Revista Brasileira de Farmacognosia

https://doi.org/10.1007/s43450-020-00090-w

ORIGINAL ARTICLE

Potentialities of Cashew Nut (Anacardium occidentale) By-Product

for Pharmaceutical Applications: Extraction and Purification
Technologies, Safety, and Anti-inflammatory and Anti-arthritis
Activities
Ariane Teixeira dos Santos 1 & Gerlane Coelho Bernardo Guerra 2 & José Ivan Marques 1 &
Manoela Torres-Rêgo 1,3 & Jovelina Samara Ferreira Alves 1 & Roseane Carvalho Vasconcelos 4 &
Daline Fernandes de Souza Araújo 5 & Lucas Silva Abreu 6 & Thaís Gomes de Carvalho 7 &
Daniel Rodrigo Cavalcante de Araújo 8 & Josean Fechine Tavares 6 & Aurigena Antunes de Araújo 2 &
Raimundo Fernandes de Araújo Júnior 7 & Matheus de Freitas Fernandes-Pedrosa 1 &
Leandro de Santis Ferreira 1 & Silvana Maria Zucolotto 1

Received: 14 June 2020 / Accepted: 24 August 2020

# Sociedade Brasileira de Farmacognosia 2020

Abstract
The large consumption of cashew nut from Anacardium occidentale L., Anacardiaceae, generates accumulation of shells, a solid
by-product from agri-food industry. The cashew nut shell liquid is rich in phenolic lipids, recognized for its anti-inflammatory
and anti-tumor properties, and the shell of this fruit carry the great oil. For these reasons, this study aimed to investigate the
potential of the residual shells as a source of phenolic lipids and bioactive extract for pharmaceutical applications. The ethyl
acetate extract from the oil of the chestnut bark of A. occidentale (M3 extract) was optimized to obtain a phenolic lipid-rich
extract and preparative chromatography was used to increase the yield of isolated phenolic lipid derivatives. In addition, the
toxicity profile, anti-inflammatory, and acute and chronic anti-arthritis activities of the M3 extract were evaluated in in vivo
models. The M3 extract (25–100 mg/kg) on zymosan-induced air pouch and arthritis models revealed a significant anti-
edematogenic, antioxidant, and anti-inflammatory potential. The post-treatment with M3 extract at dose of 100 mg/kg depen-
dently improved a complete Freud’s adjuvant-induced arthritis model, with prevention osteoarticular degradation. Therefore,
these results report that bioactive M3 extract obtained from by-product is safe, eco-friendly, and promising for the rheumatoid
arthritis therapy.

Electronic supplementary material The online version of this article

(https://doi.org/10.1007/s43450-020-00090-w) contains supplementary
material, which is available to authorized users.

* Silvana Maria Zucolotto 4

Departamento de Odontologia, Faculdade de Odontologia,
[email protected] Universidade Federal do Rio Grande do Norte, Av. Senador Salgado
Filho, 300, Lagoa Nova, Natal, RN 59078-970, Brazil
1
Departamento de Ciências Farmacêuticas, Faculdade de Farmácia, 5
Departamento de Nutrição, Universidade Federal do Rio Grande do
Universidade Federal do Rio Grande do Norte, Av, General Gustavo Norte, Av. Trairí, s/n, Centro, Santa Cruz, RN 59200-000, Brazil
Cordeiro de Farias, s/n, Petrópolis, Natal, RN 59012-570, Brazil 6
Departamento de Ciências Farmacêuticas, Universidade Federal da
2 Paraíba, Av. Contorno da Cidade Universitária, s/n, Castelo Branco
Departamento de Biofísica e Farmacologia, Centro de Biociências, III, João Pessoa, PB 58051-085, Brazil
Campus Universitário, Universidade Federal do Rio Grande do
7
Norte, Av. Senador Salgado Filho, 3000, Lagoa Nova, Departamento de Morfologia, Centro de Biociências, Universidade
Natal, RN 59072-970, Brazil Federal do Rio Grande do Norte, Av. Senador Salgado Filho, 3000,
Lagoa Nova, Natal, RN 59072-970, Brazil
3 8
Instituto de Química, Universidade Federal do Rio Grande do Norte, Laboratorio de Fitoquímica, Instituto Nacional do Semi-Árido, Av.
Av. Senador Salgado Filho, 3000, Lagoa Nova, Francisco Lopes, s/n, Serrotão, Campina Grande, PB 58434-700,
Natal, RN 59072-970, Brazil Brazil
 Rev. Bras. Farmacogn.

Keywords Anacardic acid derivatives . Herbal medicine . Environmental conservation . Inflammation . Rheumatoid arthritis

Introduction Furthermore, these phenolic lipids have great pharmaceu-

tical potential due to the surfactant power, pharmacological
The agro-food industry produces annually large amounts of properties, and usefulness in the quality control of herbal med-
solid and liquid wastes that when accumulated, these organic icines from A. occidentale (Balachandran et al. 2013).
compounds cause serious environmental problems (Sadh et al. However, the isolation processes of these molecules, com-
2018). The almond-processing industries produce large monly recognized as the phenolic lipids from nut oil, have
amounts of waste, since nuts are the parts of interest used by shown low mass yield (Oiram-Filho et al. 2019). For these
industries and the bark is neglected. Cashew nuts remaining reasons, the present study aimed to take advantage of the
from Anacardium occidentale L., Anacardiaceae, processing agro-industrial cashew nut by-product as a source of natural
contain a resin in the interior that corresponds to 25% of the product for application in the pharmaceutical industry, in in-
total weight of this fruit (Gandhi et al. 2013). vestigating safety and its potential anti-arthritis bioactivity,
It has been known for many years that plants are empiri- and in improving the oil extraction and phenolic lipid
cally used in folk medicine for treating various diseases purification.
(Akhtar et al. 2011). According to the World Health
Organization, it is estimated that around 65–80% of the pop-
ulation in developing countries use natural products as medi-
Materials and Methods
cines (WHO 2011). As the use of medicinal plants grows,
acute and subchronic toxicity studies on these products should
Plant Material
be performed to ensure safe use by humans and the develop-
ment of new pharmaceuticals (Ukwuani et al. 2012; Saleem
Cashew nuts of Anacardium occidentale L., Anacardiaceae,
et al. 2017). Currently, the monitoring of the phytochemical
were collected at Alto Longá, Piauí, Brazil (lat: 5° 32′ 41.7″ N,
profile of their chemical markers by chromatographic analysis
long: 121° 44′ 25.9″ W) in September 2016 and was identified
of raw vegetal material is required by the worldwide regula-
by the botanist Dr. Leonardo de Melo Versieux. The voucher
tory pharmaceutical agencies to validate the quality and safety
specimen (UFRN 22361) was deposited in Herbarium of the
(Shi et al. 2014).
Federal University of Rio Grande do Norte, Brazil. The study
Rheumatoid arthritis (RA) is a chronic, autoimmune dis-
is authorized to collect material (SISBIO-64598) and access
ease of unknown origin, affects approximately 1% of the pop-
biodiversity for scientific purposes (SISGEN-A618873).
ulation, and is characterized by peripheral and symmetric
polyarthritis (Mavrogeni et al. 2013). Many potent anti-
arthritic drugs are available for the management of arthritis; Phenolic Lipid-Rich Extract Preparation
however, their prolonged use is associated with serious ad-
verse effects such as gastrointestinal ulcers, cardiovascular The cracked cashew nuts (30 g), using 10:1 (v/w: solvent/raw
problems, nephrotoxicity, pulmonary toxicity, and cirrhosis material) were submitted to four extraction methods to obtain
(Lee et al. 2013; Choudhary et al. 2015; Ruscitti et al. the CNSL employing soxhlet extraction with hexane (SH ex-
2017). Consequently, a growing number of patients with ar- tract; 50 °C; 3 h), maceration with petroleum ether (M1 ex-
thritis and other pathologies are making use of natural prod- tract; 25 °C; 120 h), maceration using ethyl ether (M2 extract;
ucts as a complementary therapeutic alternative (Bevaart et al. 25 °C; 120 h), and maceration with EtOAc (M3 extract; 25 °C;
2010; Xu et al. 2013; Dudics et al. 2018). 120 h). The extracts were concentrated in a rotary evaporator
The cashew nut shell liquid (CNSL) is a product remaining at 35 °C, solubilized in MeOH (1 mg/ml), analyzed by ultra-
from the processing of the cashew nut. Inside it, it has a dark- high-performance liquid chromatography coupled with diode
colored viscous oil, which corresponds to about 25% of the array detector (UHPLC/TOF/MS) (see the Supplementary
nut’s weight (Hamad and Mubofu 2015). The CNSL is a rich material : Section 1.2) and the areas of phenolic lipids were
source of long-chain hydrocarbon phenols, basically consisting measured for selecting the extract that showed the highest
of cardol, cardanol, 2-methylcardol, and anacardic acids content of these compounds. Added to this, more environmen-
(Gandhi et al. 2013). This oil has been the object of recent tally friendly solvent was taken into account, since that, in the
research motivated by its high biological potential mainly due literature, generally, apolar solvents are used to extraction of
to the presence of phenolic acids in its constitution, in which we cashew nuts. In view of this, M3 extract was obtained follow-
can highlight its antioxidant and anti-inflammatory activity ing the steps. Calcium hydroxide (5 g) was added to the se-
(Sung et al. 2008; Souza et al. 2018). However, the safety lected M3 extract (10 g) dissolved in 95% EtOH and stirred
profile of this oil extract has not yet been demonstrated. (50 °C; 3 h) to form calcium anacardate. Calcium anacardate
was dissolved in 50 ml of distilled water acidified with HCl
Rev. Bras. Farmacogn.

(1 M) and stirred (25 °C; 1 h). The sample obtained was Legislation Brazilian “Lei Arouca” number 11794/08 (08/
washed with ethyl acetate (2 × 15 ml; 25 °C) and concentrated 10/2008).
in a rotary evaporator at 35 °C, afforded the phenolic lipid-rich
extract from CNSL (M3 extract) (Paramashivappa et al. Acute Toxicity Model
2001).
The single-dose intra-gastric toxicity test was performed ac-
Purification and Identification of Isolated Compounds cording to the guideline for testing of chemical protocol of
OECD 423 (OECD 2001) The complete methodology was
The M3 extract (4 g) was solubilized in 4 ml of EtOAc and described in the Supplementary material (Section 1.6).
subjected to the normal silica gel column (350 g; 40 × 2.4 cm)
eluted with 400 ml of toluene/ethyl acetate/formic acid Subchronic Toxicity Model
(7:1.5:0.5; v/v/v). This procedure resulted in six fractions.
The fraction 1 (2.89 g) was solubilized in 3 ml of CH2Cl2/ The assay was performed according to the guideline for test-
EtOH (1:1; v/v) and subjected to a second column fraction- ing of chemical protocol of OECD 407 (OECD 2008). The
ation, using Sephadex LH20® (50 g; 30 × 2.5 cm) as a station- protocol test is in the Supplementary material (Section 1.7).
ary phase and CH2Cl2/EtOH (1:1; v/v) as a mobile phase
affording two fractions (Fr 1 and 2). F1 (2.89 g) was subjected Zymosan-Induced Air Pouch Model
to a purification procedure in preparative HPLC/UV/ESI/MS
(see the Supplementary material: Section 1.3). The samples The acute anti-inflammatory activity was evaluated using the
obtained were concentrated in a Rocket Evaporator (Thermo zymosan-induced air pouch model. The mice received into the
Scientific) at 35 °C and afforded the isolated compounds. back by subcutaneous route 5 ml of sterile air. After 3 days,
After, the chemical structures of the isolated compounds were 2.5 ml of sterile air were injected into air pouch. After 6 days
confirmed by HPLC/ESI/MS/MS and monodimensional and of the first administration, the animals were pretreated by
two-dimensional spectra obtained from nuclear magnetic res- intra-gastric route with 300 μl of M3 extract (25, 50, or
onance of 1H and 13C (see the Supplementary material : 100 mg/kg), saline, vehicle, or dexamethasone (2 mg/kg).
Sections 1.4 and 1.5). The animals received a solution of zymosan (1 mg/ml) in
the dorsal region after 30 min. Six hours after induction, the
mice were euthanized and washed with 2 ml of saline solution
Structural Identification for the collection of exudate (Torres-Rêgo et al. 2016;
Marques et al. 2018).
The chemical structures of the isolated phenolic lipids were
confirmed by means of MS and nuclear magnetic resonance of Zymosan-Induced Arthritis Model
1
H and 13C. The monodimensional and two-dimensional
spectra were recorded in dimethyl sulfoxide-d6 (DMSO-d6) The acute anti-inflammatory activity was evaluated using the
at 400 and 100 MHz in Bruker Avance III HD Ascend 400 zymosan-induced arthritis model. One hour prior to induction
(Bruker, Billerica, MA, USA). The chemical shifts (δ) were of the zymosan (1 mg/50 μl), the rats were treated by intra-
expressed in ppm and the coupling constants (J) in MHz. gastric gavage with 1.5 ml of saline, vehicle, diclofenac
(5 mg/kg), or M3 extract (25, 50, or 150 mg/kg) from the
Animals A. occidentale. Six hours after induction, the animals were
euthanized and the synovial membrane and articular exudates
Male and female Swiss mice (25–35 g), 6–8 weeks of age, and were harvested with 200 μl (twice) of saline (Keystone et al.
Wister rats (200–250 g), 8–10 weeks of age, provided by the 1977).
Animal Facility of the Department of Biophysics and
Pharmacology at the Federal University of Rio Grande do Complete Freund’s Adjuvant-Induced Arthritis Model
Norte (UFRN), were kept in climacteric rooms (22 ± 2 °C)
and with controlled light–dark cycle (12/12 h). The animals The chronic anti-inflammatory activity was evaluated using
were euthanized with a super dosage of thiopental and lido- the complete Freund’s adjuvant (CFA)-induced arthritis mod-
caine (90–10 mg/kg, respectively) by intraperitoneal route. el. The arthritis was induced in all animals, except in the saline
The experimental study was approved by the Committee for group, by intra-articular injection of 0.1 ml of complete
Ethics in Animal Experimentation of UFRN under protocol Freund’s adjuvant into the plantar surface of the right hind
numbers 013.003/2017 (05/06/2017) and 064.003/2017 (12/ paw. After 4 days, the rats were treated every day by intra-
12/2017), following the ethical principles adopted by the gastric gavage with 1.5 ml of saline, vehicle, dexamethasone
Brazilian Society of Animal Science and National (5 mg/kg), and M3 extract (25, 50, or 100 mg/kg) from the
 Rev. Bras. Farmacogn.

A. occidentale for 28 days. On the last day, the animals were absorbance was determined at 412 nm immediately and
euthanized and right hind paws were collected and analyzed expressed as nmol NPSH/mg (Sedlak and Lindsay 1968).
(Eissa et al. 2016).
Histopathological Analysis
Differential Cell Count in the Exudate
The histopathological analysis was realized by Ali et al.
In zymosan-induced air pouch model, the polymorphonuclear (2015) with some changes. The method is in
and mononuclear cell count was determined based on the the Supplementary material (Section 1.17).
count of 100 cells using a hemocytometer. The absolute
values were expressed as leukocytes counts per milliliter as Immunohistochemistry Analysis
previously described by Furtado et al. (2016).
Immunohistochemistry was performed of paw tissue second
Determination of Myeloperoxidase (MPO) methodology of Carvalho et al. (2018) with some changes
(see the Supplementary material : Section 1.18).
The MPO level in tissue rats in arthritis models were deter-
mined using a modified version of the method described by Statistical Analysis
Bradley et al. (1982). MPO activity was determined by a col-
orimetric method, with absorbance at 460 nm. Data are expressed as the mean ± standard deviation.
Statistical analyses were carried out by one-way analysis of
variance (ANOVA) with Tukey’s post hoc test for multiple
Determination of Total Protein
comparisons. All statistical analyses were performed using
GraphPad Prism version 5.00 (San Diego, CA, USA) and
From the subcutaneous lavage obtained from the zymosan-
statistical significance was set at ***p < 0.001, **p < 0.01,
induced air pouch model, the total protein was determined
and *p < 0.05.
using the Bradford assay. The exudates were analyzed on a
microtiter plate reader at 595 nm and the results were
expressed as μg/ml (Bradford 1976).
Results
Determination of Cytokine Levels Profile of Extracts by UHPLC/DAD Analysis
The IL-1β and TNF-α cytokines were determined from the An initial study was carried out in which this material was
subplantar and exudate tissue homogenate supernatants using subjected to two extraction methods (Soxhlet and macera-
commercial enzyme-linked immunosorbent assay (ELISA) tion) with different solvents of increasing polarity in order
kits according to the manufacturer’s protocol. Results were to obtain an extract with a higher concentration of phenolic
expressed as cytokine concentration (pg/ml). lipids and low toxicity. The chromatograms obtained by
UHPLC/DAD enabled visualizing the different sample
Determination of Malondialdehyde (MDA) profiles, in which M1, M2, and M3 maceration extracts
showed a better extraction of more apolar compounds
For the determination of malondialdehyde, samples of exu- (Fig. 1). Among the four extracts, SH presented a more
date from the animals’ air pouch and paw of chronic arthritis complex chromatographic profile. The M2 and M3 extracts
model were homogenized with 20 mM Tris-HCl and centri- showed more efficiency with respect to the extraction of
fuged. MDA reacts with the chromogen 1-methyl-2- the phenolic lipids. The LC/TOF/MS analysis of the M3
phenylindole, with a maximum absorbance at 586 nm extract showed signals with m/z 347 (M-H), m/z 345 (M-
(Gérard-Monnier et al. 1998). H), m/z 343 (M-H), and m/z 341 (M-H), associated with
UV spectra obtained by UHPLC/DAD, suggesting that the
Determination of Glutathione constituents present in M3 extract are phenolic lipids, con-
sidering that these substances were previously described in
NPSH is considered an antioxidant and glutathione is the CNSL (Gandhi et al. 2013). From these data obtained by
NPSH present in the highest amount (Nassar et al. 2014). MS together with the UV spectra, it was possible to assign
Samples of exudate and articular tissue were homogenized the chromatogram signals obtained by UHPLC/DAD, in
1:10 (v/v and w/v, respectively) in 0.1 M PBS buffer (pH which the tri-unsaturated phenolic lipid is the major com-
7.4) and centrifuged. After this step, the samples were plated pound, followed by two and one unsaturated acids, and
onto 96-well microplates with Tris/EDTA (1 mM) and finally the saturated acid.
Rev. Bras. Farmacogn.

6-[8(Z),11(Z),14-pentadecatrienyl] salicylic acid; AAC2:

6-[8(Z),11(Z), pentadecadienyl] salicylic acid; AAC3:
6-[8(Z)-pentadecenyl] salicylic acid (Supporting
Information; Table S1).

Acute and Subchronic Toxicity of Anacardium

occidentale M3 Extract

In the acute toxicity test, animals observed for 14 consecutive

days after administration at dose of 2000 mg/kg of
A. occidentale M3 extract did not show any behavioral chang-
es or death, and they did not exhibit a significant change in
body weight during the monitoring period. The serum levels
in relation to the biochemical and hematological dosages were
not significant in relation to the groups treated with saline
solution, vehicle, or extract at dose of 2000 mg/kg. The sub-
chronic administration of repeated doses of saline, vehicle, or
extract at doses of 50 and 100 mg/kg caused no change in
behavior, to body weight (data not shown), or to biochemical
and hematological measurements. The results can be visual-
ized in the Supporting Information (Tables S2–S5).

Effect of Anacardium occidentale M3 Extract in Acute

Zymosan-Induced Air Pouch Model

The animals treated by intra-gastric gavage with

A. occidentale M3 extract at doses of 25, 50, and 100 mg/kg
showed a significant inhibition of leukocyte migration com-
pared with the zymosan group (Fig. 2a). Similarly, the differ-
ent cashew nut shell extract doses revealed a significant re-
Fig. 1 UHPLC/DAD chromatograms of Anacardium occidentale CNSL duction in the MPO levels (Fig. 2b). Moreover, inhibition was
extracts obtained for Soxhlet extraction (SH) and maceration (M1, M2, observed for the mononuclear (MN) and polymorphonuclear
and M3) (PMN) cells (Fig. 2c, d, respectively) for all the groups treated
with M3 extract. In addition, the reduction in leukocyte mi-
Isolation and Structural Elucidation of Compounds gration for the group treated with dexamethasone was closely
similar to those treated with A. occidentale M3 extract.
Phenolic lipids have a basic structure consisting of a hydroxyl The pro-inflammatory IL-1β cytokine levels showed a sig-
functional group and carboxylic acid attached to their aromat- nificant reduction in the levels (Fig. 3a), as well as decreased
ic ring and a C15 terpene chain, with the different number of the IL-1β cytokine production (Fig. 3b) when compared with
unsaturations. The M3 extract was fractionated in classical the zymosan group. Similarly, the mice treated with different
column chromatography, resulting in three isolate compounds M3 extract doses reduced the MDA levels (Fig. 3c); however,
named AAC1 (56.6 mg), AAC2 (50.1 mg), and AAC3 it increased the NPSH levels (Fig. 3d) when compared with
(88 mg). It was possible to estimate the purity of the AAC1, the zymosan group.
AAC2, and AAC3 samples at more than 94% by the software The inflammation induced in this model is characterized
of UHPLC/DAD when the chromatogram was visualized at a for histologic changes in the thickness of the air pouch mem-
wavelength of UV 248 nm. The isolated compounds were brane with the presence of leukocyte infiltrate in strip
analyzed by 1H and 13C NMR spectra, LC/MS, and compar- (Fig. 4b). The mice treated by intra-gastric gavage with sub-
ison with data in the literature (VanBeek and Wintermans stances showed a condensed and thin air pouch membrane
2001; Omanakuttan et al. 2012; Morais et al. 2017). Taken with severe presence of polymorphonuclear cells when com-
together, it was possible to conclude by NMR and mass spec- pared with the groups that only received saline (Fig. 4a). On
tral analysis that AAC1, AAC2, and AAC3 compounds are the other hand, the mice treated with all the M3 extract doses
triene, diene, and monoene acids, respectively; AAC1: (Fig. 4d–f) showed an increase in thickness of the air pouch
 Rev. Bras. Farmacogn.

Fig. 2 Effect of Anacardium occidentale M3 extract on leukocyte air pouch model. ***p < 0.001, **p < 0.01, and *p < 0.05 compared with
migration (a), MPO levels (b), and mononuclear (c) and the zymosan group (black bar). Sal, saline (0.9 mg/ml); Dex, dexameth-
polymorphonuclear (d) cell subpopulation count in zymosan-induced asone (2 mg/kg)

membrane, as well as few leukocyte cells, being similar to the Effect of Anacardium occidentale M3 Extract in Acute
dexamethasone group (Fig. 4c). Zymosan-Induced Arthritis Model
The cyclooxygenase-2 (COX-2) expression in the zymosan
group (Fig. 5a) was more significantly intense when compared The anti-inflammatory acute effect of treatment with
with standard drug-treated group (Fig. 5b), which exhibited A. occidentale M3 extract was evaluated in a zymosan-
discrete immunoexpression to the inflammatory skin in the induced arthritis model. It is possible to observe the intense
screened protein. Similarly, the factor nuclear kappa B inflammatory infiltrate in this model, with increased vascular
(NF-κB) immunostaining significantly increased at the mem- permeability and edema formation. The rats treated with M3
brane margin of the pouch (Fig. 5d), whereas dexamethasone extract at the doses of 25, 50, and 100 mg/kg showed suppres-
(Fig. 5e) was able to reduce the expression. The groups treated sion in edema formation (Fig. 6a). In addition, the groups
with extract (25 mg/kg) demonstrated COX-2 (Fig. 5c) and treated with extract at the doses of 50 and 100 mg/kg were
NF-κB (Fig. 5f) immunostaining, similar to the group treated effective in decreasing cell migration into the joint when they
with dexamethasone. were compared with the zymosan group, as shown in Fig. 6b.
Rev. Bras. Farmacogn.

Fig. 3 Effect of Anacardium occidentale M3 extract on total proteins (a), the zymosan group (black bar). Sal, saline (0.9 mg/ml); Dex, dexameth-
IL-1β cytokine (b), MDA (c), and NPSH (d) levels in zymosan-induced asone (2 mg/kg)
air pouch model. ***p < 0.001, **p < 0.01, and *p < 0.05 compared with

Similarly, a decrease in the groups treated with the extract at cytokine levels. On the other hand, the rats treated with dif-
the dose of 50 and 100 mg/kg was observed in quantifying the ferent M3 extract doses presented suppression in edema for-
MPO level (Fig. 6c). Diclofenac showed significant inhibition mation during 28 days when compared with the CFA group
in all assays. (Fig. 7a). Similarly, treatment with all doses of the extract
enabled reducing MPO production (Fig. 7b).
Effect of M3 Extract from Anacardium occidentale in a The rats treated with different extract doses showed a sig-
Chronic CFA Arthritis Model nificant reduction in the TNF-α (Fig. 7c) and IL-1β cytokine
levels (Fig. 7d) when compared with the CFA group. In addi-
Considering the rodent arthritis models, the CFA-induced tion, a reduction of the edema and decrease in the production
model has several similarities with the human arthritis, being of MPO and cytokines in the group treated with dexametha-
considered a suitable model for arthritis induction (Khader sone were closely similar to those treated with the extract.
et al. 2019; Gupta et al. 2020; Zhang et al. 2020). As expected, Moreover, the mice treated with M3 extract showed reduced
the rats received the complete Freud’s adjuvant (CFA) and MDA levels (Fig. 7e); however, they also showed increased
4 days after were treated with vehicle (CFA group) and exhib- NPSH levels (Fig. 7f) when compared with the CFA group.
ited paw edema during 28 days and increased the The paw histological changes were observed in the com-
myeloperoxidase and pro-inflammatory (IL-1β and TNF-α) plete Freud’s adjuvant-induced arthritis model. As expected,
 Rev. Bras. Farmacogn.

Fig. 4 Effect of Anacardium occidentale M3 extract on the air pouch tissue histopathologic changes in zymosan-induced air pouch model. Saline (a);
zymosan (b); dexamethasone at 2 mg/kg (c); and A. occidentale M3 extract at 25 (d), 50 (e), and 100 mg/kg (f). APM, air pouch membrane

the rats who received CFA and were treated with vehicle ex- dexamethasone groups (Fig. 8c). The 25-mg/kg dose did not
hibited severe inflammatory infiltrate, extensive areas of bone show anti-inflammatory effects (Fig. 8d).
and cartilage degradation caused by pannus formation, atro- Immunolabeled cells for IL-17 were those that showed
phy and degeneration of chondrocytes, and area suggestive of brownish staining in the nuclei, cytoplasm, or membranes.
synovial polyp and edema (Fig. 8b), when compared with the The animals that received CFA and were treated with vehicle
saline group which showed intact joint space, cartilage, and showed an intense immunoreactivity with mononuclear, fibro-
bones without leukocyte cells (Fig. 8a). The rats treated with blast, and endothelial inflammatory cells in the pannus region.
A. occidentale M3 extract at the doses of 50 mg/kg (Fig. 8e) Immunolabeled osteocytes were also observed in bone trabec-
and 100 mg/kg (Fig. 8f) showed discrete areas of cartilaginous ular medium (Fig. 9a). The group treated with extract at the
degradation, synovial membrane hyperplasia, chondrocyte dose of 100 mg/kg demonstrated discrete tibiotarsal bone
degeneration, and inflammatory infiltrate arranged in focal structure in the adjacent connective tissue and moderate im-
areas by the specimen when compared with the saline and munoblotting for the mononuclear inflammatory cells (Fig.

Fig. 5 Effect of Anacardium occidentale M3 extract on COX-2 and NF- A. occidentale M3 extract at 25 mg/kg (c). For the NF-κB expression,
κB expression in zymosan-induced air pouch model. For the COX-2 zymosan (d), dexamethasone at 2 mg/kg (e), and A. occidentale M3
expression, zymosan (a), dexamethasone at 2 mg/kg (b), and extract at 25 mg/kg (f)
Rev. Bras. Farmacogn.

Fig. 6 Effect of Anacardium occidentale M3 extract on joint *p < 0.05 compared with the zymosan group (black bar). Sal, saline
circumference (a), leukocyte migration (b), and MPO levels (c) in a (0.9 mg/ml); Dcf, diclofenac (5 mg/kg)
zymosan-induced arthritis model. ***p < 0.001, **p < 0.01, and

9c). The animals treated with dexamethasone presented dis- double bonds. In this study, based on the chromatographic
creet immunostaining in chondrocytes and fibroblasts analysis, it was possible to suggest that the substances present
(Fig. 9b). in the four oil extracts from the chestnut bark of A. occidentale
have more apolar compounds, while the maceration extraction
showed to be the most satisfactory for obtaining more content
Discussion of phenolic acids (observed by the peak’s area), corroborating
the results obtained by Tyman (1975). The performance of the
Cashew nut liquid is a rich source of long-chain hydrocarbon separation process when related to economic factors shows
phenols, basically being composed of cardol, cardanol, 2- great feasibility when compared with the available products.
methylcardol, and phenolic lipids (Gandhi et al. 2013). The isolation of these substances used a fast and modern tech-
Phenolic lipids have been isolated from different extracts nique, obtaining a higher yield when compared with other
and parts of A. occidentale. These substances were first report- techniques used for the isolation of these substances (Kubo
ed in Gingko biloba extract, in which they belong to the group et al. 1986). It has been known for many years that plants are
of 6-alkylsalicylic acids, having 13–19 C-atoms with three empirically used in folk medicine for treating various
 Rev. Bras. Farmacogn.

Fig. 7 Effect of Anacardium occidentale M3 extract on paw edema (a), compared with the CFA group (black bar). Sal, saline (0.9 mg/ml); Dex,
MPO (b), TNF-α (c) and IL-1β (d) cytokines, MDA (e), and NPSH (f) dexamethasone (5 mg/kg)
levels in a CFA arthritis model. ***p < 0.001, **p < 0.01, and *p < 0.05

pathologies (Akhtar et al. 2011). However, using these prod- 2017). Therefore, the phytochemical profile, toxicity, and
ucts without proven safety for their use has a disadvantage, anti-inflammatory activity of A. occidentale M3 extract in
with the fact being that many of these popularly used plants do animal models was evaluated. The present study showed that
not have an established toxicity study (Christapher et al. the minimum lethal dose of M3 extract is greater than
Rev. Bras. Farmacogn.

Fig. 8 Effect of Anacardium occidentale M3 extract in paw histopathologic changes in a complete Freud’s adjuvant-induced arthritis model. Saline (a);
CFA (b); dexamethasone at 5 mg/kg (c); and A. occidentale M3 extract at 25 (d), 50 (e), and 100 mg/kg (f)

2000 mg/kg by the intra-gastric route, since no animal died Therefore, the objective of this study was to investigate the
after administering the samples, and this result is in agreement activity of an extract obtained from the CNSL in an acute and
with the findings of Konan and Bacchi (2007) and Carvalho chronic arthritis model for the first time. In this study, it was
et al. (2013). The information in the short-term and long-term demonstrated that the pre- and post-treatment with M3 extract
toxic effects of nut oil has already been reported in the litera- can act in the models tested herein on reducing joint edema by
ture (Morais et al. 2010; Daswanto et al. 2016). The evaluation blocking leukocyte influx and inhibition of MPO enzymatic
of organ weight is a frequently used parameter in the toxicity activity. In the inflammatory process of arthritis, the first cells
studies to verify if there was any alteration in the organs, as to migrate to the joint are neutrophils, which are responsible
well as to confirm the findings of the biochemical and hema- for releasing proteoglycans and reactive oxygen species
tological analyses (Parasuraman 2011). (ROS), causing tissue and joint destruction (Srirangan and
In the literature, there are several studies proving the ben- Choy 2010). The antioxidant effects of the extract obtained
efits of phenolic lipids, among which we can highlight its from A. occidentale bark oil may be related to the inhibition
antioxidant and anti-inflammatory potential (Masuoka and potential of a series of pro-oxidant enzymes involved in the
Kubo 2004; Ha and Kubo 2005; Omanakuttan et al. 2012; production process of the oxygen reactive species (Ha and
Fusco et al. 2020; Siracusa et al. 2020). Furthermore, hepato- Kubo 2005; Kubo et al. 2006).
protective, gastroprotective, anti-parasitic, and anti-bacterial In the pathogenesis of rheumatoid arthritis, the cells infil-
properties have already been evaluated (Cui et al. 2008; trating the intra-articular space stimulate production of inflam-
Pereira et al. 2008; Morais et al. 2010). Studies have shown matory mediators, such as cytokines, which perform impor-
that other species belonging to the Anacardiaceae family also tant biological processes (Siebert et al. 2015). Macrophages
have anti-inflammatory potential (Orhan et al. 2006). play a central role in the release of cytokines such as IL-1, IL-

Fig. 9 Effect of Anacardium occidentale M3 extract on IL-17 expression in paw in a complete Freud’s adjuvant-induced arthritis model. CFA (a),
dexamethasone at 5 mg/kg (b), and A. occidentale M3 extract at 100 mg/kg (c)
 Rev. Bras. Farmacogn.

6, TNF-α, IL-10, and IL-18, which coordinate immune and Furthermore, the oil extract obtained is safe in rodents and
inflammatory responses (Alunno et al. 2017). These cytokines A. occidentale M3 extract presents anti-inflammatory and
also stimulate the proliferation and differentiation of T lym- anti-arthritic effects, in which their effects suggest to be relat-
phocytes in TH-17 which produce IL-17 (Hashimoto 2017). ed to antioxidant and immunoregulatory mechanisms. In ad-
The inhibitory effects of IL-17 expression add further infor- dition, our results suggest that the bioactive compounds pres-
mation to the anti-arthritic mechanism of the M3 extract from ent in the extract may play an important role in controlling the
A. occidentale. These cytokines play an important role in joint inflammatory process, thus assigning an anti-inflammatory
and cartilaginous degeneration in RA, and suppression of potential to the nutshell oil. Moreover, this study also explored
these cytokines reveals an immunomodulatory role of this some potential for using a by-product from the agri-food in-
extract (Robert and Miossec 2019). Although there is a large dustry, promoting a reduction in solid waste that pollutes cities
number of studies demonstrating the immunoregulatory role and harms the various biomes of the environment.
of phenolic lipids in vivo and in vitro inflammation models,
their role in arthritis has not been described. The main results Acknowledgments The authors acknowledge all contributors for their
valuable time and commitment to the study. This research received no
obtained in this study support a protective effect of both anti-
external funding in part by the Coordenação de Aperfeiçoamento de
gens used with an improvement of clinical and histopatholog- Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. We also
ical signs. thank to Conselho Nacional de Desenvolvimento Científico e
The biological effects of compounds isolated from Tecnológico - Brasil (CNPq) for the financial support (478661/2010-0)
and the Federal University of Rio Grande do Norte (UFRN).
A. occidentale cashew nut bark oil have been widely evaluated
and have indicated that these substances reduce and induce
Author Contributions ATS, SMZ, and GCBG planned and designed the
cell death in tumor cells such as in the breasts, kidneys, pros- experiments. ATS, JIM, MT-R, JSFA, RCV, and DFSA performed and
tate, colon, lungs, and melanomas (Huang et al. 2010; designed of experiments. ATS, LSA, MT-R, TGC, RCV, and DFSA
Hemshekhar et al. 2012; Seong et al. 2013; Legut et al. performed data analysis. SMZ, GCBG, JFT, MGA, AAA, RFAJ, MFF-
P, and LSF collaborated with the preparation of materials/reagents/kits
2014). The mechanism by which these substances present
and analytical tools. ATS, SMZ, GCBG, MFF-P, JSFA, and MT-R col-
anti-tumor potential is by inhibiting histone acetyltransferase laborated with the preparation of the manuscript and critical analyses of
p300, which acts on DNA transcription (Balasubramanyam the text.
et al. 2003). Added to this, Sung et al. (2008) found that tri-
unsaturated anacardic acid blocked the transcription factor Compliance with Ethical Standards
NF-κB responsible for expressing more than 400 gene prod-
ucts which are important for the survival and growth of cancer Conflict of Interest The authors declare that they have no conflict of
cells. In this study, the NF-κB immunoexpression was in- interest.
creased in mice treated with CFA, whereas post-treatment
with the M3 extract was able to suppress the effects.
Negative regulation of NF-κB by the M3 extract reduced the References
immunoexpression of NF-κB-regulated protein products such
Akhtar N, Miller MJS, Haqqi TM (2011) Effect of a herbal-leucine mix
as COX-2 (Lee et al. 2011). Similarly, Paramashivappa et al. on the IL-1β-induced cartilage degradation and inflammatory gene
(2003) found that anacardic acid has the potential to inhibit expression in human chondrocytes. BMC Complement Altern Med
human COX-2. Recently, four anacardic acids were identified 11:1–10. https://doi.org/10.1186/1472-6882-11-66
in the Knema hoookeriana species, and an anti-apoptotic po- Ali EAI, Barakat BM, Hassan R (2015) Antioxidant and angiostatic effect
of Spirulina platensis suspension in complete Freund’s adjuvant-
tential was found in these substances when antagonizing the induced arthritis in rats. PLoS One 10:1–13. https://doi.org/10.
Blc-xL/Bak and Mcl-1/Bid association (Gény et al. 2016). 1371/journal.pone.0121523
Although there is a large number of studies demonstrating Alunno A, Carubbi F, Giacomelli R, Gerli R (2017) Cytokines in the
the immunoregulatory role of anacardic acids in in vivo and pathogenesis of rheumatoid arthritis: new players and therapeutic
targets. BMC Rheumatol 1:1–13. https://doi.org/10.1186/s41927-
in vitro inflammation models, their role in arthritis has not 017-0001-8
been described. The main results obtained in this study sup- Balachandran VS, Jadhav SR, Vemula PK, John G (2013) Recent ad-
port a protective effect of both antigens used with an improve- vances in cardanol chemistry in a nutshell: from a nut to
ment of clinical and histopathological signs. nanomaterials. Chem Soc Rev 42:427–438. https://doi.org/10.
1039/C2CS35344J
Balasubramanyam K, Swaminathan V, Ranganathan A, Kundu TK
(2003) Small molecule modulators of histone acetyltransferase
Conclusions p300. J Biol Chem 278:19134–19140. https://doi.org/10.1074/jbc.
M301580200
Bevaart L, Vervoordeldonk MJ, Tak PP (2010) Evaluation of therapeutic
These findings demonstrated that the extraction and isolation targets in animal models of arthritis: how does it relate to rheumatoid
processes developed increased the yield of isolated com- arthritis? Arthritis Rheum 62:2192–2205. https://doi.org/10.1002/
pounds, which can be applied to industrial processes. art.27503
Rev. Bras. Farmacogn.

Bradford MM (1976) A rapid and sensitive method for the quantitation of interactions. J Nat Prod 79:838–844. https://doi.org/10.1021/acs.
microgram quantities of protein utilizing the principle of protein-dye jnatprod.5b00915
binding. Anal Biochem 72:248–254. https://doi.org/10.1016/0003- Gérard-Monnier D, Erdelmeier I, Régnard K, Moze-Henry N, Yadan J-C,
2697(76)90527-3 Chaudière J (1998) Reactions of 1-methyl-2-phenylindole with
Bradley PP, Priebat DA, Christensen RD, Rothstein G (1982) malondialdehyde and 4-hydroxyalkenals. Analytical applications
Measurement of cutaneous inflammation: estimation of neutrophil to a colorimetric assay of lipid peroxidation. Chem Res Toxicol
content with an enzyme marker. J Invest Dermatol 78:206–209. 11:1176–1183. https://doi.org/10.1021/tx9701790
https://doi.org/10.1111/1523-1747.ep12506462 Gupta S, Mishra KP, Kumar B, Singh SB, Ganju L (2020)
Carvalho ALN, Annoni R, Torres LHL, Durão ACCS, Shimada ALB, Andrographolide attenuates complete Freund’s adjuvant induced
Almeida FM, Hebeda CB, Lopes FDTQS, Dolhnikoff M, Martins arthritis via suppression of inflammatory mediators and pro-
MA, Silva LFF, Farsky SHP, Saldiva PHN, Ulrich CM, Owen RW, inflammatory cytokines. J Ethnopharmacol 261:113022. https://
Marcourakis T, Trevisan MTS, Mauad T (2013) Anacardic acids doi.org/10.1016/j.jep.2020.113022
from cashew nuts ameliorate lung damage induced by exposure to Ha TJ, Kubo I (2005) Lipoxygenase inhibitory activity of anacardic acids.
diesel exhaust particles in mice. Evid Based Complement Altern J Agric Food Chem 53:4350–4354. https://doi.org/10.1021/
Med 2013:1–13. https://doi.org/10.1155/2013/549879 jf048184e
Carvalho TG, Garcia VB, Araújo AA, Gasparotto LHS, Silva H, Guerra Hamad FB, Mubofu EB (2015) Potential biological applications of bio-
GCB, Miguel EC, Leitão RFC, Costa DVS, Cruz LJ, Chan AB, based anacardic acids and their derivatives. Int J Mol Sci 16:8569–
Araújo-Júnior RF (2018) Spherical neutral gold nanoparticles im- 8590. https://doi.org/10.3390/ijms16048569
prove anti-inflammatory response, oxidative stress and fibrosis in Hashimoto M (2017) Th17 in animal models of rheumatoid arthritis. J
alcohol-methamphetamine-induced liver injury in rats. Int J Pharm Clin Med 6:1–13. https://doi.org/10.3390/jcm6070073
548:1–14. https://doi.org/10.1016/j.ijpharm.2018.06.008 Hemshekhar M, Santhosh MS, Kemparaju K, Girish KS (2012)
Choudhary M, Kumar V, Malhotra H, Singh S (2015) Medicinal plants Emerging roles of anacardic acid and its derivatives: a pharmaco-
with potential anti-arthritic activity. J Intercult Ethnopharmacol 4: logical overview. Basic Clin Pharmacol Toxicol 110:122–132.
147–179. https://doi.org/10.5455/jice.20150313021918 https://doi.org/10.1111/j.1742-7843.2011.00833.x
Christapher PV, Parasuraman S, Asmawi MZ, Murugaiyah V (2017) Huang WY, Cai YZ, Zhang Y (2010) Natural phenolic compounds from
Acute and subchronic toxicity studies of methanol extract of medicinal herbs and dietary plants: potential use for cancer preven-
Polygonum minus leaves in Sprague Dawley rats. Regul Toxicol tion. Nutr Cancer 62:1–20. https://doi.org/10.1080/
Pharmacol 86:33–41. https://doi.org/10.1016/j.yrtph.2017.02.005 01635580903191585
Cui L, Miao J, Furuya T, Fan Q, Li X, Rathod PK, Su X-Z, Cui L (2008) Keystone E, Schorlemmer H, Pope C, Allison A (1977) Zymosan-
Histone acetyltransferase inhibitor anacardic acid causes changes in induced arthritis: a model of chronic proliferative arthritis following
global gene expression during in vitro Plasmodium falciparum de- activation of the alternative pathway of complement. Arthritis
velopment. Eukaryot Cell 7:1200–1210. https://doi.org/10.1128/ Rheum 20:1396–1401
EC.00063-08 Khader SZA, Ahmed SSZ, Arunachalam T, Radhakrishnan K (2019)
Daswanto H, Satuti N, Sagi M, Astuti P (2016) Acute toxicity of cashew Therapeutic effect of Parmotrema tinctorum against complete
nut shell extract (Anacardium occidentale L.) in albino rat (Rattus Freund’s adjuvant-induced arthritis in rats and identification of nov-
norvegicus Berkenhout 1769). Pak J Biol Sci 19:89–94. https://doi. el isophthalic ester derivative. Biomed Pharmacother 112:108646.
org/10.3923/pjbs.2016.89.94 https://doi.org/10.1016/j.biopha.2019.108646
Dudics S, Langan D, Meka R, Venkatesha S, Berman B, Che C-T, Konan NA, Bacchi EM (2007) Antiulcerogenic effect and acute toxicity
Moudgil K (2018) Natural products for the treatment of autoimmune of a hydroethanolic extract from the cashew (Anacardium
arthritis: their mechanisms of action, targeted delivery, and interplay occidentale L.) leaves. J Ethnopharmacol 112:237–242. https://doi.
with the host microbiome. Int J Mol Sci 19:1–21. https://doi.org/10. org/10.1016/j.jep.2007.03.003
3390/ijms19092508 Kubo I, Komatsu S, Ochi M (1986) Molluscicides from the cashew
Eissa MM, Mostafa DK, Ghazy AA, El MZ, Boulos M, Younis LK Anacardium occidentale and their large-scale isolation. J Agric
(2016) Anti-arthritic activity of Schistosoma mansoni and Food Chem 34:970–973. https://doi.org/10.1021/jf00072a010
Trichinella spiralis derived-antigens in adjuvant arthritis in rats: role Kubo I, Masuoka N, Ha TJ, Tsujimoto K (2006) Antioxidant activity of
of FOXP3+ treg cells. PLoS One 11:1–20. https://doi.org/10.1371/ anacardic acids. Food Chem 99:555–562. https://doi.org/10.1016/j.
journal.pone.0165916 foodchem.2005.08.023
Furtado AA, Torres-Rêgo M, Lima MCJS, Bitencourt MAO, Estrela AB, Lee S, Shin S, Kim H, Han S, Kim K, Kwon J, Kwak J-H, Lee C-K, Ha
Silva NS, Siqueira EMS, Tomaz JC, Lopes NP, Silva-Júnior AA, N-J, Yim D, Kim K (2011) Anti-inflammatory function of arctiin by
Zucolotto SM, Fernandes-Pedrosa MF (2016) Aqueous extract from inhibiting COX-2 expression via NF-κB pathways. J Inflamm 8:1–
Ipomoea asarifolia (Convolvulaceae) leaves and its phenolic com- 9. https://doi.org/10.1186/1476-9255-8-16
pounds have anti-inflammatory activity in murine models of edema, Lee S-G, Kim J-M, Lee S-H, Kim K-H, Kim J-H, Yi J-W, Jung W-J, Park
peritonitis and air-pouch inflammation. J Ethnopharmacol 192:225– Y-E, Park S-H, Lee J-W, Baek S-H, Lee J-H, Kim G-T (2013) Is the
235. https://doi.org/10.1016/j.jep.2016.07.048 frequency of metabolic syndrome higher in South Korean women
Fusco R, Siracusa R, Peritore AF, Gugliandolo E, Genovese T, D’Amico with rheumatoid arthritis than in healthy subjects? Korean J Intern
R, Cordaro M, Crupi R, Mandalari G, Impellizzeri D, Cuzzocrea S, Med 28:206–215
Paola R (2020) The role of cashew (Anacardium occidentale L.) Legut M, Lipka D, Filipczak N, Piwoni A, Kozubek A, Gubernator J
nuts on an experimental model of painful degenerative joint disease. (2014) Anacardic acid enhances the anticancer activity of liposomal
Antioxidants 9:1–12. https://doi.org/10.3390/antiox9060511 mitoxantrone towards melanoma cell lines - in vitro studies. Int J
Gandhi TS, Dholakiya BZ, Patel MR (2013) Extraction protocol for Nanomedicine 9:653–668. https://doi.org/10.2147/IJN.S54911
isolation of CNSL by using protic and aprotic solvents from cashew Marques JI, Alves JSF, Torres-Rêgo M, Furtado AA, Siqueira EMS,
nut and study of their physico-chemical parameter. Pol J Chem Galinari E, Araújo DFS, Guerra GCB, Azevedo EP, Fernandes-
Technol 15:24–27. https://doi.org/10.2478/pjct-2013-0062 Pedrosa MF, Zucolotto SM (2018) Phytochemical analysis by
Gény C, Rivière G, Bignon J, Birlirakis N, Guittet E, Awang K, Litaudon HPLC– HRESI-MS and anti-infl ammat ory activity of
M, Roussi F, Dumontet V (2016) Anacardic acids from Knema Tabernaemontana catharinensis. Int J Mol Sci 19:1–19. https://
hookeriana as modulators of Bcl-xL/Bak and Mcl-1/Bid doi.org/10.3390/ijms19020636
 Rev. Bras. Farmacogn.

Masuoka N, Kubo I (2004) Characterization of xanthine oxidase inhibi- Sadh PK, Duhan S, Duhan JS (2018) Agro-industrial wastes and their
tion by anacardic acids. Biochim Biophys Acta Mol basis Dis 1688: utilization using solid state fermentation: a review. Bioresour
245–249. https://doi.org/10.1016/j.bbadis.2003.12.010 Bioprocess 5:1–15. https://doi.org/10.1186/s40643-017-0187-z
Mavrogeni S, Dimitroulas T, Sfikakis PP, Kitas GD (2013) Heart in- Saleem U, Amin S, Ahmad B, Azeem H, Anwar F, Mary S (2017) Acute
volvement in rheumatoid arthritis: multimodality imaging and the oral toxicity evaluation of aqueous ethanolic extract of Saccharum
emerging role of cardiac magnetic resonance. Semin Arthritis munja Roxb. roots in albino mice as per OECD 425 TG. Toxicol
Rheum 43:314–324. https://doi.org/10.1016/j.semarthrit.2013.05. Rep 4:580–585. https://doi.org/10.1016/j.toxrep.2017.10.005
001 Sedlak J, Lindsay RH (1968) Estimation of total, protein-bound, and
Morais TC, Pinto NB, Carvalho KMMB, Rios JB, Ricardo NMPS, nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Anal
Trevisan MTS, Rao VS, Santos FA (2010) Protective effect of Biochem 25:192–205. https://doi.org/10.1016/0003-2697(68)
anacardic acids from cashew (Anacardium occidentale) on 90092-4
ethanol-induced gastric damage in mice. Chem Biol Interact 183: Seong YA, Shin PG, Kim G (2013) Anacardic acid induces
264–269. https://doi.org/10.1016/j.cbi.2009.10.008 mitochondrial-mediated apoptosis in the A549 human lung adeno-
Morais S, Silva K, Araujo H, Vieira I, Alves D, Fontenelle R, Silva A carcinoma cells. Int J Oncol 42:1045–1051. https://doi.org/10.3892/
(2017) Anacardic acid constituents from cashew nut shell liquid: ijo.2013.1763
NMR characterization and the effect of unsaturation on its biological Shi ZQ, Song DF, Li RQ, Yang H, Qi LW, Xin GZ, Wang DQ, Song HP,
activities. Pharmaceuticals 10:1–10. https://doi.org/10.3390/ Chen J, Hao H, Li P (2014) Identification of effective combinatorial
ph10010031 markers for quality standardization of herbal medicines. J
Nassar M, Hiraishi N, Shimokawa H, Tamura Y, Otsuki M, Kasugai S, Chromatogr A 1345:78–85. https://doi.org/10.1016/j.chroma.2014.
Ohya K, Tagami J (2014) The inhibition effect of non-protein thiols 04.015
on dentinal matrix metalloproteinase activity and HEMA cytotoxic- Siebert S, Tsoukas A, Robertson J, McInnes I (2015) Cytokines as ther-
ity. J Dent 42:312–318. https://doi.org/10.1016/j.jdent.2013.11.023 apeutic targets in rheumatoid arthritis and other inflammatory dis-
OECD (2001) Guidelines for the testing of chemicals - test No. 423: eases. Pharmacol Rev 67:280–309. https://doi.org/10.1124/pr.114.
Acute oral toxicity - acute toxic class method. OECD Press, Paris, 009639
p 14. https://doi.org/10.1787/9789264070943-en Siracusa R, Fusco R, Peritore AF, Cordaro M, D’Amico R, Genovese T,
OECD (2008) Guidelines for the testing of chemicals - test No. 407: Gugliandolo E, Crupi R, Smeriglio A, Mandalari G, Cuzzocrea S,
repeated dose 28-day oral toxicity study in rodents. OECD Press, Paola R, Impellizzeri D (2020) Anacardium occidentale L. cashew
Paris, p 13. https://doi.org/10.1787/9789264070684-en nuts in a mouse model of colitis. Nutrients 12:1–20
Oiram-Filho F, Zocolo GJ, Canuto KM, Silva-Júnior IJ, Brito ES (2019) Souza MQ, Teotônio IMSN, Almeida FC, Heyn GS, Alves PS, Romeiro
Productivity of a preparative high-performance liquid chromatogra- LAS, Pratesi R, Nóbrega YKM, Pratesi CB (2018) Molecular eval-
phy isolation of anacardic acids from cashew nut shell liquid. Sep uation of anti-inflammatory activity of phenolic lipid extracted from
Sci Plus 2:192–199. https://doi.org/10.1002/sscp.201900014 cashew nut shell liquid (CNSL). BMC Complement Altern Med 18:
Omanakuttan A, Nambiar J, Harris RM, Bose C, Pandurangan N, 1–11. https://doi.org/10.1186/s12906-018-2247-0
Varghese RK, Kumar GB, Tainer JA, Banerji A, Perry JJP, Nair Srirangan S, Choy EH (2010) The role of interleukin 6 in the pathophys-
BG (2012) Anacardic acid inhibits the catalytic activity of matrix iology of rheumatoid arthritis. Ther Adv Musculoskelet Dis 2:247–
metalloproteinase-2 and matrix metalloproteinase-9. Mol Pharmacol 256. https://doi.org/10.1177/1759720X10378372
82:614–622. https://doi.org/10.1124/mol.112.079020 Sung B, Pandey MK, Ahn KS, Yi T, Chaturvedi MM, Liu M, Aggarwal
Orhan E, Esitken A, Ercisli S, Turan M, Sahin F (2006) Effects of plant BB (2008) Anacardic acid (6-nonadecyl salicylic acid), an inhibitor
growth promoting rhizobacteria (PGPR) on yield, growth and nutri- of histone acetyltransferase, suppresses expression of nuclear factor-
ent contents in organically growing raspberry. Sci Hortic 111:38– kappaB-regulated gene products involved in cell survival, prolifer-
43. https://doi.org/10.1016/j.scienta.2006.09.002 ation, invasion, and inflammation through inhibition of the inhibi-
Paramashivappa R, Kumar PP, Vithayathil PJ, Rao AS (2001) Novel tory subunit of nuclear factor-kappaBalpha kinase, leading to poten-
method for isolation of major phenolic constituents from cashew tiation of apoptosis. Blood 111:4880–4891. https://doi.org/10.1182/
(Anacardium ccidentale L.) nut shell liquid. J Agric Food Chem blood-2007-10-117994
49:2548–2551 Torres-Rêgo M, Furtado AA, Bitencourt MAO, Lima MCJS, Andrade
Paramashivappa R, Kumar PP, Rao PVS, Rao AS (2003) Design, syn- RCLC, Azevedo EP, Soares TC, Tomaz JC, Lopes NP, Silva-Júnior
thesis and biological evaluation of benzimidazole/benzothiazole and AA, Zucolotto SM, Fernandes-Pedrosa MF (2016) Anti-
benzoxazole derivatives as cyclooxygenase inhibitors. Bioorg Med inflammatory activity of aqueous extract and bioactive compounds
Chem Lett 13:657–660. https://doi.org/10.1016/S0960-894X(02) identified from the fruits of Hancornia speciosa Gomes
01006-5 (Apocynaceae). BMC Complement Altern Med 16:1–10. https://
Parasuraman S (2011) Toxicological screening. J Pharmacol doi.org/10.1186/s12906-016-1259-x
Pharmacother 2:74–79. https://doi.org/10.4103/0976-500X.81895 Tyman JHP (1975) Quantitative determination of the olefinic composi-
Pereira JM, Severino RP, Vieira PC, Fernandes JB, Silva MFGF, Zottis tion of the component phenols in cashew nut-shell liquid. J
A, Andricopulo AD, Oliva G, Corrêa AG (2008) Anacardic acid Chromatogr A 111:277–284. https://doi.org/10.1016/S0021-
derivatives as inhibitors of glyceraldehyde-3-phosphate dehydroge- 9673(00)99275-6
nase from Trypanosoma cruzi. Bioorg Med Chem 16:8889–8895. Ukwuani AN, Abubakar M, Hassan SW, Agaie BM (2012)
https://doi.org/10.1016/j.bmc.2008.08.057 Toxicological studies of hydromethanolic leaves extract of Grewia
Robert M, Miossec P (2019) IL-17 in rheumatoid arthritis and precision crenata. Int J Pharm Sci Drug Res 4:245–249
medicine: from synovitis expression to circulating bioactive levels. VanBeek TA, Wintermans MS (2001) Preparative isolation and dual
Front Med 6:1–10. https://doi.org/10.3389/fmed.2018.00364 column high-performance liquid chromatography of ginkgolic acids
Ruscitti P, Cipriani P, Masedu F, Romano S, Berardicurti O, Liakouli V, from Ginkgo biloba. J Chromatogr A 930:109–117. https://doi.org/
Carubbi F, Benedetto P, Alvaro S, Penco M, Valenti M, Giacomelli 10.1016/S0021-9673(01)01194-3
R (2017) Increased cardiovascular events and subclinical atheroscle- WHO (2011) United-residue force field for off-lattice protein-structure
rosis in rheumatoid arthritis patients: 1 year prospective single simulations: III. Origin of backbone hydrogen-bonding
Centre study. PLoS One 12:1–15. https://doi.org/10.1371/journal. cooperativity in united-residue potentials. The world medicines sit-
pone.0170108 uation 2011 - medicines prices, availability and affordability. World
Rev. Bras. Farmacogn.

Health Organization. Geneva WHO Press. p.30. https://doi.org/10. Zhang Z, Chinnathambi A, Ali SA, Bai L (2020) Copper oxide nanopar-
1002/(SICI)1096-987X(199802)19:3<259::AID-JCC1>3.0.CO;2-S ticles from Rabdosia rubescens attenuates the complete Freund’s
Xu WD, Zhang M, Zhang YJ, Ye DQ (2013) IL-33 in rheumatoid arthri- adjuvant (CFA) induced rheumatoid arthritis in rats via suppressing
tis: potential role in pathogenesis and therapy. Hum Immunol 74: the inflammatory proteins COX-2/PGE2. Arab J Chem 13:5639–
1057–1060. https://doi.org/10.1016/j.humimm.2013.06.029 5650. https://doi.org/10.1016/j.arabjc.2020.04.005