South African Journal of Botany 161 (2023) 472 481

Contents lists available at ScienceDirect

South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Multielement/proximate composition, chemical and cytotoxic profile to

assess the Crinum americanum L. as an unconventional raw material of
nutritional, chemical, and pharmaceutical potential

zara de Arau
Renata La
joa, Carolina Lilibeth Carvalho de Pinhoa, Fabiane Oliveira Fariasa,
Wanderlei do Amarala, Alberto Wisniewski Juniorb, Adenilda Cristina Hono
rio-Françac,
a a,
Luciana Igarashi-Mafra , Marcos R. Mafra *
a
Department of Chemical Engineering, Federal University of Parana, Av. Francisco Hera
clito dos Santos, n. 100, PC 81531-980, Brazil
b
Post-Graduate Program in Chemistry, Petroleum and Energy from Biomass Research Group (PEB), Federal University of Sergipe (UFS), Av. Marechal Rondon, S / n
- Jardim Rosa Elze, Sa ~o Cristo ~o, SE 49100-000, Brazil

va
c
Institute of Biological and Health Science, Federal University of Mato Grosso, Barra do Garças, Mato Grosso, 78600-000, Brazil

A R T I C L E I N F O A B S T R A C T

Article History: This work aimed to characterize the nutritional and multielement profile of leaves, bulbs and roots of C.
Received 22 January 2023 americanum L., as well as to optimize a mixture of solvents (ethanol, water and ethyl acetate) to extract the
Revised 3 August 2023 highest content of bioactive compounds and evaluate its ex vivo cytotoxicity against human peripheral blood
Accepted 21 August 2023
mononuclear (MN) cells. With the nutritional and multielement analysis, it was possible to determine a safe
Available online xxx
dosage for direct consumption of this plant. The optimized solvent system resulted in high antioxidant capac-
Edited by Dr J. Gruz ity (DPPH and FRAP), total phenolic compounds (TPC) and total alkaloids (TAC). These results may be associ-
Keywords:
ated with the number of bioactive compounds identified by Mass Spectrometry with Electrospray Ionization
Medicinal plants and Fourier Transform (ESI-FT-MS). In total, 89 compounds were identified, including alkaloids, phenolic
Cytotoxicity compounds, fatty acids and others never before identified for this plant. Furthermore, the optimized extracts
Natural products did not show ex vivo cytotoxicity against MN cells at the tested concentrations. To the best of our knowledge,
Amaryllidaceae there is no data in the literature concerning the C. americanum L. composition in its totality. Thus, the follow-
Bioactive compounds ing data are unprecedented and corroborate to establish the C. americanum L. as an unconventional plant
nutritionally viable alternative for human consumption.
© 2023 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction Among the genera of this family, Crinum has been used in tradi-
tional medicine to treat fever, pain, swelling, wounds, cancer, and
Plants of the Amaryllidaceae family are known to produce a vari- malaria (de Arau
jo et al., 2022). Within this genus, the species Crinum
ety of pharmacologically active secondary metabolites, such as phe- americanum L., although poorly studied, has some alkaloids such as
nolic compounds, lecithins, peptides, and especially alkaloids (de crinine, lycorine, and phenanthridine (Gomes et al., 2022), and essen-
Arau
jo et al., 2022). Regarding the latter, they have cytotoxic (Gomes tial fatty acids (Barra et al., 2021) with potential for pharmaceutical
et al., 2022), antiviral activities (Narayanan et al., 2022), and a wide and medicinal applications. This fact can be observed in the biological
range of other physiological effects such as antibacterial, anti-inflam- potential presented by the tea of this plant, used in traditional medi-
matory (Jin and Yao, 2019) and acetylcholinesterase inhibitor (Lekhak cine in coastal communities in the interior of Parana
, Brazil, for the
et al., 2021). treatment of cancer (Silva et al., 2020).
Although this use is performed without scientific basis, recent
studies indicate that the ethanolic extract of leaves and bulbs of this
Abbreviations: AChE, acetylcholinesterase; ATR, atropine; DF, dietary fiber; ESI-FT-
MS, mass spectrometry with electrospray Ionization and Fourier transform; HCA, hier-
species showed cytotoxic effects against strains of human neuroblas-
archical clustering; MN, mononuclear cells; OEB, optimized extracts of bulbs; OEL, toma and promising activity in inhibiting acetylcholinesterase (Gomes
optimized extracts of leaves; OER, optimized extracts of roots; PCA, principal compo- et al., 2022), demonstrating a scientific relation with popular knowl-
nent analysis; RSM, response surface methodology; SDF, soluble dietary fiber; SLE, edge. In addition, C. americanum L. extracts were found to have antifun-
solid-liquid extraction; TAC, total alkaloid content; TE, Trolox; TPC, total phenolic con-
gal activity against various Candida species, which is another interesting
tent; UEP, unconventional food plants
* Corresponding author. biological activity, since Candida species are hospital patients’ main fun-
E-mail address: [email protected] (M.R. Mafra). gal agents causing infectious conditions (Silva et al., 2022).

https://doi.org/10.1016/j.sajb.2023.08.042
0254-6299/© 2023 SAAB. Published by Elsevier B.V. All rights reserved.

C.L.C. de Pinho, F.O. Farias et al.
R.L. de Araujo, South African Journal of Botany 161 (2023) 472 481

The biological potential of C. americanum L. observed by popular Methods (AOAC International, 2012). The difference between 100
medicine and in scientific studies can be attributed to the alkaloids and the sum of the percentage of the approximate composition of
present in its composition (de Arau
jo et al., 2022). Alkaloids are sec- plant material was designated as carbohydrates.
ondary metabolites that can be used in medicines, cosmetics, antimi-
crobial films, and even agricultural insecticides (Rachkeeree et al., 2.3. Multi-element analysis
2020). In this sense, the application of this species in different prod-
ucts can be diverse. Thus, studies that verify the toxicological safety, Digestion of material for multi-element determination was per-
yield, phytochemical profiles, and nutritional values of this plant or formed according to Silva et al. (2021), with some modifications.
their extracts are important to develop new technological applica- Briefly, 0.7 g of each sample was weighed and digested with HNO3,
tions in the chemical, pharmaceutical, and, possibly, food sectors under heating at 70 °C. The obtained solution was filtered with a
(Moura et al., 2021). 0.45 mm syringe filter and injected into an inductively plasma optical
The toxicity of a plant extract can be studied using different chem- emission spectrometer (ICP OES iCAP 6000 series - Thermo Scien-
ical and cell-based in vitro and ex vivo assays. Mononuclear cells (MN) tific, Massachusetts, USA) in the experimental conditions (plasma gas
from human peripheral blood (lymphocytes and monocytes) are flow: 12 L/min; auxiliar gas flow: 0.5 L/min; carrier gas flow: 0.7 L/
examples of cells used for cytotoxicity tests in extracts and vegetable min; radio frequency power (RF): 1.15 kW; and stabilization time:
oils (Arau
jo et al., 2019; Cruz et al., 2020). The study of cytotoxicity in 10 s). The wavelengths of the analyzed elements ranged from
these cells is interesting, as they do not have biotransformation 182.034 to 766.490 nm.
enzymes, so they do not activate indirect mutagens, but inform on
the action of direct mutagens, that is, they have toxic effects by them- 2.4. Extraction procedure and experimental design
selves, without the need for metabolization (Mehra and Chadha,
2021). Also, investigating the cell proliferation effect of the natural The extractions were carried out at 40 °C for 45 min under mag-
product-treated immune cell is the preliminary study for discovering netic stirring, and a 1:20 (sample: solvent, m/v) ratio. Three different
new immunomodulators from medicinal plants. Mononuclear cells solvents were tested: ultrapure water, ethyl alcohol (EtOH), and ethyl
(MN) from human peripheral blood is commonly used as target cell acetate (EtOAc) and their mixtures. The extracts obtained were rotae-
for this study (Yeap et al., 2007). vaporated, lyophilized, and diluted with ultrapure water at the time
Furthermore, due to the intrinsic nature and stability of chemical of use. An augmented simplex centroid mixture design containing 10
compounds, obtaining them requires several process variables, direc- combinations of solvents was used to evaluate the isolated effects,
tion, and individual consideration. Since the functional properties binary and ternary mixtures of solvents (Table 1). DPPH, FRAP, TPC,
(e.g. antioxidant, antimicrobial, and antiproliferative activities) of the and TAC analyzes were performed to select the optimal extraction
plant extract depend on the chemical composition, the study of dif- conditions.
ferent solvents is a relevant and demanding factor to be considered In combinations in which a binary mixture was formed (assays 6,
(Fidelis et al., 2020). 7, and 8), only the phase that presented the highest values for total
In this sense, studying the chemical composition and obtaining alkaloids (TAC) and antioxidant capacity was considered for the
compatible processes for the extraction of bioactive compounds from study.
C. americanum L. in a simple, low-toxic way and with advantageous
characteristics, such as sustainability, biodegradability and accept- 2.5. Total phenolics and chemical antioxidant activity
able toxicity profiles, would lead to economic and sustainable devel-
opment coastal communities in the interior of Brazil, as well as the The total phenolic content (TPC) was determined by colorimetric
appreciation of this species and its products. Therefore, this study analysis with Folin-Ciocalteu reagent (FC), according to Singleton et
evaluated the nutritional and multielement composition of C. ameri- al. (1999). The free-radical scavenging activity of DPPH was analyzed
canum L., as well as the biological composition and ex vivo toxicity of according to Brand-Williams et al. (1995). The extracts’ ferric reduc-
extracts obtained by an optimized solvent system. ing antioxidant power (FRAP) was performed according to Benzie &
Strain (1996). All data were compared with a standard curve (gallic
2. Material and methods acid for TPC, Trolox for DPPH, and ferric sulfate for FRAP) and the
results were expressed as mg of equivalent per 100 g of dry sample.
2.1. Collection and preparation of plant material
2.6. Total alkaloids content (TAC)
Samples of C. americanum L. were collected in the coastal man-
grove region, located in Bahia do Guaratuba, municipality of Guara- The total alkaloid content (TAC) was determined by colorimetric
tuba, Parana
, Brazil (geographical coordinates S 25° 530 000 and W analysis according to Ghane et al. (2018) with modifications. The
48° 340 340 ’), in March 2021. Samples were separated into leaves, extract of the three parts of C. americanum L. (500 mL) was treated
bulbs, and roots, and dried in an oven at 40 °C with air circulation separately with 500 mL of 2 N HCl. This solution was transferred to
until constant weight. Afterward, they were ground in a laboratory 50 mL falcon tubes. 2.5 mL of bromocresol green solution and 2.5 mL
mill (Requipal, MR 320, Sa ~o Paulo, Brazil), sieved to obtain particle of phosphate buffer were added. Afterward, 1, 2, 3, and 4 mL of chlo-
sizes <0.2 mm, vacuum packed, and frozen for further application at roform were successively added by vigorous stirring. At each addi-
10 °C. This research was registered in the National Genetic Heritage tion, the chloroform phase was collected. In the end, the volume of
Management System (SisGen) under the number A58F1B2. The exsic- all samples was adjusted to 10 mL. Atropine was used as a reference.
cates of the species were herborized, identified, and registered in the The results were expressed as mg of Atropine per 100 g of dry sam-
Herbarium of UFPR UPCB under the number UPCB 101062. The ple.
plant name has been checked with http://www.theplantlist.org on
December 5, 2023. 2.7. Optimization of extraction conditions

2.2. Proximate composition of plant material To evaluate the effects of the solvents on the extraction of bioac-
tive compounds from extracts from leaves, bulbs, and roots of C.
The contents of moisture, ash, protein, lipids, soluble and insolu- americanum L., the response surface methodology was used (RSM)
ble fiber, and total sugar were determined by AOAC International following the recommendations outlined by Granato & Arau
jo Calado

473

C.L.C. de Pinho, F.O. Farias et al.
R.L. de Araujo, South African Journal of Botany 161 (2023) 472 481

Table 1
Antioxidant capacity, total phenolic compounds, and total alkaloids of extracts from leaves, bulbs, and roots of C. americanum L.

Leaf

Assay Ethanol (A) Water (B) Ethyl acetate (C) DPPH (mg TE/100 g) FRAP (mg Fe (II)/100 g) TPC (mg GAE/100 g) TAC (mg ATR/100 g)
c f
1 1 0 0 1474 § 3 1455 § 6 g 1154 § 6 1212 § 18d
2 0 1 0 1873 § 4bc 2748 § 3e 2034 § 5d 860 § 28ef
3 0 0 1 1037 § 4e 2069f § 3 926 § 5h 811 § 15f
4 0.5 0.5 0 1840 § 9bc 2619 § 4e 2229 § 16cd 1163 § 32d
5 0.5 0 0.5 1143 § 4d 1352 § 2h 1015 § 5 g 979 § 17e
6 0 0.5 0.5 2420 § 8ab 3260 § 6b 3046 § 15a 1833 § 19c
7 0.333 0.333 0.334 2234 § 5b 2900 § 3d 2367 § 16c 1636 § 18cd
8 0.666 0.167 0.167 2608 § 9a 3368 § 4a 2865 § 11b 3081 § 17b
9 0.167 0.66 0.167 1915 § 6bc 2622 § 5e 2246 § 15c 5953 § 45a
10 0.167 0.167 0.666 2392 § 8ab 3098 § 2c 1981 § 15e 1887 § 31c
p-value (normality) 0.713 0.305 0.561 0.450
p-value (homoscedasticity) 0.013 0.567 0.743 0.149
p-value (one-way ANOVA) <0.001 <0.001 <0.001 <0.001

Bulbs
c
1 1 0 0 620 § 1 751 § 36c 792 § 15d 398 § 11d
2 0 1 0 481 § 3e 562 § 6e 743 § 3e 172 § 12fg
3 0 0 1 172 § 2j 566 § 3e 278 § 2j 162 § 11 g
4 0.5 0.5 0 450 § 3f 76 § 2i 708 § 13f 389 § 15d
5 0.5 0 0.5 263 § 1i 671 § 17d 398 § 12i 175 § 3f
6 0 0.5 0.5 409 § 3h 383 § 6 g 551 § 21h 613 § 9c
7 0.333 0.333 0.334 543 § 3d 662 § 3d 916 § 14c 249 § 3e
8 0.666 0.167 0.167 633 § 5b 833 § 7b 1094 § 13b 1236 § 18b
9 0.167 0.66 0.167 442 § 4 g 327 § 6h 679 § 19 g 1984 § 16a
10 0.167 0.167 0.666 818 § 3a 1890 § 13a 1306 § 21a 649 § 10c
p-value (normality) 0.703 0.391 0.941 0.436
p-value (homoscedasticity) 0.852 0.743 0.723 0.303
p-value (one-way ANOVA) <0.001 <0.001 <0.001 <0.001

Root

1 1 0 0 330 § 3h 615 § 14c 339 § 12f 584 § 10bc

2 0 1 0 337 § 13 g 2571 § 5d 490 § 4c 134 § 3 g
3 0 0 1 150 § 8k 2899 § 34a 183 § 2h 147 § 6f
4 0.5 0.5 0 560 § 12b 543 § 6c 675 § 12b 496 § 7c
5 0.5 0 0.5 279 § 4i 513 § 7c 241 § 8gh
6 0 0.5 0.5 354 § 14f 166 § 3e 468 § 7d 239 § 11e
7 0.333 0.333 0.334 606 § 8a 758 § 6b 748 § 14a 611 § 4bc
8 0.666 0.167 0.167 454 § 18c 206 § 3e 538 § 4c 846 § 10b
9 0.167 0.66 0.167 358 § 11e 57 § 0.3 g 367 § 3e 297 § 6d
10 0.167 0.167 0.666 448 § 5d 47§ 0.3h 482 § 9cd 1355 § 11a
p-value (normality) 0.934 0.601 0.802 0.755
p-value (homoscedasticity) 0.303 0.352 0.909 0.342
p-value (one-way ANOVA) <0.001 <0.001 <0.001 <0.001
Note: Trolox (TE); ferric sulfate (Fe III); gallic acid (GAE); Atropine (ATR). Different letters in the same column indicate a statistically significant difference
(p  0.05).

(2013), and modifications suggested by Fidelis et al. (2020). To opti- 2.9. Cytotoxicity ex vivo
mize the extraction conditions (e.g., solvent mixture), the desirability
function was used to maximize the extraction of TPC, TAC, and anti-
2.9.1. Cell preparation
oxidant capacity (DPPH and FRAP).
Separation of mononuclear phagocytes from human (MN) periph-
eral blood was performed accordingly to Cruz et al. (2020). For the
study, 10 mL of blood was collected from voluntary donors who
2.8. Mass spectrometry analysis
signed a consent form approved by the local ethics committee of the
Federal University of Mato Grosso (Protocol Number CAAE:
Mass spectrometry (MS) characterization was performed with
1.415.375). Then, the methyl thiazolyl tetrazolium (MTT) cytotoxicity
optimized extracts of leaves (OEL), bulbs (OEB), and roots (OER) of C.
assay was performed.
americanum L. Samples were injected and data were collected for ESI
(-), according to Fraga et al. (2021). For ESI (+) data were collected
under the following conditions: heating of the vaporization region, 2.9.2. MTT assay
50 °C; capillary temperature, 300 °C; spray voltage, 3.0 kV; coating The MTT reduction assay by mitochondria of viable cells was per-
gas stream 15; and sweep gas 5. The structural formulas were sug- formed according to Mosmann (1983), with some modifications, and
gested from information from previous works involving species of cell viability was determined after 24 h of reaction. Briefly, 100 mL of
the Amaryllidaceae family and from the NIST and Chemspider data- MN cells and extracts were pipetted into 96-well plates and incu-
base. Altogether, 89 compounds were identified in ESI (+) and ESI (-) bated (5% CO2) for 24 h at 37 °C. Subsequently, 20 mL of MTT (5 mg/
(Table S1 - Supplementary Material). Table 2 identifies the main alka- mL) were added. After 3 h of incubation, 50 mL of 20% Sodium
loids determined by ESI (+) and phenolic compounds and saturated Dodecyl Sulfate (SDS) solution was added and the reading was per-
and unsaturated fatty acids determined by ESI (-). formed in a microplate reader at 492 nm.
474

C.L.C. de Pinho, F.O. Farias et al.
R.L. de Araujo, South African Journal of Botany 161 (2023) 472 481

Table 2
Accurate mass measurement for molecular ions of parent compounds in C. americanum L. optimized extracts.

N Compound name ESI (+) Formula Theoretical OEL OEB OER References
mass+
Experimental data

Mass %* Mass %* Mass %*

ALKALOIDS
1 Crinine C16H17NO3 271.1208 271.1279 19.69 271.1278 1.09 271.1278 3.28 (Gomes et al., 2022)
2 Lycorine C16H17NO4 287.1158 287.1228 100 287.1235 100 287.1227 82.68
3 Crinamine C17H19NO4 301.1314 301.1385 14.74 301.1383 1.06 301.1383 0.34
3.1 Haemanthamine C17H19NO4 301.1314 301.1385 14.74 301.1383 1.06 301.1383 0.34
3.2 Epipowelline C17H19NO4 301.1314 301.1385 14.74 301.1383 1.06 301.1383 0.34
4 1,2-didehydrocrinan-3-one C16H15NO3 269.1052 269.0980 0.1
5 Dihydrolycorine C16H19NO4 289.1314 289.1382 1.73 289.1382 2.85 289.1383 6.05
6 Galanthaminone C17H19NO3 285.1365 285.1435 0.32 (Harvey, 1995)
7 Tazettine C18H21NO5 331.1420 331.1489 30.6 331.1491 19.3 331.1488 5.92 (Mahomoodally et al., 2020)
7.1 Criwelline C18H21NO5 331.1420 331.1489 30.6 331.1491 19.3 331.1488 5.92
7.2 Ambelline C18H21NO5 331.1420 331.1489 30.6 331.1491 19.3 331.1488 5.92
8 Norgalanthamine C16H19NO3 273.1365 273.1435 12.06 273.1434 2.28 273.1434 4.23
9 Yemenine C C16H17NO5 303.1107 303.1177 1.33 303.1176 0.41 303.1176 0.12
10 Lycorine-1-O-glucoside C22H27NO9 450.1775 450.1755 0.13
11 Pratorimine C16H11NO3 265.0739 265.0809 0.56 265.0808 1.11
12 Oxocrinine C16H15NO3 269.1052 269.1123 0.14 (Ali et al., 1986)

N° Compound name ESI (-) Formula Theoretical OEL OEB OER References
Mass
Experimental data

Mass %* Mass %* Mass %*

PHENOLICS
13 (E)-ferulic acid C10H10O4 194.0580 194.0501 4.46 194.0401 2.31 (Ghane et al., 2018)
14 Kaempferol C15H10O6 286.0477 286.0404 0.11
15 Caffeic acid 3-glucoside C15H18O9 342.0951 342.0885 0.18 342.0882 0.13 (Mahomoodally et al., 2020)
16 7,40 -dihydroxy-8-methylflavan C16H16O3 256.1099 256.1028 2.12 (Moyo et al., 2017)
17 Naringenin C15H12O5 272.0685 272.0613 0.27
18 Quinic acid C7H12O6 192.0634 192.0556 0.11 (Chahal et al., 2022)
19 Quercetrin C21H20O11 448.1006 448.0935 0.42
20 Sinapic acid C11H12O5 224.0685 224.0609 4.18
21 4-o-beta-D-glucosyl-4-coumaric acid C15H18O8 326.1002 326.0931 0.24 326.0932 0.26 326.0931 0.12
22 Caffeic acid phenethyl ester C17H16O4 284.1049 284.0977 0.11 284.0977 0.11
23 Gluconic acid C6H12O7 196.0583 196.0505 0.75
LIPID PROFILE
Saturated Fatty Acids
24 Hexanedioic acid C9H18O6 222.1103 222.1031 0.12 (Barra et al., 2021)
25 Pentadecanoic acid C15H30O2 242.2246 242.2171 0.69 242.2172 17.3 242.2172 0.36
26 Dodecanoic acid C12H24O2 200.1776 200.1698 0.55 200.1699 2.36 200.1698 0.33
27 Heptadecanoic acid C17H34O2 270.2559 270.2489 0.55 270.2488 9.65 270.2488 0.23
28 Octadecanoic acid C18H36O2 284.27153 284.2641 3.35 284.2644 23.9 284.2644
29 Docosanoic acid C22H44O2 340.3341 340.3269 1.29 340.3270 1.19 228.2014
30 Tetradecanoic acid C14H28O2 228.2089 228.2014 19.3 228.2014 0.81
31 3-oxohexadecanoic acid C16H30O3 270.2195 270.2124 0.58
32 Palmitic acid C16H32O2 256.2402 256.2330 100
33 Heneicosanoic acid C21H42O2 326.3185 326.3114 0.29
34 10-oxooctadecanoic acid C18H34O3 298.2508 298.2435 0.38 298.2436 0.9 284.2644 6.32
35 Phytanic acid C20H40O2 312.3028 312.2957 0.94 312.2958 1.27
36 Octadecanedioic acid C18H34O4 314.2457 314.2386 0.75 314.2386 0.53
37 Nonadecanedioic acid C19H36O4 328.2614 328.2543 0.24 328.2544 0.36
38 2-oxoundecanoic acid C11H20O3 200.1412 200.1334 0.35 200.1334 0.28
Unsaturated Fatty Acids
39 Octadec-9-enoic acid C18H34O2 282.2559 282.2486 11.23 282.2487 27.6 282.2487 3.9 (Barra et al., 2021)
40 (9Z,12Z,15Z)-octadeca-9,12,15-trienoate C18H30O2 278.2246 278.2175 31.74 278.2174 2.12 278.2174 0.1
41 Ethyl linoleate C20H36O2 308.2715 308.2644 0.77 308.2645 1.01 (Mahomoodally et al., 2020)
42 Methyl 9-octadecenoate C19H36O2 296.2715 296.2644 0.7
43 2-hexadecenoic acid C16H30O2 254.2246 254.2174 28.9 254.2174 0.89
* Relative Abundance.

Optimized extracts of leaves (OEL), bulbs (OEB) and roots (OER) of (ANOVA) and differences between treatments were identified by
C. americanum L. were dried and resuspended in DMSO for MTT anal- Tukey’s test (p<0.05). The data set homoscedasticity was verified by
ysis. The final concentration, after the addition of MN cells, was Levene’s test and normality by the Shapiro-Wilk test. Chemometrics
100 mg or 100 ng of extract (dry weight - SD)/mL of DMSO. was applied using principal component analysis (PCA) and hierarchical
cluster analysis (HCA) to assess similarities between the morphological
2.9.3. Statistical analysis parts of C. americanum L. For PCA, eigenvalues > 1.0 were adopted and
All analyses were performed in triplicate and results were expressed the two-dimensional analysis was based on linear correlations. For
by means followed by standard deviation. The comparison of means HCA, the Euclidean metric and Ward’s method were used. The software
between the groups was performed by one-factor analysis of variances StatisticaÒ , version. 10.0 (StatSoft, Tulsa, EUA) was used in the analyses.
475

C.L.C. de Pinho, F.O. Farias et al.
R.L. de Araujo, South African Journal of Botany 161 (2023) 472 481

3. Results Of these, 6 correspond to macroelements (Ca, K, Mg, Na, S, and P), 7

to microelements (Co, Cu, Fe, Mn, Mo, Se, and Zn), and 3 are poten-
3.1. Proximate composition tially toxic elements (Al, Cr and Pb). The results are expressed on a
dry basis and the samples showed mineral concentrations (mg/g)
The C. americanum L. nutritional composition in terms of mois- ranging from 0.53 for Se (micro) and 81.8 for K (macroelement).
ture, ash, protein, lipids, dietary fiber, total sugar, reducing and non- Macroelements are relevant to various physiological functions in
reducing sugars, and other carbohydrates, on a dry basis, is shown in plants and animals. In this study, all parts of the plant matrix pre-
Table 3. sented relevant proportions of these elements. The leaves showed
Comparing the proximate composition of the three parts of C. higher concentrations of Ca (18.8 mg/g) and Mg (33.7 mg/g) when
americanum L., the leaves showed higher ash, protein, and lipid con- compared to the other parts of the plant. The bulbs had the highest
tent. On the other hand, the bulbs had a higher content of total sugars content of K (81.8 mg/g), Na (24.5 mg/g) and P (9.44 mg/g). In the
and carbohydrates. The roots had the highest concentration of mois- roots, Mg (28.7 mg/g) and S (76.5 mg/g) were the major macroele-
ture, ash, and total dietary fiber. This variability in the composition of ments found. In addition, the bulb and root had equivalent levels of
the parts of the same plant can be associated with multiple factors, Ca (11.5 and 10.3 mg/g, respectively); while leaves and roots had
such as how each part is exposed to the sun, temperature, rain, and equivalent levels of Mg (33.7 and 28.7 mg/g, respectively) and Na
interaction with other plants or animals in the ecosystem. (4.58 and 11.6 mg/g, respectively).
The protein content was higher in leaves (13.26%) than in bulbs Micronutrients (Co, Cu, Fe, Mn, Mo, Se, and Zn) and potentially
(5.49%) and roots (5.66%). The macromolecules that stand out in the toxic elements (Al, Cr, and Pb) were also evaluated. Leaves and bulbs
composition of C. americanum L. are carbohydrates, with emphasis on showed similar concentrations of Se (0.59 and 0.53 mg/g, respec-
the subclass of dietary fiber (DF) which represent approximately tively) and Mo (0.50 and 0.54 mg/g, respectively). However, the
37.30; 34.88, and 42.12% for leaves, bulbs, and roots, respectively. leaves did not show significant concentrations of Co and Cu. The bulb
Another expressive result is related to the soluble dietary fiber (SDF) had a higher content of Zn (0.79 mg/g) when compared to the other
content, which corresponds to about 5% of the total DF for the leaves parts of the plant. The root had the highest content of Fe (51.9 mg/g),
and roots and 18% for the bulbs. Mn (15.4 mg/g), Mo (1.01 mg/g) and Zn content (0.31 mg/g) similar
to that presented by the leaves (0.33 mg/g). The mineral content in
plant parts depends on the characteristics, availability, and absorp-
3.2. Multi-element analysis
tion capacity of the soil. Thus, the roots and bulbs, which are the parts
of the plant that are most in contact with the soil, had a higher con-
Of the 18 minerals investigated in the leaves, bulbs, and roots of C.
tent of trace elements (Fe, Mn, Mo, and Zn).
americanum L., 16 elements were detected and are shown in table 3.
Concerning potentially toxic elements, all parts of the plant had
Al, Cr, and Pb in their composition. The leaves had a lower Al content
(0.90 mg/g), when compared to the bulb (6.1 mg/g) and the root
Table 3 (5.74 mg/g). Bulbs and roots had equivalent concentrations of Pb
Proximate composition (%; g/100 g) and determinations of elements (mg/g) of
(10.5 and 10.1 mg/g, respectively), however, the roots had a higher
leaves, bulb, and root of C. americanum L.
content of Cr (4.80 mg/g).
PROXIMATE COMPOSITION (%; g/100 g)

Leaf Bulb Root

3.3. C. americanum L. extracts obtaining and characterization
b c a
Moisture 7.19 § 0.10 6.87 § 0.36 8.97 § 0.30
To assess the bioactivity potential of the C. americanum L. parts,
Ash 12.12a § 0.02 5.64b § 0. 01 11.5a § 0.02
Protein 13.26a § 0.01 5.49b § 0.01 5.66b § 0.01
they were submitted to a solid-liquid extraction (SLE). The SLE was
Lipids 2.12a § 0.07 0.80c § 0.001 1.05b § 0.01 performed according to the experimental design (Table 1). TPC,
Total Sugar 4.65b § 0.02 7.05a § 0.02 3.98c § 0.01 DPPH, FRAP, and TAC were used as screening methods for the elabo-
Reducing Sugar 0.83 § 0.001 0.86 § 0.001 0.85 § 0.01 ration of an optimized extract, rich in alkaloids, phenolic compounds,
Non-Reducing Sugar 3.82 § 0.001 6.19 § 0.03 3.13 § 0.02
and other chemical compounds. The optimized extract of leaves
Dietary Fiber 37.30b § 0.13 34.88c § 0.04 42.12a § 0.21
Soluble Dietary Fiber 1.71 § 0.001 6.35 § 0.03 2.32 § 0.01 (OEL), bulb (OEB), and root (OER) were analyzed by ESI-FT-MS to
Insoluble Dietary Fiber 35.39 § 0.03 28.53 § 0.09 39.8 § 0.12 determine its chemical compounds. In addition, the cytotoxicity of
Carbohydrate 23.36c § 0.001 39.27a § 0.15 26.72b § 0.14 these extracts was tested. One of the main purposes of this step is to
ELEMENT (mg/g of sample) suggest an effective form to take advantage of the C. americanum L.
benefits, even in regions where the plant is not available, or even as a
Leaf Bulb Root
natural additive to be used in industrial processes. All results for
Ca 18.8a § 0.02 11.5b § 0.04 10.3b § 0.06 these approaches are described in the next topics.
K 3.06c § 0.01 81.8a § 0.76 18.3b § 0.12
Mg 33.7a § 0.13 15.4b § 0.12 28.7a § 0.38
Na 4.58b § 0.03 24.5a § 0.22 11.6b § 0.56
3.3.1. Screening methods
P 2.39b § 0.02 9.44a § 0.08 0.13c § 0.01 In the present study, two mechanisms of antioxidant action were
S 0.40c § 0.01 33.3b § 0.29 76.5a § 0.79 tested in C. americanum L. extracts: DPPH scavenging activity and
Co 0.35 § 0.01 iron reduction capacity (FRAP) (Table 1). The results showed that the
Cu 0.36 § 0.02
extracts were able to scavenge the DPPH radical and the antioxidant
Fe 2.02c § 0.03 3.32b § 0.02 51.9a § 0.23
Mn 0.59c § 0.03 10.9b § 0.04 15.4a § 0.09 activity was higher in the ternary mixture for leaf extracts (assay 8:
Mo 0.50b § 0.03 0.54b § 0.04 1.01a § 0.04 2608 § 9 mg TE/100 g), bulb (assay 10: 818 § 3 mg TE/100 g) and
Se 0.59a § 0.03 0.53a § 0.03 root (assay 7: 606 § 8 mg TE/100 g). This demonstrates that C. ameri-
Zn 0.33b § 0.01 0.79a § 0.02 0.31b § 0.01 canum L. has biomolecules with high antioxidant content in its com-
Al 0.90b § 0.01 6.1a § 0.03 5.74a § 0.07
Cr (mg/g) 1.63b § 0.05 1.74b § 0.07 4.80a § 0.03
position and that the ternary mixture of solvents (ethanol:water:
Pb 7.54b § 0.02 10.5a § 0.16 10.1a § 0.19 ethyl acetate) was efficient for the extraction of these compounds.
Statistical analysis was carried out by the Tukey test at 95% confidence and statisti-
Ferric reducing antioxidant power (FRAP) was also more signifi-
cal significance was accepted at p < 0.05. The superscript letter a, b, c d, e, and f cant in the ternary mixture for leaf extracts (assay 8: 3368 § 4 mg Fe
denotes significant differences on the same line. (III)/100 g) and bulb (assay 10: 1890 § 13 mg Fe (III)/100 g). For the
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R.L. de Araujo, South African Journal of Botany 161 (2023) 472 481

Fig. 1. Principal component analysis (PCA) and dendrogram were obtained for the determination of multielement compounds, approximate composition, antioxidant capacity
(DPPH and FRAP), TPC, and TAC in leaves, bulbs, and roots of C. americanum L. (A) Graph for the loading components in PC1 and PC2; (B) Score plot of the three components; (C)
Euclidean distances from the analysis of multielement compounds, approximate composition, antioxidant capacity (DPPH and FRAP), TPC and TAC (d) Euclidean distances from the
morphological parts from C. americanum L.

root extract, ethyl acetate was the solvent that stood out the most As can be seen from the score chart (Fig. 1B), the plant parts were
(assay 3: 2899 § 34 mg Fe (III)/100 g) Table 1. divided into three separate groups based on their similarities. Leaves
The maximum TPC for the extract of the leaves was presented in were in the positive left quadrant and presented the highest values
the mixture of solvent water and ethyl acetate (assay 6: of lipids, moisture, proteins, DPPH, TPC, and Ca and the lowest Mn
3046 § 15 mg GAE/100 g). On the other hand, the highest TPC for the and S values. The bulb was in the negative right quadrant and pre-
bulb and root extracts (1306 § 21 and 748 § 14 mg GAE/100 g, sented the highest values of Zn, Cu, P, Na, total sugar, carbohydrate,
respectively), was observed in the tests of the ternary mixture of sol- and soluble fiber and lower ash and TAC values. Finally, the root was
vents (Table 1). in the positive right quadrant and showed higher values of TAC, Mo,
The TAC was higher for all extracts in the tests with the ternary Cr, Fe, Co, and insoluble fiber and lower values of Al, Se, and FRAP.
mixture of solvents (5953 § 45; 1984 § 16; 1355 § 11 mg ATR/100 g,
for extracts of leaves, bulb, and root, respectively) (Table 1). 3.3.3. SLE optimization
The proposed models, which explain the effects of water, ethanol,
and ethyl acetate on the chemical composition, antioxidant capacity,
3.3.2. Multivariate analysis and total alkaloids of extracts of leaves, bulbs, and roots of C. ameri-
Principal component analysis (PCA) and hierarchical clustering canum L. are shown in Table S2 - Supplementary Material. Only the
(HCA) were used as exploratory analysis to evaluate multielement model proposed for the FRAP of the root extract showed R2< 0.7 and
compounds, approximate composition, antioxidant capacity (DPPH was not used to predict the values of the response variables. Further-
and FRAP), TPC, and TAC of leaves, bulbs, and roots of C. americanum more, all models were significant (p<0.001) and R2 values explained
L. As a result of this multivariate analysis (Fig. 1), PC1 represented more than 70% of the variability of the results.
52.32% and PC2 47.38% of the total variance. Considering the two PCs, The effect of the interaction between water and ethyl acetate was
loading graphs (Fig. 1A) and scores (Fig. 1B) were generated. All vari- the most significant (p  0.05) for the increase in antioxidant capacity
ables analyzed were dominant and contributed to the greater vari- and total alkaloids of extracts from leaves, bulbs, and roots of C. amer-
ability of data on these PCs. icanum L. The same was observed in ternary mixtures for root and
477

C.L.C. de Pinho, F.O. Farias et al.
R.L. de Araujo, South African Journal of Botany 161 (2023) 472 481

Table 4 extracts. In technological terms, this is the first work to report the
Validation of the optimization procedure: predicted and experimental values for anti- optimization of the C. americanum L. solvent extraction system.
oxidant capacity (DPPH and FRAP), TPC, and TAC of the optimized extract of leaves
(OEL), bulb (OEB), and root (OER) of C. americanum L.
3.3.4. Extracts characterization
Responses Predicted 95% +95% Experimental [P-E]** The optimized extracts of leaves, bulbs, and roots of C. ameri-
(mg equivalent*/ mean value Prediction Prediction value canum L. were submitted to ESI-FT-MS analysis to identify their com-
100 g of
pounds (Table 2). Among hundreds of detected compounds, we
dry sample)
identified a total of 89 common molecules of the Amaryllidaceae fam-
OEL ily, such as saturated and unsaturated fatty acids, amino acids, sugars,
DPPH 2655 2492 2813 2466 § 32.23 189
phenolic compounds, and alkaloids. However, most of these com-
FRAP 3625 3451 3793 3650 § 57.02 25
TPC 3215 3042 3388 3302 § 86.85 87 pounds were first reported for C. americanum L. Of the 32 alkaloids
TAC 1731 1677 1785 1727 § 58.59 4 detected in the extracts, 24 were identified for the first time in C.
OEB americanum L. (Table S1 - Supplementary Material).
DPPH 722 603 845 669 § 12.17 53
A total of 23 phenolic compounds were identified among the opti-
FRAP 2774 2139 3524 3451 § 156.9 677
TPC 1132 995 1275 1240 § 60.49 108
mized extracts, including (E)-ferulic acid, Kaempferol, Caffeic acid 3-
TAC 584 622 619 598 § 29.34 14 glucoside, Naringenin and others. In addition to phenolic compounds,
OER 16 saturated fatty acids were identified, such as pentadecanoic acid,
DPPH 713 652 775 658 § 5.46 55 dodecanoic acid, heptadecanoic acid and octadecanoic acid, and 5
TPC 1040 897 1195 990 § 94.67 50
unsaturated fatty acids, such as octadec-9-enoic acid and (9Z,
TAC 2980 1946 4562 4210 § 97.95 1230
12Z,15Z)-octadeca-9,12,15-trienoate (Table 2).
* equivalent: Trolox (TE); ferric sulfate (Fe III); gallic acid (GAE); Atropine (ATR).
** absolute difference in modulus between predicted and experimental values.
3.3.5. Cytotoxicity
The maximum concentration that determines whether a product,
natural or synthetic is toxic for a given cell must be known to define
leaf extracts, except for FRAP in the latter. The interaction between the possibility of in vivo tests and establish a safety margin of the
ethyl acetate and ethanol only increased the FRAP of the leaf extract product concerning its biological activities. In this work, the cytotoxic
and the DPPH of the root extract, as shown in Figure S1 - Supplemen- dosage of the optimized extracts was tested in two concentrations
tary Material. (100 mg/mL and 100 ng/mL). It can be seen in Fig. 2 that the percent-
To maximize the values of DPPH, FRAP, TPC and TAC, the simulta- age of viability is not dose-dependent and there are no significant dif-
neous optimization suggested a solvent system containing 56.04% ferences in cytotoxicity between the concentrations tested. Cell
water and 43.96% ethyl acetate for the OEL (d-value = 0.995); 56.51% viability was higher than 80% at all concentrations of the optimized
water and 43.59% ethyl acetate for the OEB (d-value = 0.926) and extracts without significant differences to the control (p< 0.05).
76.97% water and 18.51% ethanol for the OER (d-value = 1.00) (Figure
S2- Supplementary Material). External validation was performed 4. Discussion
with the aforementioned solvent proportions to assess the predictive
capacity of the RSM models. The predicted results are shown in As previously mentioned, tea from C. americanum L. has been used
Table 4 and it is possible to observe that the experimental data were in traditional medicine in coastal communities in the interior of Par-
within the § 95% prediction intervals proposed by the RSM models. ana
, Brazil, to treat cancer (Silva et al., 2020). This direct use may
Thus, the generated models can be considered predictive to maxi- characterize C. americanum L. as an unconventional edible plant
mize the extraction of alkaloids, TPC, and antioxidant activity of the (UEP), since UEPs have an indescribable medicinal value, and can be

Fig. 2. Effect of optimized extract of leaves (OEL), bulbs (OEB), and root (OER) of C. americanum L. on human peripheral blood MN cell viability. The effect of optimized extracts on
cell viability was evaluated by the MTT assay. Values are expressed as mean § standard deviation (n = 3) and are representative of three independent experiments with similar
results. Different letters differ statistically from each other (p< 0.05), as determined by ANOVA.

478

C.L.C. de Pinho, F.O. Farias et al.
R.L. de Araujo, South African Journal of Botany 161 (2023) 472 481

used not only as spices, in popular cuisine, but also in teas, and baths, Expressive values of FRAP for methanolic extracts of leaves of C. asiat-
among other methods (Milia ~o et al., 2022). In this sense, the determi- icum (462.81 § 1.98 mg Fe (II)/g of extract) were verified in the work
nation of the nutritional profile and inorganic components of these of Ghane et al. (2018). In another study, FRAP values of methanolic
plants and their extracts are of great interest for the public well- root extracts of Crinum species ranged from 6751 § 0.06 for C. mene-
being, due to the role that certain elements and nutrients play in hune, 5677 § 0.14 for C. viviparum and 3380 § 0.08 mg Fe (II)/g for C.
human and animal health (Castro Alba et al., 2019). asiaticum (Lekhak et al., 2021).
Normative Instruction n.° 75 of the National Health Surveillance The C. americanum L. extracts showed lower TPC levels than other
Agency (ANVISA, 2020) determines that a food source of protein is Crinum species, such as C. jagus, C. latifolium and C. pratense
one that contains at least 10% of the recommended daily value of this (8177 § 0.75; 77.32 § 0.56; 70.61 § 0.62 mg TAE/100 g of extract,
macromolecule per serving. As the document recommends that this respectively) (Ghane et al., 2018).
value correspond to 50 g for proteins and, considering that the por- The TAC levels found in the extracts were higher than those
tion of leaves, bulb or root is equivalent to that attributed to the por- reported in the work by Ghane et al. (2018) for aqueous extracts
tion of dried legumes (60 g), it can be considered that C. americanum from the leaves of C. jagus and C. asiaticum (930 § 0.02 and
L. is a possible source of protein for human consumption, since the 860 § 0.03 mg colchicine/ 100 g, respectively) and for the methanolic
protein content observed in the leaves (13.26%), bulbs (5.49%) and extract of the roots of C. solapurense and C. asiaticum (1280 § 0.11
roots (5.66%) presented 10% of the recommended daily value and 1153 § 0.02 mg Brucine/ 100 g, respectively) in the work by
(Table 3). Lekhak et al. (2021).
In addition to the significant protein content, the macromolecules Principal component analysis (PCA) and hierarchical clustering
that stand out in the composition of C. americanum L. are carbohy- (HCA) were used as exploratory analysis to classify the parts of C.
drates, with emphasis on the subclass of dietary fiber (DF) and the americanum L. and help to better understand their specific character-
content of soluble dietary fiber (SDF) (Table 3). These results are istics. Plant leaves generally contain higher amounts of Ca and anti-
promising, as fiber consumption has demonstrated a positive role in oxidants, including secondary metabolites such as phenolic
the prevention of inflammatory bowel diseases, increasing the diver- compounds. The high amount of Na found in the bulbs is related to
sity of the intestinal microbiota and modulating its effects on the the regulation of osmotic pressure and water balance within the
mucosal immune system (Liu et al., 2022). plant (Silva et al., 2021). Furthermore, the P content in the bulbs may
Among the determined micronutrients, Fe showed significant lev- be related to the formation of ATP and subsequent accumulation of
els, mainly for the root fraction (Table 3). According to the National energy in the form of starch, which is correlated with the high
Health Surveillance Agency (ANVISA), the daily consumption of Fe for amount of carbohydrates (Song et al., 2019).
adults is 14 mg. In this sense, 0.3 g of the dry root of C. americanum L. Hierarchical cluster analysis revealed the same three main groups
would be enough to supply the daily need for this microelement (Bra- (Fig. 1C and D) and confirmed, to a high degree, the results extracted
zil, 2005). Given this context and taking into account that foods forti- from PCA, providing an overview of all samples and their similarities.
fied with iron are one of the most effective methods for preventing In addition, the PCA provided a better interpretation of the groups
its deficiency in the body and that the delivery of this mineral that formed naturally in the HCA. In this way, using multivariate sta-
through the diet is a practical, economical and sustainable approach tistical methods, the main components were extracted, which pro-
(Man et al., 2022), C. americanum L. can be considered as an unex- vided a reduction in the number of original variables and both
plored source of Fe and, depending on its bioavailability, these plants techniques proved to be very useful.
can be used as an ingredient rich in this mineral. The optimized extract of each part of C. americanum L. was evalu-
Regarding potentially toxic elements, all parts of the plant had Al, ated by ESI-FT-MS. So far, only two studies in the literature reported
Cr and Pb in their composition. The National Health Surveillance the presence of alkaloids in C. americanum L. The first described the
Agency (ANVISA) recommends that, for adults weighing 70 kg, the presence of oxocrinine, crinine, hippadine, and lycorine in extracts
daily consumption of these potentially toxic elements, in mg, is: Al from the bulbs of this species(Ali et al., 1986). The second described
(7); Cr (0.035) and Pb (42) (Brazil, 2005). Thus, it is within the limits the presence of 13 alkaloids in extracts from leaves and bulbs of C.
recommended by ANVISA up to 5.6 g of leaves, 1.0 g of bulb and americanum L. (Gomes et al., 2022). Of the 13 alkaloids found by the
1.20 g of dry roots of C. americanum L. per day (Table 3), either in the authors, 7 were also identified in our work, among them: crinine,
form of tea, plasters or baths. The dissemination of this information is lycorine, dihydrolycorine, crinamine, haemanthamine, epipowelline
important, since people consume tea from this plant directly in popu- and 1,2-didehydrocrinan-3-one (Table 2).
lar medicine and could avoid its excessive use. The alkaloid galanthaminone, reported for the first time in the
Extracts from the leaves, bulbs and roots of C. americanum L. were extract of leaves of C. americanum L., is a derivative of galanthamine
obtained and the antioxidant capacity (DPPH and FRAP), TPC and TAC (Harvey, 1995). This alkaloid has stood out in recent years as a class
were used as screening methods for the elaboration of an optimized of compounds used in drugs for the treatment of Alzheimer’s disease
extract, rich in alkaloids, phenolic compounds and others chemical (Ghane et al., 2018). Other alkaloids, also identified in our work, such
compounds. The DPPH levels obtained in this study (2608 § 9; as crinine and lycorine have already been cited in the literature for
818 § 3; 606 § 8 mg TE/100 g for leaf, bulb and root extract, respec- having potentially useful cytotoxic and apoptosis-inducing properties
tively) were higher than those determined by Gomes et al. (2022). for the treatment of cancers (Gomes et al., 2022).
These authors evaluated hexanic and methanolic extracts of leaves, So far, the presence of phenolic compounds has not been reported
bulbs, and roots of C. americanum L. for DPPH scavenging activity and for C. americanum L. Although most of the 23 phenolic compounds
expressed the results in EC50. The best values found by the authors found in this work have been identified in plants of the Amaryllida-
ranged from 156 § 6 to 191 § 18 mg/mL for leaf extracts. ceae family, the data in this work are unpublished for C. americanum
The considerable content obtained for the DPPH scavenging activ- L. (Chahal et al., 2022; Moyo et al., 2017). The compounds commonly
ity in this work may be related to the presence of isoquinoline alka- identified in plants of the Amaryllidaceae family are Kaempferol, (E)-
loids in the extracts of C. americanum L., since the antioxidant ferulic acid (Ghane et al., 2018), Caffeic acid 3-glucoside (Mahomoo-
potential of these compounds can be driven mainly by the proton dally et al., 2020), 7,40 -dihydroxyflavan (Coxon et al., 1980) among
affinity (PA), consistent in the mechanism of sequential proton loss others.
electron transfer (SPLET) (Dung et al., 2020). The presence of saturated and unsaturated fatty acids in C. ameri-
The iron reducing capacity test (FRAP) also showed significant canum L. was previously reported in the work by Barra et al. (2021).
results, mainly for the ternary mixture of solvents (Table 1). These authors characterized the lipid profile in C. americanum L.
479

C.L.C. de Pinho, F.O. Farias et al.
R.L. de Araujo, South African Journal of Botany 161 (2023) 472 481

leaves using the HRGC-MS technique. They identified 11 of the 16 CRediT authorship contribution statement
saturated fatty acids found in our work, including dodecanoic, tetra-
decanoic, hexadecanoic, heptadecanoic, octadecanoic acid, and the Renata La
zara de Arau
jo: Conceptualization, Methodology, Inves-
unsaturated fatty acid: (9Z, 12Z, 15Z)-octadeca-acid-9,12,15- tri- tigation, Data curation, Writing original draft, Writing review &
enoic. editing. Carolina Lilibeth Carvalho de Pinho: Methodology, Investi-
Even though ESI-FT-MS is a qualitative technique, the promising gation, Data curation, Writing review & editing. Fabiane Oliveira
results observed in the topics of this work demonstrate that all parts Farias: Conceptualization, Writing review & editing, Supervision.
of C. americanum L. can present considerable levels of bioactive com- Wanderlei do Amaral: Methodology, Investigation. Alberto Wis-
pounds. niewski Junior: Funding acquisition, Methodology, Data curation.
Cell viability was higher than 80% at all concentrations of the opti- Adenilda Cristina Hono
rio-França: Funding acquisition, Methodol-
mized extracts. According to Moura et al. (2021), a sample is consid- ogy, Data curation. Luciana Igarashi-Mafra: Conceptualization,
ered cytotoxic when the percentage of viability of exposed cells is Funding acquisition, Resources, Writing review & editing. Marcos
less than 70%. Therefore, this demonstrates that there was no change R. Mafra: Conceptualization, Funding acquisition, Project administra-
in cell viability at both tested concentrations of the optimized tion, Resources, Supervision, Writing review & editing.
extracts. This percentage may be related to the mechanism of action
of these extracts, since medicinal plant extracts induce the activation Funding
of the immune system, without altering cell viability, and provide
effective benefits for the treatment of diseases (Arau
jo et al., 2019). R. L. Araujo and C. L. C. Pinho are grateful for the scholarship pro-
Furthermore, stimuli such as medicinal plants can increase the vide by CAPES (Coordination for the Improvement of Higher Educa-
release of superoxide anions from MN phagocytes, causing a change tion Personnel - Finance Code 001). Professors M.R. Mafra and L.
in immune system function and activating oxidative metabolism Igarashi-Mafra are grateful to the Brazilian National Council for Scien-
(Honorio-França et al., 2013). Current results support the hypothesis tific and Technological Development (CNPq - Grant 315667/2021-4
that plant extracts can preserve normal MN cells in a dose-dependent and 316815/2021-7, respectively).
manner. These results may be secondary to the presence of alkaloids,
phenolic compounds, and unsaturated fatty acids, among others, Acknowledgments
which were identified in the optimized crinum americanum extracts,
as evidenced by the results of the chemical identification-ESI-FT-MS This work was supported by the Laboratory of Thermodynamics
analysis (Table 2). and Separation Operations (LATOS), the Graduate Program in Food
Thus, analyzing the cytotoxicity of these extracts with different Engineering (PPGEAL - UFPR), and the Federal University of Paran
a
chemical compositions is of fundamental importance for future appli- (UFPR).
cations. This is because phenolic compounds, such as Kaempferol,
identified in the extract of C. americanum L. leaves, have already been
Supplementary materials
cited in the literature for having benefits related to reducing the risk
of chronic non-communicable diseases (Lin et al., 2014). Galanthami-
Supplementary material associated with this article can be found,
none, an alkaloid identified in the leaf extract, is known due to its in the online version, at doi:10.1016/j.sajb.2023.08.042.
properties as an acetylcholinesterase (AChE) inhibitor (Ghane et al.,
2018). In addition, lycorine, an alkaloid found in extracts from leaves,
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