Anacardic Acid Mixture: Utilization of a Byproduct for the Sustainable Development of a Potential Antichagasic Agent Against Trypanosoma cruzi

Full text

Vol.:(0123456789)
1 3
Revista Brasileira de Farmacognosia
https://doi.org/10.1007/s43450-023-00453-z
SHORT COMMUNICATION
Anacardic Acid Mixture: Utilization ofaByproduct fortheSustainable
Development ofaPotential Antichagasic Agent Against Trypanosoma
cruzi
TiagoRochaNogueira1 · IgorMesquitaFigueredo2 · JoãoHenriqueSilvaLuciano3 · AntonioCalixtoLima4·
AluisioMarquesdaFonseca5 · LuziaKalyneAlmeidaMoreiraLeal6 · MaryAnneSousaLima1 ·
AliceMariaCostaMartins7 · EmmanuelSilvaMarinho8 · JacksondeQueirozMalveira9· PedrodeLimaNeto10 ·
FátimaMirandaNunes1 · MariaAlexsandradeSousaRios2 · AntôniaTorresÁvilaPimenta1
Received: 27 March 2023 / Accepted: 19 August 2023
© The Author(s) under exclusive licence to Sociedade Brasileira de Farmacognosia 2023
Abstract
The work presents an optimization of the extraction and purification processes of the anacardic acids mixture and its evalu-
ation as a potential antichagasic drug. The purified mixture was obtained by chromatography techniques and characterized
by 1H and 13C NMR. The cytotoxicity was evaluated in Rhesus monkey Kidney Epithelial Cells (LLC-MK2) after 24h of
exposure in concentrations between 400 and 6.25µg/ml. The new procedure was efficient for the purification of the anacardic
acid mixture and separating them, with a 31.6% yield, considering a 3-step process. The mixture presented cytotoxicity at
concentrations of 400 and 200µg/ml, with CC50/24h of 286µg/ml and IC50 of 12.9µg/ml These are promising initial results,
since the epimastigotes proliferate alone demonstrating a potential trypanocidal effect on Trypanosoma cruzi strain Y, higher
than isolated anacardic acids. Results show of the anacardic acid mixture as a bioproduct with potential antichagasic, a
without isolation steps necessary to obtain of the pure compounds and, therefore, more economically viable and sustainable.
Keywords Cashew nut shell liquid· Antichagasic· Anacardic acid· Chromatographic column· Cytotoxicity
Introduction
The cashew tree (Anacardium occidentale L., Anacar-
diaceae) is native to Brazil and its fruit and pseudofruit
are used to obtain cashew nuts and produce juice and
pulp, respectively. The main byproduct of the cashew
nut processing is the cashew nut shell liquid (CNSL),
formed mainly by anacardic acids, cardanols, and cardols
(Mazzetto etal. 2009).
Anacardic acids are a mixture of 6-alkyl-salicylic acids,
where the alkyl groups can be saturated or unsaturated
(one, two, or three unsaturations), the latter being more
frequent. These compounds have several pharmacologi-
cal effects, such as antimicrobial effect on Streptococcus
mutans (Lima etal. 2020), preventing the acute respira-
tory distress syndrome caused by viral pathogens (Gondim
etal. 2021), specific covalent inhibitors of SARS-CoV-2
cysteine proteases (Chen etal. 2021), and activities against
cancer stem cells (Vien etal. 2022). These compounds
are also reported in the literature for having action against
Trypanosoma cruzi, the parasite that causes Chagas’ dis-
ease (Freitas etal. 2009; Umehara etal. 2020).
According to World Health Organization (WHO),
Chagas disease, caused by the protozoan parasite Trypa-
nosoma cruzi, is found mainly in endemic areas of con-
tinental Latin American countries and possesses a great
power of infection (WHO 2023). About 6–7 million people
worldwide are estimated to be infected with this parasite
(WHO 2023; Echeverría etal. 2020). As reported by Bar-
bosa etal. (2023), this disease, also known as American
trypanosomiasis, is classified by the WHO as one of the
seventeen neglected tropical illnesses affecting regions of
Latin America, but because of international immigration
has spread to other countries such as Australia, Canada,
Japan, Spain, and the USA (Ribeiro etal. 2020; Barbosa
etal. 2023).
Despite the diverse pharmacological activities pre-
sented by such anacardic acids, there are few stud-
ies on their purification. The current work presents an
Extended author information available on the last page of the article

Revista Brasileira de Farmacognosia
1 3
optimization of the extraction and purification processes of
the anacardic acids mixture and proposes the evaluation of
the mixture, instead of the isolated compounds, as a poten-
tial antichagasic drug, a bioproduct without isolation steps
and, therefore, more economically viable and sustainable.
Materials andMethods
The cashew nut shells were obtained from the Experimen-
tal Farm of the Brazilian Agricultural Research Corpora-
tion (Embrapa).
The cashew nut shells (1.5kg) were cut into pieces and
exhaustively extracted with hexane at room temperature. Sol-
vent evaporation under vacuum yielded 230g of natural CNSL.
The separation of the anacardic acid mixture from
the natural CNSL was carried out according to Para-
mashivappa etal. (2001) with some modifications. This
procedure led to obtaining a fraction rich in anacardic
acid mixture (monoene, diene, and triene), but not pure.
Through analysis by thin layer chromatography, the pres-
ence of other compounds was verified.
The natural CNSL (23.5g) was submitted to sil-
ica gel column chromatography. The glass column
(45cm × 9.5cm) was packing using silica gel (height of
8.5cm) and hexane (500ml). The eluents used were hex-
ane, CH2Cl2, CH2Cl2:EtOAc (1:1), and EtOAc, 500ml of
each in that order of polarity to afford four fractions: C1F-1
(hexane; 300mg), C1F-2 (CH2Cl2; 1.9g), C1F-3 (CH2Cl2:
EtOAc; 2.3g) and C1F-4 (EtOAc; 18.4g). The C1F-4 was
re-chromatographed on silica gel column using the sol-
vents CH2Cl2 and EtOAc afforded two fractions: C2F-1
(CH2Cl2) and C2F-2 (EtOAc). The C2F-1 (11g) fraction
was re-chromatographed on silica gel column succes-
sively eluted with hexane, CH2Cl2, CH2Cl2/EtOAc (1:1)
and EtOAc to afford four subfractions: C3F-1, C3F-2, C3F-3,
C3F-4. The C3F-3 fraction provided 8.7g of the anacardic
acid mixture (AAM).
The purification of anacardic acid triene (1), anacardic acid
diene (2), and anacardic acid monoene (3) was performed by
using the methodology previously described by our research
group (Gondim etal. 2021). The spectra of 1H and 13C NMR
were obtained in Bruker Avance DRX-300 spectrometer.
The cytotoxic activity of the anacardic acid mixture on
mammalian cells was performed in the LLC-MK2 lineage
(Rhesus monkey Kidney Epithelial Cells). The cells were
cultured in sterile 96-well plates at a concentration of 105
cells/ml in DMEM medium at 10% FBS, penicillin (100IU/
ml), and streptomycin (100µg/l) at 37°C in an atmosphere
with 5% CO2 for 24h (Nwaka and Hudson 2006).
The MTT assay was performed to determine cell viability,
verifying the oxidative capacity of cells. For that, they were
treated with different concentrations of the AAM and benz-
nidazole (400, 200, 100, 50, 25, 12.5, 6.25µg/ml) as a refer-
ence drug, and incubated at 37°C for 24h. MTT (5mg/ml
in PBS) was added to each well, and the plates were left to
stand in the dark for 4h at 37°C. With the formazan crystals
formed, they were solubilized in 10% sodium dodecyl sulfate
(SDS) in 0.01 N HCl. After 17h, the absorbance at 570nm
was read in a microplate reader (Biochrom® ASYS Expert
Plus). These values were used to determine cell viability and
estimate the CC50, using the statistical program GraphPad
Prism® (Swift 1997).
The toxicity of substances on LLC-MK2 served as a basis
for defining the concentrations used in vitro assays (Nwaka
and Hudson 2006).
The epimastigote forms of Trypanosoma cruzi (strain Y)
were grown in LIT medium (Rodrigues etal. 2014), sup-
plemented with 10% fetal bovine serum (FBS), penicillin
(100IU/ml), and streptomycin (100µg/l), and kept in a BOD
incubator at 28°C. To evaluate the antiproliferative effect of
the infusion in these forms, parasites from the exponential
phase of the initial culture were used.
Epimastigote forms at a density of 1 × 106 cells/ml in
the log growth phase were plated and incubated in 96-well
plates with different concentrations of the AAM (200, 100,
50, 25, 12.5, 6.25µg/ml). After 24, 48, and 72h, quantifica-
tions of viable cells were performed in a Neubauer chamber
(Rodrigues etal. 2014). As a negative control, cells treated
with sterile PBS were used, a solution that has a pH balance
and presence of ions commonly present in the human physi-
ological system, ensuring a greater degree of similarity with
the human biological system. The percentage of cell viabil-
ity was calculated in relation to the negative control group
(PBS). The IC50 was determined by non-linear regression.
The selectivity index (SI) was computed to establish a con-
nection between the toxic concentration of a sample and its
effective concentration for anti-T. cruzi activity, using Eq.1:
In general, the formula is intended to assess the accuracy
and efficacy with which a sample or compound acts in combat-
ing the parasite, minimizing the damage that may occur to the
healthy cells of the host. This implies that a considerably high
SI signals a greater potential to develop a safe and effective
treatment against Chagas disease (Villamizar etal 2017).
All experiments in vitro were performed in triplicate
(n = 3) in three independent experiments. The results are
expressed as mean ± standard error mean (SEM), and the
comparison between experimental groups was performed
by one-way or two-way ANOVA, with Bonferroni’s posttest,
using p < 0.05 as significance criteria. Microsoft Excel 2016
and GraphPad Prism 5.0 were used for analysis and graphs.
(1)
SI
=
IC
50
(LLC −MK2cells)
IC
50
(extracellularformsofT.cruzi
)

Revista Brasileira de Farmacognosia
1 3
Results andDiscussion
In the methodology described here for the incremental
improvement of the antichagasic agents, the anacardic acid
mixture was obtained with a yield of 37% in just three steps
with filtering chromatographic columns. To verify the purity
and identity of the AAM acids, the mixture was analyzed by
HPLC. The chromatogram showed three peaks (Fig.S1) that
were isolated and identified as tri-unsaturated anacardic acid
(1), di-unsaturated anacardic acid (2), and mono-unsaturated
anacardic acid (3) by analysis of their spectra 1H and 13C
NMR and by comparing their spectroscopy data with those
previously reported (Morais etal. 2017).
The cytotoxicity of the anacardic acid mixture was evalu-
ated in LLC-MK2 renal tubular cells after 24h of exposure
to different concentrations in µg/ml (400, 200, 100, 50, 25,
12.5, 6.25) and subjected to the MTT reduction to verify cell
viability (Fig.1A). It was observed that the AAM causes cyto-
toxicity only in high concentrations (400 and 200µg/ml), with
CC50/24h 286 ± 28.1µg/ml, showing better results than the
reference drug benznidazole (CC50/24h 211.1 ± 16.9µg/ml).
After the cytotoxicity test, concentrations were determined
for the tests with epimastigote forms, one of the main evolu-
tionary forms of Trypanosoma cruzi. Epimastigote forms were
treated with the anacardic acid mixture after 24, 48, and 72h
(Fig.1B, C, and D) of exposure to different concentrations
(200, 100, 50, 25, 12.5, 6.25, and 3.125µg/ml). Quantification
was performed in a Neubauer chamber for the incubation peri-
ods of 24, 48, and 72h. The percentage of cell viability was
calculated in relation to the control, whose quantification was
considered 100%. The results showed that there was no statis-
tical difference between the times of 24h (IC50 12.9 ± 2.4µg/
ml), 48h (13.2 ± 2.1µg/ml), and 72h (15.4 ± 3.2µg/ml) when
the epimastigotes were treated with the anacardic acid mix-
ture, causing an antiproliferative effect on epimastigote forms.
Fig. 1 Cytotoxic effect of the
anacardic acid mixture (AAM)
(A) and benznidazole (B) on
LCC-MK2 cells; Effect of
anacardic acid mixture (AAM)
(C), and benznidazole (D) on
epimastigote forms after 72h
of incubation. The data are pre-
sented as mean ± SEM of three
independent experiments per-
formed in triplicate. *p < 0.05
vs. control group

Revista Brasileira de Farmacognosia
1 3
The anacardic acid mixture under study showed an IC50 value
(12.9µg/ml) slightly higher than that of saturated anacardic
acid (9.75µg/ml) (Pereira etal. 2008) and lower than that of
monoene anacardic acid (55.7µg/ml), diene (23.77µg/ml),
and triene (13.01µg/ml), demonstrating a potential trypano-
cidal effect of the anacardic acid mixture. As epimastigote
forms proliferate alone, these initial results are very promis-
ing, being an indication of the trypanocidal potential of the
anacardic acid mixture, since the mixture showed better activ-
ity than the isolated compounds, probably due to synergistic
effects. Umehara etal. (2020) showed that anacardic acid
diene exhibited in vitro activity against trypomastigote forms
of T. cruzi, which can be caused by altered plasma membrane
permeability, and its hydrogenated reaction derivative also
had a lethal effect on trypomastigote forms of T. cruzi pro-
moting changes in the parasite's mitochondria instead of the
plasma membrane.
Thus, an SI of around 22.17 is observed within an opti-
mal range of selectivity (SI > 20) (Vahermo etal. 2016),
expressed as the ratio between the toxic and effective con-
centrations of AAM, showing that the mixture constitutes a
bioproduct with potential antichagasic, a without isolation
steps necessary to obtain of the pure compounds and, there-
fore, more economically viable and sustainable.
In conclusion,
the new procedure was efficient for the separation of the
anacardic acids. The method was composed of adsorption
chromatographic techniques and showed a yield of 31.6% con-
sidering a process of 3 steps. Regarding the antichagasic poten-
tial, the mixture showed a potential trypanocidal effect superior
to the isolated unsaturated anacardic acids, being indicative of
the development of a pharmacological tool, with the advantage
of the reduction of isolation steps for the sustainable purifica-
tion of the antichagasic mixture of anacardic acids.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s43450- 023- 00453-z.
Author Contribution TRN: methodology, investigation, writing —
original draft. IMF: methodology. JHSL: formal analysis and meth-
odology. AMF: software, formal analysis. LKAML: investigation
and formal analysis. MASL: resources and formal analysis. AMCM:
investigation and formal analysis. ESM: writing — review and edit-
ing, investigation and formal analysis. PLN: investigation and formal
analysis. FMN: formal analysis, writing — review and editing. MASR:
conceptualization supervision, investigation, writing — original draft.
ATÁP: conceptualization, data curation, writing — review and edit-
ing, supervision.
Funding This work was supported by Conselho Nacional de Desen-
volvimento Científico e Tecnológico (CNPq); Fundação Cearense
de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP),
Financiadora de Estudos e Projetos (FINEP), and Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
Data Availability The authors declare that the data supporting the find-
ings of this study are available within the paper and itsSupplementary
Information files.
References
Barbosa H, Thevenard F, Quero RJ, Tempone AG, Honorio KM, Lago
JHG (2023) The potential of secondary metabolites from plants as
drugs or leads against Trypanosoma cruzi-an update from 2012 to
2021. Curr Top Med Chem 23:159–213. https:// doi. org/ 10. 2174/
15680 26623 66622 12121 11514
Chen Z, Cui Q, Cooper L, Zhang P, Lee H, Chen Z, Wang Y, Liu
X, Rong L, Du R (2021) Ginkgolic acid and anacardic acid are
specific covalent inhibitors of SARS-CoV-2 cysteine proteases.
Cell Biosci 11:45. https:// doi. org/ 10. 1186/ s13578- 021- 00564-x
Echeverría LE, Marcus R, Novick G, Estani SS, Ralston K, Zaidel EJ,
Forsyth C, Ribeiro ALP, Mendoza I, Falconi ML, Mitelman J,
Morillo CA, Pereiro AC, Pinazo MJ, Salvatella R, Martinez F,
Pere P, Liprandi AS, Piñeiro DJ, Molina GR (2020) WHF IASC
roadmap on Chagas disease. Glob Heart 15:26. https:// doi. org/
10. 5334/ GH. 484
Freitas RF, Prokopczyk IM, Oliva G, Andricopulo AD, Trevisan MTS,
Vilegas W, Silva MGV (2009) Discovery of novel Trypanosoma
cruzi glyceraldehyde-3-phosphate dehydrogenase inhibitors.
Bioorganic Med Chem 17:2476–2482. https:// doi. org/ 10. 1016/j.
bmc. 2009. 01. 079
Gondim FL, Ferreira RM, Nogueira TR, Serra DS, Rios SMA, Pimenta
ATA, Cavalcante FSA (2021) Effects of anacardic acid monoene
on the respiratory system of mice submitted to acute respiratory
distress syndrome. Rev Bras Farmacogn 31:232–238. https:// doi.
org/ 10. 1007/ s43450- 021- 00151-8
Lima RA, de Souza SLX, Lima LA, Batista ALX, de Araujo JTC,
Sousa FFO, Rolim JPML, Bandeira TJPG (2020) Antimicrobial
effect of anacardic acid-loaded zein nanoparticles loaded on
Streptococcus mutans biofilms. Braz J Microbiol 51:1623–1630.
https:// doi. org/ 10. 1007/ s42770- 020- 00320-2
Mazzetto SE, Lomonaco D, Mele G (2009) Óleo da castaha do caju:
Oportuidades e desafios no contexto do desenvolvimento e sus-
tentabilidade industrial. Quim Nova 32:732–741
Morais SM, Silva KA, Araujo H, Vieira IGP, Alves DR, Fontenelle
ROS, Silva AMS (2017) Anacardic acid constituents from cashew
nutshell liquid: NMR characterization and the effect of unsatura-
tion on its biological activities. Pharmaceuticals 10:31. https:// doi.
org/ 10. 3390/ ph100 10031
Nwaka S, Hudson A (2006) Innovative lead discovery strategies for
tropical diseases. Nat Rev Drug Discov 5:941–955. https:// doi.
org/ 10. 1038/ nrd21 44
Paramashivappa R, Phani KP, Vithayathil PJ, Srinivasa RA (2001)
Novel method for isolation of major phenolic constituents from
cashew (Anacardium occidentale L.) nut shell liquid. J Agric Food
Chem 49:2548–2551. https:// doi. org/ 10. 1021/ jf001 222j
Pereira JM, Severino RP, Vieira PC, Fernandes JB, da Silva MFGF,
Zottis A, Andricopulo AD, Oliva G, Corrêa AG (2008) Anac-
ardic acid derivatives as inhibitors of glyceraldehyde-3-phosphate
dehydrogenase from Trypanosoma cruzi. Bioorganic Med Chem
16:8889–8895. https:// doi. org/ 10. 1016/j. bmc. 2008. 08. 057
Ribeiro V, Dias N, Paiva T, Hagström-Bex L, Nitz N, Pratesi R, Hecht
M (2020) Current trends in the pharmacological management of
Chagas disease. Int J Parasitol Drugs Drug Resist 12:7–17. https://
doi. org/ 10. 1016/j. ijpddr. 2019. 11. 004

Revista Brasileira de Farmacognosia
1 3
Rodrigues JHS, Ueda-Nakamura T, Correa AG, Sangi DP, Nakamura
CV (2014) A quinoxaline derivative as a potent chemotherapeutic
agent, alone or in combination with benznidazole, against Trypa-
nosoma cruzi. PLoS ONE 9:e85706. https:// doi. org/ 10. 1371/ journ
al. pone. 00857 06
Swift ML (1997) GraphPad prism, data analysis, and scientific graph-
ing. J Chem Inf Comput Sci 37:411–412
Umehara E, Silva TAC, Mendes VM, Guadagninc RC, Sartorellic P,
Tempone AG, Lagoa JHG (2020) Differential lethal action of
C17:2 and C17:0 anacardic acid derivatives Trypanosoma cruzi
– a mechanistic study. Bioorg Chem 102:104068. https:// doi. org/
10. 1016/j. bioorg. 2020. 104068
Vahermo M, Krogerus S, Nasereddin A, Kaiser M, Brun R, Jaffe CL,
Moreira VM (2016) Antiprotozoal activity of dehydroabietic acid
derivatives against Leishmania donovani and Trypanosoma cruzi.
Med Chem Comm 7:457–463
Vien LT, Nga NT, Hue PTK, Kha THB, Hoang NH, Hue PT,Thien PN,
Huang CYF, Van Kiem P, Thao Do T (2022) A new liposomal
formulation of hydrogenated anacardic acid to improve activities
against cancer stem cells. Nat Prod Commun 17. https:// doi. org/
10. 1177/ 19345 78X22 11056 96
Villamizar LH, Cardoso MDG, Andrade JD, Teixeira ML, Soares MJ
(2017) Linalool, a Piper aduncum essential oil component, has
selective activity against Trypanosoma cruzi trypomastigote forms
at 4 C. Mem Inst Oswaldo Cruz 112:131–139. https:// doi. org/ 10.
1590/ 0074- 02760 160361
WHO (2023) Chagas disease (also known as American trypanosomi-
asis). https:// www. who. int/ news- room/ fact- sheets/ detail/ chagas-
disea se- (ameri can- trypa nosom iasis). Accessed on July 14 2023
Authors and Aliations
TiagoRochaNogueira1 · IgorMesquitaFigueredo2 · JoãoHenriqueSilvaLuciano3 · AntonioCalixtoLima4·
AluisioMarquesdaFonseca5 · LuziaKalyneAlmeidaMoreiraLeal6 · MaryAnneSousaLima1 ·
AliceMariaCostaMartins7 · EmmanuelSilvaMarinho8 · JacksondeQueirozMalveira9· PedrodeLimaNeto10 ·
FátimaMirandaNunes1 · MariaAlexsandradeSousaRios2 · AntôniaTorresÁvilaPimenta1
* Antônia Torres Ávila Pimenta
antonia.pimenta@ufc.br
1 Departamento de Química Orgânica e Inorgânica,
Universidade Federal doCeará, Campus do Pici, Bl. 933,
Fortaleza, CECEP60021-940, Brazil
2 Departamento de Engenharia Mecânica, Universidade
Federal doCeará, Campus do Pici, Bl. 715, Fortaleza,
CECEP60440-554, Brazil
3 Departamento de Ensino, Instituto Federal doCeará,
CampusCaucaia, CE, Brazil
4 Laboratório de Tecnologia de Biomassas, Embrapa
Agroindústria Tropical, Planalto do Pici, Fortaleza,
CECEP60511-110, Brazil
5 Instituto de Engenharia e Desenvolvimento Sustentável,
Universidade da Integração Internacional da Lusofonia
Afro-Brasileira, Redenção, CECEP62790-970, Brazil
6 Centro de Estudos Farmacêuticos e Cosméticos,
Departamento de Farmácia, Faculdade de Farmácia,
Universidade Federal doCeará, Fortaleza, CE, Brazil
7 Departamento de Análises Clínicas e Toxicológicas,
Universidade Federal doCeará, Fortaleza,
CECEP60430-170, Brazil
8 Grupo de Química Teórica e Eletroquímica, Faculdade de
Filosofia Dom Aureliano Matos, Universidade Estadual
doCeará, LimoeirodoNorte, CECEP62930-000, Brazil
9 Laboratório de Referência em Biocombustíveis, Fundação
Nucleo de Tecnologia Industrial doCeará, Fortaleza,
CECEP60440-552, Brazil
10 Departamento de Química Analítica e Físico-Química,
Universidade Federal doCeará, Campus do Pici, Fortaleza,
CECEP60455-760, Brazil