁹9 Ayres, M. C. C. A.; Escórcio, S. P.; da Costa, D. A.; Chaves, M. H.; Vieira Jr., G. M.; Cavalheiro, A. J.; Quim. Nova 2008, 31, 1481. Flavanones have been identified in the stems of this species, as well. Flavones, flavanones and dihydroflavonols are usually found in the wood of Qualea species.¹¹11 Neto, F. C.; Pilon, A. C.; Silva, D. H.; da Silva, V. B.; Castro-Gamboa, I.; Phytochem. Rev. 2011, 10, 413. In this regard, this is the first report of the occurrence of flavonol and flavone in the fruits of the genus Qualea.

Note that most of the abovementioned compounds, e.g., all the gallotannins and gallotannin derivatives and the flavonoids isorhamnetin acetyl-hexoside and apigenin-O-hexoside were identified for the first time in Q. grandiflora (Table 2). On the other hand, gallic acid and some ellagic acid (EA) derivatives from Q. grandiflora stems, such as 3,3’-di-O-methylellagic acid-4-O-β-D-glucopyranoside, 3-O-methylellagic acid-4’-O-α-L-rhamnopyranoside, 3,3’,4-tri-O-methylellagic acid-4’-O-β-D-glucopyranoside, and 3,3’-di-O-methylellagic acid, have been identified in previous studies.¹¹11 Neto, F. C.; Pilon, A. C.; Silva, D. H.; da Silva, V. B.; Castro-Gamboa, I.; Phytochem. Rev. 2011, 10, 413.^,²²22 Nasser, A. L. M.; Carli, C. B. A.; Rodrigues, C. M.; Maia, D. C. G.; Carlos, I. Z.; Eberlin, M. N.; Hiruma-Lima, C.; Vilegas, W.; Z.Naturforsch. 2008, 63, 794.

Antiplasmodial activity and chemical profiling

Our findings suggest that Q. grandiflora fruit and stems polar extracts contain active components with antiplasmodial activity. These results led us to ascertain the chemical fingerprint of the active extracts in order to identify the key components of the plant’s antiplasmodial activity. LC-HRMS proved to be a comprehensive and robust method and an excellent tool for the prospection of known and unknown substances in natural product extracts. This approach combines the separation ability of high-performance liquid chromatography with high-resolution mass spectra from which fragment ions are obtained through different collision energies provided by soft ionization techniques.²⁹29 Allard, P. M.; Genta-Jouve, G.; Wolfender, J. L.; Curr. Opin. Chem. Biol. 2017, 36, 40.

30 https://www.intechopen.com/books/tandem-mass-spectrometry-applications-and-principles/electrospray-ionization-tandem-mass-spectrometry-as-a-tool-for-the-structural-elucidation-and-derepl, accessed in February 2020.
https://www.intechopen.com/books/tandem-...

31 Henke, M. T.; Kelleher, N. L.; Nat. Prod. Rep. 2016, 33, 942.^-³²32 Stylos, E.; Chatziathanasiadou, M. V.; Syriopoulou, A.; Tzakos, A. G.; J. Chromatogr. B 2017, 1047, 15.

The LC-HRMS analysis enabled the identification of gallotannins, ellagic acid (EA) derivatives and flavonoids. We identified galloyl-hexose (8A,6F) and gallotannin pedunculagin (10A,1F) in both the QgEAs and QgEAfr active extracts, as well as EA analogues (1A,2F and 3Hi) and EA bound to sugar moiety (pentose or hexose) (2A,3A,9F,11F,1Hi and 2Hi) in the QgEAs, QgEAfr and QgHs extracts. Previous studies³³33 Soh, P. N.; Witkowski, B.; Gales, A.; Huyghe, E.; Berry, A.; Pipy, B.; Benoit-Vical, F.; PLoS One 2012, 7, e45906. found that ellagic acid derivatives exhibited in vitro antiplasmodial activity (IC₅₀ values ranging from 105 to 330 nM) and in vivo activity (median effective dose (ED₅₀) activity lower than 1 mg kg^-1 day^-1, i.p.). Given that EA is the constituent normally identified in active stem and fruit extracts (QgEAs and QgEAfr, respectively), it could be the main component of the mixture that contributed favorably to the in vitro and in vivo efficacy of the extracts observed in this study.

The biological activities mainly related to the consumption of ellagitannins in natural sources could be consequence of the EA release as well.³⁴34 Seeram, N. P.; Lee, R.; Heber, D.; Clin. Chim. Acta 2004, 348, 63. Previous studies³⁵35 Long, J.; Guo, Y.; Yang, J.; Henning, S. M.; Lee, R. P.; Rasmussen, A.; Zhang, L.; Lu, Q. Y.; Heber, D.; Li, Z.; Food Funct. 2019, 10, 6582. investigating absorption and bioavailability of bioactive ellagitannins and EA in vivo dietary consumption of fruits showed that human ingestion of 237 mL of pomegranate (23 mg free EA) and 500 mg of free EA lead to the same plasma concentration of EA. The EA from natural source was more bioavailable and bioactive than free EA related to anti-inflammatory response. The promising result found on fruit extract (QgEfr) evaluated in P. bergheiin vivo also may be due to a role of the other constituents with EA that would be further investigated.

Additionally, we identified the presence of isorhamnetin acetyl-hexoside and apigenin-O-hexoside (compounds 4F and 10F, respectively) in the Q. grandiflora fruit ethyl acetate extract (QgEAfr). The in vitro antiplasmodial activity of the fruit ethyl acetate extract (QgEAfr) observed here also may be due to the inhibitory property of the apigenin aglycone. The compound isolated from Melampyrum arvense L. displayed a significant inhibitory effect against P. falciparum, with an IC₅₀ value of 15.5 µg mL^-1.³⁶36 Kirmizibekmez, H.; Atay, I.; Kaiser, M.; Brun, R.; Cartagena, M. M.; Carballeira, N. M.; Yesilada, E.; Tasdemir, D.; Phytother. Res. 2011, 25, 142.

Conclusions

To the best of our knowledge, this paper offers the first report of the chemical profile of Qualea fruits. Moreover, our findings indicate that both the in vitro and in vivo inhibitory activity of the fruit ethanolic extract may be related to the presence of ellagic acid, a major component of the mixture. In summary, the collected data provided solid scientific information about the chemical profile of Qualea fruits, in addition to the in vitro and in vivo antiplasmodial activity of polar extracts of Qualea grandiflora (“pau-terra”) fruits and stems.

Supplementary Information

The MS/MS spectra data of the identified compounds (1A-13A; 1F-14F; 1Hi-5Hi) are available free of charge at http://jbcs.sbq.org.br as PDF file.

Acknowledgments

This study was financed in part by the CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil), Finance Code 001, and by CNPq (the National Council for Scientific and Technological Development, grants 405330/2016-2 and 471509/2012-4). We also thank FAPESP, the São Paulo Research Foundation (CEPID grant 2013/07600-3 and grant 2013/07600-3, 2013/01710-1 and 2014/50244-6) and Serrapilheira Institute (grant Serra-1708-16250).

References

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» http://www.who.int/malaria/publications/world-malaria-report-2017/en/
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³
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⁴
Miotto, O.; Almagro-Garcia, J.; Manske, M.; Macinnis, B.; Campino, S.; Rockett, K. A.; Amaratunga, C.; Lim, P.; Suon, S.; Sreng, S.; Anderson, J. M.; Duong, S.; Nguon, C.; Chuor, C. M.; Saunders, D.; Se, Y.; Lon, C.; Fukuda, M. M.; Amenga-Etego, L.; Hodgson, A. V.; Asoala, V.; Imwong, M.; Takala-Harrison, S.; Nosten, F.; Su, X. Z.; Ringwald, P.; Ariey, F.; Dolecek, C.; Hien, T. T.; Boni, M. F.; Thai, C. Q.; Amambua-Ngwa, A.; Conway, D. J.; Djimdé, A. A.; Doumbo, O. K.; Zongo, I.; Ouedraogo, J. B.; Alcock, D.; Drury, E.; Auburn, S.; Koch, O.; Sanders, M.; Hubbart, C.; Maslen, G.; Ruano-Rubio, V.; Jyothi, D.; Miles, A.; O’Brien, J.; Gamble, C.; Oyola, S. O.; Rayner, J. C.; Newbold, C. I.; Berriman, M.; Spencer, C. C.; McVean, G.; Day, N. P.; White, N. J.; Bethell, D.; Dondorp, A. M.; Plowe, C. V.; Fairhurst, R. M.; Kwiatkowski, D. P.; Nat. Genet. 2013, 45, 648.
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¹⁰
Hiruma-Lima, L. C.; Santos, H.; Kushima, C. H.; Pellizzon, G. G.; Silveira, P. C. P.; Vasconcelos, W.; Vilegas, A. R. M.; Brito, C. A. S.; J. Ethnopharmacol. 2006, 104, 207.
¹¹
Neto, F. C.; Pilon, A. C.; Silva, D. H.; da Silva, V. B.; Castro-Gamboa, I.; Phytochem. Rev. 2011, 10, 413.
¹²
Gaspi, F. O. G.; Foglio, M. A.; Carvalho, J. E.; Moreno, R. A.; J. Ethnopharmacol. 2006, 107, 19.
¹³
Muñoz, V.; Sauvain, M.; Bourdy, G.; Gallapa, J.; Bergeron, S.; Rojas, I.; Bravo, J. A.; Balderrama, L.; Ortiz, B.; Gimenez, A.; Deharo, E.; Ethnopharmacology 2000, 69, 127.
¹⁴
Cordeiro, T. M.; Borghetti, F.; Charneau, S.; Pharmacogn. Mag. 2017, 52, 668.
¹⁵
Lambros, C.; Vanderberg, J. P.; J. Parasitol. 1979, 65, 418.
¹⁶
Smilkstein, M.; Sriwilaijaroen, N.; Kelly, J. X.; Wilairat, P.; Riscoe, M.; Antimicrob. Agents Chemother. 2004, 48, 1803.
¹⁷
OriginPro, version 8; OriginLab Corp., Massachussets, USA, 2008.
¹⁸
Peters, W.; Exp. Parasitol. 1965, 17, 97.
¹⁹
Aquino, A. J.; Alves, T. C.; Oliveira, R. V.; Ferreira, A. G.; Cass, Q. B.; Ind. Crops Prod. 2019, 132, 59; https://massbank.eu/, accessed in February 2020; http://mona.fiehnlab.ucdavis.edu/, accessed in February 2020; https://metlin.scripps.edu, accessed in February 2020.
» https://massbank.eu/» http://mona.fiehnlab.ucdavis.edu/» https://metlin.scripps.edu
²⁰
Gu, D.; Yang, Y.; Bakri, M.; Chen, Q.; Xin, X.; Aisa, H. A.; Phytochem. Anal. 2013, 24, 661.
²¹
Singh, A.; Bajpai, V.; Kumar, S.; Sharma, K. R.; Kumar, B.; Nat. Prod. Commun. 2016, 11, 239.
²²
Nasser, A. L. M.; Carli, C. B. A.; Rodrigues, C. M.; Maia, D. C. G.; Carlos, I. Z.; Eberlin, M. N.; Hiruma-Lima, C.; Vilegas, W.; Z.Naturforsch. 2008, 63, 794.
²³
Zhou, Z. H.; Liu, M. Z.; Wang, M. H.; Qu, W.; Sun, J. B.; Liang, J. Y.; Wu, F. H.; Nat. Prod. Res. 2015, 29, 798.
²⁴
Wu, S. H.; Chen, Y. W.; Yang, L. Y.; Li, S. L.; Li, Z. Y.; Fitoterapia 2008, 79, 474.
²⁵
Bai, N.; He, K.; Roller, M.; Zheng, B.; Chen, X.; Shao, Z.; Peng, T.; Zheng, Q.; J. Agric. Food Chem. 2008, 56, 11668.
²⁶
Dutra, R. P.; Abreu, B. V. B.; Cunha, M. S.; Batista, M. C. A.; Torres, L. M. B.; Nascimento, F. R. F.; Ribeiro, M. N. S.; Guerra, R. N. M.; J. Agric. Food Chem. 2014, 62, 2549.
²⁷
Slimestad, R.; Biochem. Syst. Ecol. 2003, 31, 1247.
²⁸
Zhang, Z.; He, L.; Lu, L.; Liu, Y.; Dong, G.; Miao, J.; Luo, P.; J. Pharm. Biomed. Anal. 2015, 109, 62.
²⁹
Allard, P. M.; Genta-Jouve, G.; Wolfender, J. L.; Curr. Opin. Chem. Biol. 2017, 36, 40.
³⁰
https://www.intechopen.com/books/tandem-mass-spectrometry-applications-and-principles/electrospray-ionization-tandem-mass-spectrometry-as-a-tool-for-the-structural-elucidation-and-derepl, accessed in February 2020.
» https://www.intechopen.com/books/tandem-mass-spectrometry-applications-and-principles/electrospray-ionization-tandem-mass-spectrometry-as-a-tool-for-the-structural-elucidation-and-derepl
³¹
Henke, M. T.; Kelleher, N. L.; Nat. Prod. Rep. 2016, 33, 942.
³²
Stylos, E.; Chatziathanasiadou, M. V.; Syriopoulou, A.; Tzakos, A. G.; J. Chromatogr. B 2017, 1047, 15.
³³
Soh, P. N.; Witkowski, B.; Gales, A.; Huyghe, E.; Berry, A.; Pipy, B.; Benoit-Vical, F.; PLoS One 2012, 7, e45906.
³⁴
Seeram, N. P.; Lee, R.; Heber, D.; Clin. Chim. Acta 2004, 348, 63.
³⁵
Long, J.; Guo, Y.; Yang, J.; Henning, S. M.; Lee, R. P.; Rasmussen, A.; Zhang, L.; Lu, Q. Y.; Heber, D.; Li, Z.; Food Funct. 2019, 10, 6582.
³⁶
Kirmizibekmez, H.; Atay, I.; Kaiser, M.; Brun, R.; Cartagena, M. M.; Carballeira, N. M.; Yesilada, E.; Tasdemir, D.; Phytother. Res. 2011, 25, 142.

Publication Dates

Publication in this collection
03 July 2020
Date of issue
July 2020

History

Received
15 Sept 2019
Accepted
04 Mar 2020

This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

[1] ¹
http://www.who.int/malaria/publications/world-malaria-report-2017/en/, accessed in February 2020.
» http://www.who.int/malaria/publications/world-malaria-report-2017/en/

[2] ²
Aguiar, C. A.; de Sousa, L. R. F.; Garcia, C. R. S.; Oliva, G.; Guido, R. V. C.; Curr. Med. Chem. 2019, 26, 4380.

[3] ³
Lin, J. T.; Juliano, J. J.; Wongsrichanalai, C.; Curr. Infect. Dis. Rep. 2010, 12, 165.

[4] ⁴
Miotto, O.; Almagro-Garcia, J.; Manske, M.; Macinnis, B.; Campino, S.; Rockett, K. A.; Amaratunga, C.; Lim, P.; Suon, S.; Sreng, S.; Anderson, J. M.; Duong, S.; Nguon, C.; Chuor, C. M.; Saunders, D.; Se, Y.; Lon, C.; Fukuda, M. M.; Amenga-Etego, L.; Hodgson, A. V.; Asoala, V.; Imwong, M.; Takala-Harrison, S.; Nosten, F.; Su, X. Z.; Ringwald, P.; Ariey, F.; Dolecek, C.; Hien, T. T.; Boni, M. F.; Thai, C. Q.; Amambua-Ngwa, A.; Conway, D. J.; Djimdé, A. A.; Doumbo, O. K.; Zongo, I.; Ouedraogo, J. B.; Alcock, D.; Drury, E.; Auburn, S.; Koch, O.; Sanders, M.; Hubbart, C.; Maslen, G.; Ruano-Rubio, V.; Jyothi, D.; Miles, A.; O’Brien, J.; Gamble, C.; Oyola, S. O.; Rayner, J. C.; Newbold, C. I.; Berriman, M.; Spencer, C. C.; McVean, G.; Day, N. P.; White, N. J.; Bethell, D.; Dondorp, A. M.; Plowe, C. V.; Fairhurst, R. M.; Kwiatkowski, D. P.; Nat. Genet. 2013, 45, 648.

[5] ⁵
Parija, S. C.; Hiasindh, A. A.; Trop. Parasitol. 2016, 6, 30.

[6] ⁶
Tu, Y.; Nat. Med. 2011, 17, 1217.

[7] ⁷
Achan, J.; Talisuna, O. A.; Erhart, A.; Yeka, A.; Tibenderana, J. K.; Baliraine, F. N.; Rosenthal, P. J.; D’Alessandro, U.; Malar. J. 2011, 10, 144.

[8] ⁸
Pan, W. H.; Xu, X. Y.; Shi, N.; Tsang, S. W.; Zhang, H. J.; Int. J. Mol. Sci. 2018, 19, 1382.

[9] ⁹
Ayres, M. C. C. A.; Escórcio, S. P.; da Costa, D. A.; Chaves, M. H.; Vieira Jr., G. M.; Cavalheiro, A. J.; Quim. Nova 2008, 31, 1481.

[10] ¹⁰
Hiruma-Lima, L. C.; Santos, H.; Kushima, C. H.; Pellizzon, G. G.; Silveira, P. C. P.; Vasconcelos, W.; Vilegas, A. R. M.; Brito, C. A. S.; J. Ethnopharmacol. 2006, 104, 207.

[11] ¹¹
Neto, F. C.; Pilon, A. C.; Silva, D. H.; da Silva, V. B.; Castro-Gamboa, I.; Phytochem. Rev. 2011, 10, 413.

[12] ¹²
Gaspi, F. O. G.; Foglio, M. A.; Carvalho, J. E.; Moreno, R. A.; J. Ethnopharmacol. 2006, 107, 19.

[13] ¹³
Muñoz, V.; Sauvain, M.; Bourdy, G.; Gallapa, J.; Bergeron, S.; Rojas, I.; Bravo, J. A.; Balderrama, L.; Ortiz, B.; Gimenez, A.; Deharo, E.; Ethnopharmacology 2000, 69, 127.

[14] ¹⁴
Cordeiro, T. M.; Borghetti, F.; Charneau, S.; Pharmacogn. Mag. 2017, 52, 668.

[15] ¹⁵
Lambros, C.; Vanderberg, J. P.; J. Parasitol. 1979, 65, 418.

[16] ¹⁶
Smilkstein, M.; Sriwilaijaroen, N.; Kelly, J. X.; Wilairat, P.; Riscoe, M.; Antimicrob. Agents Chemother. 2004, 48, 1803.

[17] ¹⁷
OriginPro, version 8; OriginLab Corp., Massachussets, USA, 2008.

[18] ¹⁸
Peters, W.; Exp. Parasitol. 1965, 17, 97.

[19] ¹⁹
Aquino, A. J.; Alves, T. C.; Oliveira, R. V.; Ferreira, A. G.; Cass, Q. B.; Ind. Crops Prod. 2019, 132, 59; https://massbank.eu/, accessed in February 2020; http://mona.fiehnlab.ucdavis.edu/, accessed in February 2020; https://metlin.scripps.edu, accessed in February 2020.
» https://massbank.eu/» http://mona.fiehnlab.ucdavis.edu/» https://metlin.scripps.edu

[20] ²⁰
Gu, D.; Yang, Y.; Bakri, M.; Chen, Q.; Xin, X.; Aisa, H. A.; Phytochem. Anal. 2013, 24, 661.

[21] ²¹
Singh, A.; Bajpai, V.; Kumar, S.; Sharma, K. R.; Kumar, B.; Nat. Prod. Commun. 2016, 11, 239.

[22] ²²
Nasser, A. L. M.; Carli, C. B. A.; Rodrigues, C. M.; Maia, D. C. G.; Carlos, I. Z.; Eberlin, M. N.; Hiruma-Lima, C.; Vilegas, W.; Z.Naturforsch. 2008, 63, 794.

[23] ²³
Zhou, Z. H.; Liu, M. Z.; Wang, M. H.; Qu, W.; Sun, J. B.; Liang, J. Y.; Wu, F. H.; Nat. Prod. Res. 2015, 29, 798.

[24] ²⁴
Wu, S. H.; Chen, Y. W.; Yang, L. Y.; Li, S. L.; Li, Z. Y.; Fitoterapia 2008, 79, 474.

[25] ²⁵
Bai, N.; He, K.; Roller, M.; Zheng, B.; Chen, X.; Shao, Z.; Peng, T.; Zheng, Q.; J. Agric. Food Chem. 2008, 56, 11668.

[26] ²⁶
Dutra, R. P.; Abreu, B. V. B.; Cunha, M. S.; Batista, M. C. A.; Torres, L. M. B.; Nascimento, F. R. F.; Ribeiro, M. N. S.; Guerra, R. N. M.; J. Agric. Food Chem. 2014, 62, 2549.

[27] ²⁷
Slimestad, R.; Biochem. Syst. Ecol. 2003, 31, 1247.

[28] ²⁸
Zhang, Z.; He, L.; Lu, L.; Liu, Y.; Dong, G.; Miao, J.; Luo, P.; J. Pharm. Biomed. Anal. 2015, 109, 62.

[29] ²⁹
Allard, P. M.; Genta-Jouve, G.; Wolfender, J. L.; Curr. Opin. Chem. Biol. 2017, 36, 40.

[30] ³⁰
https://www.intechopen.com/books/tandem-mass-spectrometry-applications-and-principles/electrospray-ionization-tandem-mass-spectrometry-as-a-tool-for-the-structural-elucidation-and-derepl, accessed in February 2020.
» https://www.intechopen.com/books/tandem-mass-spectrometry-applications-and-principles/electrospray-ionization-tandem-mass-spectrometry-as-a-tool-for-the-structural-elucidation-and-derepl

[31] ³¹
Henke, M. T.; Kelleher, N. L.; Nat. Prod. Rep. 2016, 33, 942.

[32] ³²
Stylos, E.; Chatziathanasiadou, M. V.; Syriopoulou, A.; Tzakos, A. G.; J. Chromatogr. B 2017, 1047, 15.

[33] ³³
Soh, P. N.; Witkowski, B.; Gales, A.; Huyghe, E.; Berry, A.; Pipy, B.; Benoit-Vical, F.; PLoS One 2012, 7, e45906.

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Extract	IC₅₀ ± SD / (ng mL^-1)			Selectivity index (SI)^a a Selectivity index = IC503d7 / IC50HepG2. IC50: half-maximal inhibitory concentration; QgEs: ethanolic extracts from stems; QgEfr: ethanolic extracts from fruits; QgHs: hydroalcoholic extracts from stems; QgHfr: hydroalcoholic extracts from fruits; QgEAs: ethyl acetate extract from stems; QgEAfr: ethyl acetate extract from fruits.
	P. falciparum		HepG2
	3d7	k1	HepG2
QgEs	4.5 ± 0.5	4.2 ± 0.5	≥ 400	≥ 96
QgEfr	1.2 ± 0.7	1.2 ± 0.7	≥ 400	≥ 322
QgHs	7 ± 1	7 ± 1	≥ 400	≥ 58
QgHfr	3 ± 2	3 ± 2	≥ 400	≥ 150
QgEAs	3 ± 2	5 ± 2	≥ 400	75
QgEAfr	7.0 ± 0.1	6 ± 1	370 ± 19	97
Artesunate	11 ± 0.008 nM	7 ± 0.002 nM	279 ± 24	25.36

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