319

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 70: 319-331, January-December 2022 (Published May 18, 2022)

Amoebicidal and trichomonicidal capacity of polymeric nanoparticles

loaded with extracts of the plants Curcuma longa (Zingiberaceae)

and Berberis vulgaris (Berberidaceae)

Alejandra Pacheco-Ordaz1; https://orcid.org/0000-0002-4144-5685

Eduardo Sánchez-García1; https://orcid.org/0000-0001-5751-9848

Ramiro Quintanilla-Licea1; https://orcid.org/0000-0002-4379-6913

Aldo Fabio Bazaldúa-Rodríguez1; https://orcid.org/0000-0003-2913-1437

Raymundo Alejandro Pérez-Hernández1; https://orcid.org/0000-0002-3981-8737

Magda Elizabeth Hernández-García1; https://orcid.org/0000-0003-4532-2604

Juan Gabriel Báez-González2; https://orcid.org/0000-0003-0509-4678

Rocío Castro-Ríos3; https://orcid.org/0000-0001-8753-9065

Joel Horacio Elizondo-Luévano*1; https://orcid.org/0000-0003-2954-5939

Abelardo Chávez-Montes*1; https://orcid.org/0000-0002-3948-4247

1. Department of Chemistry, Faculty of Biological Sciences, Autonomous University of Nuevo León (U.A.N.L.).

Ciudad Universitaria. C.P. 66455, San Nicolás de los Garza, Nuevo León, México;

alejandra.pachecord@uanl.edu.mx, eduardo.sanchezgrc@uanl.edu.mx, ramiro.quintanillalc@uanl.edu.mx,

aldo.bazalduarg@uanl.edu.mx, raymundo.perezhrz@uanl.edu.mx, magda.hernandezgr@uanl.edu.mx,

joel.elizondolv@uanl.edu.mx (*Correspondence), abelardo.chavezmn@uanl.edu.mx (*Correspondence)

2. Department of Food Science and Technology, Faculty of Biological Sciences, Autonomous University of Nuevo

León (U.A.N.L.). Ciudad Universitaria. C.P. 66455, San Nicolás de los Garza, Nuevo León, México;

juan.baezgn@uanl.edu.mx

3. Department of Analytical Chemistry, Faculty of Medicine, Autonomous University of Nuevo León (U.A.N.L.). C.P.

64460, Monterrey, Nuevo León, México; rocio.castrors@uanl.edu.mx

Received 04-V-2021. Corrected 11-I-2022. Accepted 17-V-2022.

ABSTRACT

Introduction: Pathogenic protozoans, like Entamoeba histolytica and Trichomonas vaginalis, represent a major

health problem in tropical countries; and polymeric nanoparticles could be used to apply plant extracts against

those parasites.

Objective: To test Curcuma longa ethanolic extract and Berberis vulgaris methanolic extracts, and their main

constituents, against two species of protozoans.

Methods: We tested the extracts, as well as their main constituents, curcumin (Cur) and berberine (Ber), both

non-encapsulated and encapsulated in polymeric nanoparticles (NPs), in vitro. We also determined nanoparticle

characteristics by photon correlation spectroscopy and scanning electron microscopy, and hemolytic capacity by

hemolysis in healthy erythrocytes.

Results: C. longa consisted mainly of tannins, phenols, and flavonoids; and B. vulgaris in alkaloids.

Encapsulated particles were more effective (P < 0.001); however, curcumin and berberine nanoparticles were the

most effective treatments. CurNPs had IC50 values (μg/mL) of 9.48 and 4.25, against E. histolytica and T. vagi-

nalis, respectively, and BerNPs 0.24 and 0.71. The particle size and encapsulation percentage for CurNPs and

https://doi.org/10.15517/rev.biol.trop..v70i1.48746

BIOMEDICINE

320 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 319-331, January-December 2022 (Published May 18, 2022)

Parasitosis is a problem that affects a huge

number of the human population; likewise,

their transcendence is linked to secondary

diseases, there are different types of parasites

that affect humans, which are closely linked to

poverty, socio-geographic and the lack of fund-

ing for their care (Theel & Pritt, 2016). Among

the most common parasitosis are amebiasis

caused mainly by Entamoeba histolytica and

trichomoniasis caused by Trichomonas vagi-

nalis. E. histolytica is a human pathogen that

mainly colonizes the large intestine through

fecal-oral contamination and sexual transmis-

sion through anal-oral contact has also been

described (Theel & Pritt, 2016). Its prevalence

increases in geographical areas with inadequate

environmental sanitation, as occurs in develop-

ing countries and it is estimated that 10 % of

the world’s population is infected with approxi-

mately 100 000 deaths per year (Turkeltaub et

al., 2015). There are 500 million new infections

per year, of which 50 million suffer from inva-

sive amebiasis, meaning that the manifestation

is intestinal and extraintestinal (Kantor et al.,

2018). T. vaginalis is one of the most prevalent

non-viral sexually-transmitted pathogens in

the world; in 2016, World Health Organization

(WHO) estimated 156 million cases of tricho-

moniasis around the world (WHO, 2018). After

colonization, this parasite causes vaginitis, ure-

thritis, and prostatitis (Cudmore et al., 2004).

Also, the pathogen has been associated with

other serious consequences such as adverse

pregnancy outcomes and preterm delivery,

infertility, predisposition to cervical cancer,

and pelvic inflammatory disease. Evidence has

demonstrated the potential of T. vaginalis as a

cofactor for increased HIV transmission (Wen-

del & Workowski, 2007).

For decades, the treatment for parasitic

protozoal infections has been metronidazole

among other nitroimidazoles, however, it is

now well established that extensive use of this

mono-therapeutic treatment regimen fosters

drug resistance, leading to unsuccessful treat-

ment and relapse of the disease (Dingsdag &

Hunter, 2018). Resistance to metronidazole

has been observed in T. vaginalis and has been

suggested in E. histolytica due to the increased

frequency of unsuccessful treatments, like-

wise, the use of this drug frequently presents

side effects that force the patient to abandon

the treatment, due to this, the need to identify

new compounds as antiparasitic drugs, natural

sources can be an alternative for the treatment

of parasitic infections (Davoodi & Abbasi-

Maleki, 2018; Elizondo-Luévano et al., 2021c).

Plants represent an important source and

diversity of biomolecules with unique proper-

ties and many of these compounds are attrac-

tive candidates for the development of new

antiparasitic agents in addition some plant

extracts have shown interesting antiparasitic

properties without the serious side effects of

cytotoxic agents (Elizondo-Luevano et al.,

2020a; Molina-Garza et al., 2014). Among

the most relevant plants are Curcuma longa

(Zingiberaceae), a tuberous herbaceous peren-

nial plant member of the ginger family that

grows in tropical climates (Prasad et al., 2014),

whose main ingredient is the lipophilic poly-

phenolic substance curcumin (Cur), which is

a valuable compound in the food and pharma-

ceutical industries due to its wide range of ben-

eficial health effects with mainly antioxidant,

antimicrobial, antiparasitic, anti-inflammatory,

and antiangiogenic properties (Cárdenas Garza

et al., 2021; Kocaadam & Şanlier, 2017; Kumar

et al., 2018). Berberis vulgaris (Berberidaceae)

is another plant that is currently relevant due to

its various health-promoting properties (Raju

et al., 2019). The isoquinoline alkaloid, i.e.

BerNPs were 66.5 and 73.4 nm, and 83.59 and 76.48 %, respectively. The NPs were spherical and significantly

reduced hemolysis when compared to non-encapsulated extracts.

Conclusions: NPs represent a useful and novel bioactive compound delivery system for therapy in diseases

caused by protozoans.

Key words: antiparasitic activity; berberine; curcumin; drug discovery; nanoparticles; natural products.

321

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 70: 319-331, January-December 2022 (Published May 18, 2022)

berberine (Ber), is the main compound of B.

vulgaris, whose pharmacological properties

include anticonvulsant, antidepressant, anti-

arrhythmic, anti-inflammatory, antiparasitic,

antiviral, antibacterial, antineoplastic and anti-

diabetic (Verma & Sharma, 2018). In general,

the application of natural products can be prob-

lematic because they can be deteriorated by

different factors such as light, pH, oxygen, and

enzymes (Barrera-Ruiz et al., 2020). In addi-

tion to presenting low solubility in water as in

the case of Cur and Ber (Fig. 1), which hinders

their bioavailability, these characteristics limit

their potential pharmaceutical uses (Mehra et

al., 2016). Therefore, to overcome those effects

nano-encapsulation into polymeric nanopar-

ticles (NPs) has become an alternative (Pina-

Barrera et al., 2019).

Among the nano-carriers used, the syn-

thetic polymers Eudragit® (polymethacrylates

by Evonik Industries AG, Germany) are widely

used for the preparation of oral drug deliv-

ery systems designed for controlled-release

and delivery of drugs to specific sites in the

gastrointestinal tract distinguished by their

versatility and ease of handling (Chong-Cerda

et al., 2020). Particularly, the polymer used

in this research was the pH-sensitive cationic

polymer Eudragit® EPO (cationic terpolymer

of N,N-dimethyl amino ethyl methacrylate

with methylmethacrylate and butylmethacry-

late), which has been used for the preparation

of hydrophobic drug dispersions that have

a dissolution rate-dependent bioavailability

(Khachane et al., 2011).

In order to introduce a novel strategy for

the administration of natural products such

as extracts and metabolites. In this study, the

activity of the extracts of C. longa (ExtCl), B.

vulgaris (ExtBv), and the main components

Ber and Cur in free form as well as encap-

sulated into NPs against trophozoites of E.

histolytica and T. vaginalis was investigated in

in vitro assays.

MATERIALS AND METHODS

2.1. Chemicals: Berberine (Ber), curcum-

in (Cur), absolute ethanol (EtOH), absolute

methanol (MeOH), ethylenediaminetetraacetic

acid (EDTA), and phosphate buffer solution

were purchased from Sigma-Aldrich (Merck

KGaA, Germany). Decomplemented Adult

Bovine Serum (DABS) was donated by the

Center for Biomedical Research of the North-

east (C.B.R.N.) of the Instituto Mexicano del

Seguro Social (I.M.S.S.) Monterrey, México.

Polymer Eudragit® EPO was donated by Helm

de México S.A. All chemicals and solvents were

of analytical grade.

Fig. 1. Chemical structures, molecular formula (MF), molecular weight (MW), CAS number (CAS No.), and PubChem

compound identifier (PubChem CID) of Ber: Berberine and Cur: Curcumin.

322 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 319-331, January-December 2022 (Published May 18, 2022)

2.2. Ethicals: The hemolysis test was

performed using human erythrocytes, under

the approval of the ethics committee of the

U.A.N.L. Under the Official Mexican Technical

Standard: NOM-253-SSA1-2012.

2.3. Plant materials and extraction: Plant

materials, i.e., C. longa rhizome (SKU: 205400-

54) and B. vulgaris roots (SKU: 209142-54)

originated from India were purchased in pow-

dered form from Starwest Botanicals (Sacra-

mento, CA, United States). The extraction of C.

longa was performed with EtOH and the extrac-

tion of B. vulgaris was with MeOH. The extrac-

tions were performed with 100 g of each plant

material, which were deposited in a 1 000 mL

Erlenmeyer flask, in which 400 mL of solvent

were added. The flasks were hermetically sealed

to avoid evaporation of the solvent, kept at room

temperature for 24 hr and in constant agita-

tion away from light. After the time required

for extraction, the solvent was separated from

the rest of the plant material by filtration with

Whatman No.1 paper (Merck® KGaA, Ger-

many), then the extracts were concentrated in a

rotary evaporator system (Heidolph Instruments

GmbH & CO. KG, Germany) at a temperature

of 30 °C and 100 rpm. The extraction yield (%

Y) was calculated with the following formula:

2.4. Phytochemical tests: The following

phytochemical tests were performed to identify

the functional groups of each extract: Unsatura-

tions, carbonyl group, sesquiterpene lactones,

lactones, tannins, triterpenes-sterols, phenols,

saponins, Shinoda (flavonoids), carbohydrates,

and Dragendorff (alkaloids).

2.5. Strains: The E. histolytica HM1:IMSS

and T. vaginalis GT15 strains were provided by

C.B.R.N - I.M.S.S. and were grown and main-

tained in vitro in TYI-S-33 culture medium

supplemented with 10 % DABS at 37 °C.

2.6. Preparation of polymer nanopar-

ticles: The NPs was prepared by the

nanoprecipitation technique (Fessi et al., 1989).

For that purpose, 50 mg of Eudragit® EPO

polymer plus 5 mg of the extracts or main

components were dissolved in 15 mL of EtOH,

which was constantly injected in an aqueous

phase (10 mL of bidistilled water), and then the

solvent was removed by a rotary evaporator

system (100 rpm / 30 ºC) to obtain an aqueous

suspension of NPs. The particle size and poly-

dispersity index (PDI) of the suspension were

determined by photon correlation spectroscopy.

An aliquot of the suspension was mixed with

bi-distilled water and read in a Zetasizer Nano

ZS (Malvern Instruments, United Kingdom)

at 25 °C to determine its average particle size

and PDI. The size of the nanoparticles was

confirmed by scanning electron microscopy

(SEM), where the conforming nanostructure

and the degree of aggregation of the NPs were

determined. Using the methodology previously

described by Chong-Cerda et al., the percent-

age of extract or active trapped inside and

outside the NPs was estimated (Chong-Cerda

et al., 2020), for which the formulations were

ultracentrifuged (Beckman-Coulter. United

States. Allegra 64R Benchtop Centrifuge) at

20 000 rpm (4 °C / 3 hr). The encapsulation

efficiency (% EE) was determined by the fol-

lowing formula:

Where EA corresponds to the entrapped amount

into the NPs, and TA corresponds to the total

amount of the extract or active ingredient.

2.7. Biological assays: The in vitro anti-

protozoal activity of the extracts and the main

components in free form and encapsulated into

NPs was based on the methodology described

by Elizondo et. al. (Elizondo-Luévano et al.,

2018; Elizondo-Luévano et al., 2021c), in which

they were tested against log-phase cultures of

E. histolytica (20 000 trophozoites/mL), and T.

vaginalis (100 000 trophozoites/mL) in TYI-S-

33 culture medium with 10 % DABS at 37 °C.

The concentrations of all treatments were evalu-

ated at different concentrations to determine the

323

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 70: 319-331, January-December 2022 (Published May 18, 2022)

IC50 of each treatment, respectively. The posi-

tive control consisted of metronidazole (refer-

ence drug) and only culture medium as negative

control. IC50 was determined by the Probit test

with a 95 % confidence interval.

2.8. Hemolysis test: Hemolysis was evalu-

ated based on the hemolytic activity in erythro-

cytes (Elizondo-Luevano et al., 2020a). For this,

100 μL of the erythrocyte solution was added to

a 96-well microplate after 30 min incubation,

treated with 100 μL of the treatments (extracts

and main components in free and nano-encap-

sulated form) diluted in PBS at a concentration

range of 10 to 100 μg/mL. Distilled water was

used as a positive control for hemolysis (C+)

and untreated culture medium as a negative

control (C-). The microplate was read at 540

nm in a microplate spectrophotometer (Thermo

Fisher Scientific Inc., United States. Multiskan

SkyHigh Spectrophotometer). The absorbance

of the untreated erythrocyte suspension was

used as blank. Percentage of hemolysis was

calculated using the next formula:

2.9. Statistical analysis: Results expressed

as the mean and standard error (SEM). Sig-

nificant differences between groups were

determined by the one-way ANOVA test, the

fifty percent inhibitory concentration (IC50)

was determined by the Probit test. The post-

hoc Tukey´s test was performed to determine

any statistical differences between treated and

untreated controls. Student’s t-test was used to

determine the differences between encapsulated

and non-encapsulated treatments. Differences

were significant at P < 0.05 with a 95 % con-

fidence interval using the IBM-SPSS (IBM

Corp, 2013). All experiments were performed

in triplicate.

RESULTS

3.1. Phytochemical analysis: Phytochem-

ical analysis indicates the presence of tannins,

phenols, and flavonoids in the extract of C.

longa and alkaloids in B. vulgaris. The extract

yields of C. longa and B. vulgaris were 8.86

and 2.33 %, respectively.

3.2. In vitro antiprotozoal activity: All

the treatments tested were biologically active

analyzed by the Probit test (Table 1). The

nanoencapsulated treatments were significantly

(P < 0.001) more effective than the non-

encapsulated treatments, it was observed that

ExtBv was more effective than ExtCl either in

free form or incorporated into NPs. However,

by Tukey’s test, it was determined that BerNPs

were the most effective treatment compared to

the reference drug metronidazole showing IC50

TABLE 1

Antiprotozoal activity of C. longa and B. vulgaris extracts

as well as curcumin and berberine in non-encapsulated

form and encapsulated into NPs compared to the effects

of the reference drug

IC50 (μg/mL)

Treatment E. histolytica T. vaginalis

Metronidazole

(Reference drug) 0.13 ± 0.03a0.07 ± 0.01a

ExtCl 361.43 ± 8.07h496.36 ± 10.52g

ExtClNPs 26.03 ± 2.31e102.29 ± 13.06e

P t-student (F=12.71) 0.009 0.025

ExtBv 134.19 ± 11.20g192.08 ± 8.04f

ExtBvNPs 19.02 ± 1.16d46.30 ± 5.51d

P t-student (F=12.71) 0.042 0.034

Cur 65.60 ± 5.17f51.11 ± 3.55d

CurNPs 9.48 ± 1.23c4.25 ± 1.04c

P t-student (F=12.71) 0.037 0.002

Ber 3.28 ± 0.51b2.60 ± 1.12c

BerNPs 0.24 ± 0.10a0.71 ± 0.22b

P t-student (F=12.71) 0.004 0.003

P Anova (F = 27.91) < 0.001 < 0.001

Values are shown as mean ± SD. Different letters within

the same column are significantly different analyzed via the

post-hoc Tukey’s test (P < 0.05).

324 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 319-331, January-December 2022 (Published May 18, 2022)

values of 0.24 and 0.71 μg/mL respectively

against E. histolytica and T. vaginalis.

3.3. Characterization of polymeric

nanoparticles: Fig. 2 shows the mean particle

size and morphology of each of the formula-

tions, which were obtained with the previously

mentioned parameters. A significant change

in size was observed for each of the formula-

tions tested and an increase in size (BerNPs >

CurNPs > ExtBvNPs > ExtClNPs > BlankNPs)

was observed when pure metabolites were

encapsulated. Also, the determination of % EE

was carried out, which was 34.81, 25.94, 83.59,

and 76.48 for ExtClNPs, ExtBvNPs, CurNPs,

and BerNPs, respectively (Table 2).

3.4. Hemolytic activity test: The hemo-

lytic activity assay in human erythrocytes

showed that none of the treatments (ExtCl,

ExtBv, Cur, and Ber in free or nano-encapsu-

lated form) were significantly (P < 0.001) less

hemolytic than the positive control (C+) which

showed 100 % hemolysis. The encapsulated

Fig. 2. Morphology and size of the NPs by SEM at 1.0 kV. Their spherical structures are shown. A: NPsBlank, B.1:

NPsExtCl, B.2: NPsExtBv, C.1: NPsCur, and C.2: NPsBer.

325

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 70: 319-331, January-December 2022 (Published May 18, 2022)

treatments were less hemolytic than the non-

encapsulated ones, and none of the treatments

(non-encapsulated or NPs) showed hemolysis

greater than 10 % even at 100 mg/mL (Table 3).

DISCUSSION

Currently, some reports are indicating the

antiparasitic activity of plant extracts like C.

longa, Elettaria cardamomum, and Kalanchoe

daigremontiana as well as their components

such as alkaloids, essential oils, and flavonoids

among other phytochemicals (Cárdenas Garza

et al., 2021; D’Andrea, 2015; Elizondo-Luéva-

no et al., 2021a; Rosmalena et al., 2019). The

activity of these compounds is well document-

ed against a wide range of diseases such as par-

asitosis (Elizondo-Luévano et al., 2020). These

products have also been shown to possess

fewer side effects in humans. As an alterna-

tive, several medicinal plant extracts had been

investigated to develop a new drug for human

parasites (Castañeda et al., 2021; Pérez et al.,

2017). E. histolytica and T. vaginalis have been

commonly treated with metronidazole, despite

being an effective molecule, increased treat-

ment failure has been observed in these patho-

gens due to increased resistance, which is a

cause for concern (Jarrad et al., 2016). Another

option for the treatment of metronidazole-resis-

tant strains is a longer treatment or with higher

doses, although this drug in most cases is well-

tolerated, it can cause severe side effects, such

as mutagenesis and carcinogenesis (Kuriyama

et al., 2011). Hence the importance of finding

alternatives for the treatment of amoebiasis and

trichomoniasis. For this, two research strategies

were proposed. The first was the search for new

active principles and the second was the search

for new ways of administration and delivery

systems such as NPs.

Previous studies on the chemical composi-

tion of C. longa and B. vulgaris have indicated

TABLE 2

Size, polydispersity index (PDI) and percentage

encapsulation (% EE) of the polymeric nanoparticles

(NPs) of C. longa and B. vulgaris extracts, and their main

components curcumin and berberine

Formulation Size (nm) PDI % EE

BlankNPs 45.9a0.114b-

ExtClNPs 53.6b0.177d34.81b

ExtBvNPs 54.4b0.190e25.94a

CurNPs 66.5c0.133c83.59d

BerNPs 73.4d0.086a76.48c

P Anova (F = 5.28) < 0.05 < 0.01 < 0.01

Different letters within the same column are significantly

different analyzed via the post-hoc Tukey’s test (P < 0.05).

TABLE 3

Evaluation of the hemolysis test of extracts of C. longa, B. vulgaris, and their main components curcumin and berberine

in unencapsulated and encapsulated form in polymeric nanoparticles (NPs)

Positive control (C+) 100 ± 3.8 Negative control (C-) 1.19 ± 0.3

Hemolysis percentage (%)

μg/mL ExtCl ExtCl NPs ExtBv ExtBv NPs Cur Cur NPs Ber Ber NPs

10 0.49 ± 0.1*1.14 ± 0.4*1.23 ± 0.1 0.10 ± 0.0*0.13 ± 0.1*0.05 ± 0.0*1.43 ± 0.6 1.09 ± 0.2*

20 0.72 ± 0.1*1.19 ± 0.2 1.25 ± 0.2 0.12 ± 0.1*1.01 ± 0.4*0.05 ± 0.0*1.88 ± 0.3 1.12 ± 0.3*

40 2.19 ± 0.3 1.52 ± 0.4 2.81 ± 0.7 1.16 ± 0.1*1.17 ± 0.2 0.40 ± 0.1*2.29 ± 0.7 1.13 ± 0.1*

60 2.43 ± 0.4 1.98 ± 0.1 2.95 ± 0.3 1.38 ± 0.1 1.26 ± 0.3 0.54 ± 0.1*4.13 ± 0.5 2.10 ± 0.2

80 3.64 ± 0.3 1.99 ± 0.3 5.65 ± 0.8 2.02 ± 0.2 1.33 ± 0.2 0.70 ± 0.1*7.03 ± 0.7 2.50 ± 0.3

100 3.79 ± 0.7 1.99 ± 0.6 6.13 ± 0.9 2.04 ± 0.2 1.51 ± 0.3 0.76 ± 0.1*7.12 ± 0.7 2.77 ± 0.5

P t-student (F=2.57) 0.011 0.003 0.019 0.016

Results are given in percentage (%) of hemolysis. The values are shown as the mean ± SD. *Significant differences in

comparison with the negative control (C-) (P < 0.05).

326 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 319-331, January-December 2022 (Published May 18, 2022)

that the most important active constituents are

Cur and Ber respectively, which have docu-

mented antioxidant, anticancer, antibacterial,

antiparasitic, and other effects (Balakrishna &

Kumar, 2015; Imenshahidi & Hosseinzadeh,

2016; Karłowicz-Bodalska et al., 2017; Vaughn

et al., 2016). Cur, an active compound pres-

ent in the rhizome of the C. longa, has been

found to have in vitro antiprotozoal activity

against Leishmania spp. malaria, G. lamblia, T.

vaginalis, and Schistosoma mansoni (Elizondo-

Luévano et al., 2020; Magalhães et al., 2009).

Ber, an active compound present in B. vulgaris,

it is considered to have antiparasitic potential

against E. histolytica, G. lamblia, T. vagina-

lis, Leishmania spp., and anthelmintic against

Strongyloides venezuelensis, Schistosoma man-

soni, and Echinococcus granulosus (Elizondo-

Luévano et al., 2021a; Elizondo-Luévano et al.,

2021b; Mahmoudvand et al., 2014; Wright et

al., 2000). Although Cur and Ber are the natural

products with good antiprotozoal activity, these

are the molecules with very low bioavailability,

which limits their use; therefore, NPs seem to

be an effective route of administration. For this

purpose, firstly, the extracts of the rhizome

of C. longa and the root of B. vulgaris were

obtained, the yields obtained were 8.86 and

2.33 %, respectively. We observed that our

results coincide with what was expected since

in the case of C. longa considering the phenols

and flavonoids, and for B. vulgaris, the alka-

loids were the compounds that stood out for

their presence.

By Probit statistical analysis, the IC50 of

each treatment evaluated against E. histolytica

and T. vaginalis in comparison to the refer-

ence drug was calculated (Table 2). For the C.

longa treatments, the IC50 of the crude extract

was 361.43 (E. histolytica) and 496.36 (T.

vaginalis) μg/mL, and for its incorporation into

NPs, decreased to 26.03 (E. histolytica) and

102.29 (T. vaginalis) μg/mL. The IC50 deter-

mined evaluating the extract of B. vulgaris,

were 134.19 (E. histolytica) and 192.08 (T.

vaginalis) μg/mL, once encapsulated the NPs

presented IC50 of 19.02 (E. histolytica) and

46.30 (T. vaginalis) μg/mL. It was possible to

observe the higher activity of ExtBv compared

to ExtCl, however, when the extracts were

incorporated into NPs, the IC50 decreased sig-

nificantly (P < 0.001) in all cases. Therefore, it

was found that both C. longa and B. vulgaris

extracts presented more effective antiprotozoal

activity in nano-encapsulated form.

Nanoparticles have been considered pow-

erful tools for the treatment of infections due to

the possibility of being endocytosed by cells,

which could benefit the release of the active

in the case of intracellular infections and due

to the possibility of controlling and prolong-

ing the release of the encapsulated molecules,

nanoparticles could be very useful in the pre-

vention or treatment of infections (Sasidharan

& Saudagar, 2020). Such is the case of CurNPs

and BerNPs, which were shown to be more

effective than their non-encapsulated counter-

parts (Table 2) with IC50 of 9.48 (E. histolytica)

and 4.25 (T. vaginalis) μg/mL for CurNPs, and

0.24 (E. histolytica) and 0.71 (T. vaginalis)

μg/mL when BerNPs were evaluated, being

far superior compared to Cur and Ber in non-

encapsulated form. The most effective treat-

ment was BerNPs behaving significantly like

metronidazole. It was also determined that Cur

was more effective than ExtClNPs against T.

vaginalis, and Ber was more effective than Ext-

BvNPs when evaluated against both parasites.

Ber is present in a wide variety of medicinal

plants such as A. mexicana, B. aristata, and B.

vulgaris and possesses a variety of pharmaco-

logical properties (Raju et al., 2019). There is

also evidence that Ber is active against filarial,

protozoa, and helminths (Elizondo-Luevano

et al., 2020b; Elizondo-Luévano et al., 2018;

Mahmoudvand et al., 2014; Rana & Misra-

Bhattacharya, 2013).

There are no previous reports in the lit-

erature concerning the use of C. longa and

B. vulgaris NPs or Cur and Ber NPs for the

treatment of amoebiasis and trichomoniasis.

However, there are studies where nanoparticles

have been used as drug carriers to treat parasit-

osis in in vivo models, where efficient uptake,

sustained release and enhanced therapeutic

effects were observed. This is due to the size of

327

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 70: 319-331, January-December 2022 (Published May 18, 2022)

the NPs, which results in a high concentration

with its payload at the site of the medication

(Sun et al., 2019). The activity of NPs opens

the possibility to the combination of drugs

that promote synergistic action or minimize

the possibility of resistance of certain strains

to drugs (Panic et al., 2014). There are other

kinds of nanoparticles, such as silver nanopar-

ticles with the aqueous extract of C. longa

(Shabanzadeh et al., 2013), and nanometric

complexes using ß-cyclodextrin incorporating

the extract of B. vulgaris however, there are

few studies on the incorporation of natural

extracts into NPs (Hadaruga et al., 2010). In

this work, we were able to produce NPs incor-

porating the crude extracts of C. longa and B.

vulgaris independently, as well as Cur and Ber,

using the cationic copolymer Eudragit® EPO

of pharmaceutical-grade approved, it provides

greater purity, stability, and reproducibility to

the formulations, compared to natural polymers

(Khoee & Yaghoobian, 2009). The character-

ization included the determination of the mean

particle size, PDI, particle morphology, and the

entrapment efficiency of the actives.

A significant difference (P < 0.05) was

determined between the NPs with the extracts,

the NPs with the actives, and the blank (Table

3). BlankNPs had a size and PDI of 45.9 nm

and 0.114, respectively. The NPs containing C.

longa extract had a size of 53.6 nm with a PDI

of 0.177, those containing B. vulgaris extract

presented size of 54.4 nm and a PDI of 0.190,

while the NPs with Cur and Ber presented sizes

of 66.5 and 73.4 nm as well as PDIs of 0.133

and 0.086, respectively. The sizes increase

when the pure actives were nano-encapsulated,

however, the PDIs decrease in comparison with

the nano-encapsulated extracts. This indicates

as the sizes of the NPs increased the homo-

geneity of the nano-formulations increased

(Lancheros et al., 2014). These results were

reproducible and meet the objective of having

a particle size less than 200 nm and PDI less

than 0.2. We sought to obtain a size smaller

than 200 nm, since the smaller the particle size,

the greater the surface interaction area, and this

size allows sterilization by filtration (Banerjee

et al., 2016). SEM confirmed the hemispheri-

cal morphology and smooth surface (Fig. 2). In

the nanoparticulate systems, 34.81 (ExtClNPs),

25.94 (ExtBvNPs), 83.59 (CurNPs) and 76.48

(BerNPs) % EE were obtained (Table 3). A

significant increase (P < 0.01) is noted in the

encapsulation of Cur and Ber compared to

crude extracts. Previous studies have reported

entrapment efficiencies ranging from 74 to 98.5

% encapsulating pure actives such as curcumin,

quercetin, piperine, and silybin by nanoprecipi-

tation method (Abolhassani et al., 2020).

The investigation of the action of natural or

synthetic products on erythrocytes is important

to determine any hemolytic activity because it

is an indicator of general cytotoxicity and bio-

activity, this type of in vitro test is commonly

employed for cyto-toxicological evaluations

(Majidzadeh et al., 2020). In the present study,

we tested concentrations between 10-100 μg/

mL and none of the treatments (nano-encap-

sulated and not) were significantly (P < 0.05)

hemolytic compared to the positive control

which had 100 % of hemolytic activity (Table

3). In the hemolysis bioassay, we observed

that the low hemolytic activity depends on the

concentration of each treatment (hemolysis was

concentration-dependent), so the dose is direct-

ly proportional to the hemolytic damage. How-

ever, when we evaluated the treatments based

on nanoparticles, it was possible to observe a

significant reduction in the nanoencapsulated

treatments, this may be because the Eudragit®

polymers are innocuous, and the low hemolytic

activity is due to the release of the active ingre-

dients or extracts to the medium where they

are, or by mechanical stress during centrifuga-

tion (Ofridam et al., 2021). As can be seen in

graph 3, Ber turned out to be the most active

treatment presenting average hemolysis of 7.21

% (not encapsulated) and 2.77 % (NPs), when

evaluated at 100 ppm, however, these results

are below the data presented in Table 2 where

we determined the IC50 values, where for the

case of BerNPs it was 0.24 (E. histolytica) and

0.71 (T. vaginalis) μg/mL, respectively. Con-

sequently, the use of NPs opens the possibility

of new and better ways of administering active

328 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 319-331, January-December 2022 (Published May 18, 2022)

ingredients with biological activity (Meerasa et

al., 2012; Raju et al., 2019). Since the NPs did

not prove to be haemolytic, their evaluation can

be considered for evaluation purposes in an in

vivo model.

In this work, extracts of the rhizome of

C. longa and roots of B. vulgaris, as well as

their main components, i.e., curcumin, and

berberine were successfully nano-encapsulat-

ed by the nanoprecipitation technique. NPs

showed increased antiprotozoal activity against

T. vaginalis and E. histolytica compared to

non-encapsulated treatments giving a guideline

to their potential use as adjuvants in the release

of natural products for the treatment of parasit-

osis. None of the NPs-based treatments showed

in vitro cytotoxicity on human erythrocytes.

However, the mechanisms of action and the in

vivo efficacy of Cur and Ber-loaded NPs need

to be evaluated in an animal-infected model.

Ethical statement: the authors declare

that they all agree with this publication and

made significant contributions; that there is no

conflict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are

fully and clearly stated in the acknowledge-

ments section. A signed document has been

filed in the journal archives.

ACKNOWLEDGMENTS

A special thanks to Oscar Alberto Pérez-

Narváez and David Gilberto García-Hernández

for their technical assistance. This research was

funded by the National Council of Science and

Technology (CONACYT), grants CB176853

and CB418935.

RESUMEN

Capacidad amebicida y tricomonicida de

nanopartículas poliméricas cargadas con extractos

de las plantas Curcuma longa (Zingiberaceae)

y Berberis vulgaris (Berberidaceae)

Introducción: Los protozoos patógenos, como Entamoe-

ba histolytica y Trichomonas vaginalis, representan un

importante problema de salud en los países tropicales; y

se podrían usar nanopartículas poliméricas para aplicar

extractos de plantas contra esos parásitos.

Objetivo: Probar los extractos etanólicos de Curcuma

longa y Berberis vulgaris, y sus principales constituyentes,

contra dos especies de protozoos.

Métodos: Probamos los extractos, así como sus principales

constituyentes, curcumina (Cur) y berberina (Ber), tanto

no encapsulados como encapsulados en nanopartículas

poliméricas (NPs), in vitro. También determinamos las

características de las nanopartículas por espectroscopía

de correlación de fotones y microscopía electrónica de

barrido, y la capacidad hemolítica por hemólisis en eri-

trocitos sanos.

Resultados: C. longa tenía principalmente: taninos, feno-

les y flavonoides; y B. vulgaris, alcaloides. Las partículas

encapsuladas fueron más efectivas (P < 0.001); sin embar-

go, las nanopartículas de curcumina y berberina fueron

los tratamientos más efectivos. CurNPs tenía valores IC50

(μg/mL) de 9.48 y 4.25, contra E. histolytica y T. vagina-

lis, respectivamente, y BerNPs 0.24 y 0.71. El tamaño de

partícula y el porcentaje de encapsulación para CurNPs

y BerNPs fueron: 66.5 y 73.4 nm, y 83.59 y 76.48 %,

respectivamente. Los NP son esféricos y redujeron signifi-

cativamente la hemólisis en comparación con los extractos

no encapsulados.

Conclusiones: Las NP representan un sistema de adminis-

tración de compuestos bioactivos útil y novedoso para la

terapia enfermedades causadas por protozoos.

Palabras clave: actividad antiparasitaria; berberina; cur-

cumina; descubrimiento de fármacos; nanopartículas; pro-

ductos naturales.

REFERENCES

Abolhassani, H., Safavi, M. S., Handali, S., Nosrati, M.,

& Shojaosadati, S. A. (2020). Synergistic Effect of

Self-Assembled Curcumin and Piperine Co-Loaded

Human Serum Albumin Nanoparticles on Suppres-

sing Cancer Cells. Drug Development and Industrial

Pharmacy, 46(10), 1647–1655. https://doi.org/10.108

0/03639045.2020.1820032

Balakrishna, A., & Kumar, M. H. (2015). Evaluation

of Synergetic Anticancer Activity of Berberine

and Curcumin on Different Models of A549, Hep-

G2, MCF-7, Jurkat, and K562 Cell Lines. BioMed

Research International, 2015, 354614. https://doi.

org/10.1155/2015/354614

Banerjee, A., Qi, J., Gogoi, R., Wong, J., & Mitragotri, S.

(2016). Role of nanoparticle size, shape and surface

chemistry in oral drug delivery. Journal of Controlled

Release: Official Journal of the Controlled Relea-

se Society, 238, 176–185. https://doi.org/10.1016/j.

jconrel.2016.07.051

329

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 70: 319-331, January-December 2022 (Published May 18, 2022)

Barrera-Ruiz, D. G., Cuestas-Rosas, G. C., Sánchez-Mari-

ñez, R. I., Álvarez-Ainza, M. L., Moreno-Ibarra, G.

M., López-Meneses, A. K., Plascencia-Jatomea, M.,

& Cortez-Rocha, M. O. (2020). Antibacterial activity

of essential oils encapsulated in chitosan nanoparti-

cles. Food Science and Technology, 40(12), 568–573.

https://doi.org/10.1590/fst.34519

Cárdenas Garza, G. R., Elizondo Luévano, J. H., Bazaldúa

Rodríguez, A. F., Chávez Montes, A., Pérez Hernán-

dez, R. A., Martínez Delgado, A. J., López Villareal,

S. M., Rodríguez Rodríguez, J., Sánchez Casas,

R., Castillo Velázquez, U., & Rodríguez Luis, O.

E. (2021). Benefits of Cardamom (Elettaria carda-

momum (L.) Maton) and Turmeric (Curcuma longa

L.) Extracts for Their Applications as Natural Anti-

Inflammatory Adjuvants. Plants, 10(9), 1908. https://

doi.org/10.3390/plants10091908

Castañeda, J. S., Suta-Velásquez, M., Mateus, J., Pardo-

Rodriguez, D., Puerta, C. J., Cuéllar, A., Robles, J.,

& Cuervo, C. (2021). Preliminary chemical characte-

rization of ethanolic extracts from Colombian plants

with promising anti - Trypanosoma cruzi activity.

Experimental Parasitology, 223, 108079. https://doi.

org/10.1016/j.exppara.2021.108079

Chong-Cerda, R., Levin, L., Castro-Ríos, R., Hernández-

Luna, C. E., González-Horta, A., Gutiérrez-Soto,

G., & Chávez-Montes, A. (2020). Nanoencapsulated

Laccases Obtained by Double-Emulsion Technique.

Effects on Enzyme Activity pH-Dependence and Sta-

bility. Catalysts, 10(9), 1085. https://doi.org/10.3390/

catal10091085

Cudmore, S. L., Delgaty, K. L., Hayward-McClelland, S.

F., Petrin, D. P., & Garber, G. E. (2004). Treatment

of infections caused by metronidazole-resistant

Trichomonas vaginalis. Clinical Microbiology

Reviews, 17(4), 783–793. https://doi.org/10.1128/

CMR.17.4.783-793.2004

D’Andrea, G. (2015). Quercetin: A flavonol with multi-

faceted therapeutic applications? Fitoterapia, 106,

256–271. https://doi.org/10.1016/j.fitote.2015.09.018

Davoodi, J., & Abbasi-Maleki, S. (2018). Effect of Ori-

ganum vulgare hydroalcoholic extract on Giardia

lamblia cysts compared with metronidazole in vitro.

Iranian Journal of Parasitology, 13(3), 486–492.

Dingsdag, S. A., & Hunter, N. (2018). Metronidazo-

le: an update on metabolism, structure-cytotoxicity

and resistance mechanisms. The Journal of Antimi-

crobial Chemotherapy, 73(2), 265–279. https://doi.

org/10.1093/jac/dkx351

Elizondo-Luévano, J. H., Castro-Ríos, R., López-Abán,

J., Gorgojo-Galindo, O., Fernández-Soto, P., Vicen-

te, B., Muro, A., & Chávez-Montes, A. (2021a).

Berberine: A nematocidal alkaloid from Argemo-

ne mexicana against Strongyloides venezuelensis.

Experimental Parasitology, 220(11). https://doi.

org/10.1016/j.exppara.2020.108043

Elizondo-Luévano, J. H., Castro-Ríos, R., Vicente, B.,

Fernández-Soto, P., López-Aban, J., Muro, A., &

Chávez-Montes, A. (2021b). In vitro antischistoso-

mal activity of the Argemone mexicana methanolic

extract and its main component berberine. Iranian

Journal of Parasitology, 16(1), 91–100. https://doi.

org/10.18502/ijpa.v16i1.5518

Elizondo-Luévano, J. H., Pérez-Narváez, O. A., Sánchez-

García, E., Castro-Ríos, R., Hernández-García, M.

E., & Chávez-Montes, A. (2021c). In-vitro effect

of Kalanchoe daigremontiana and its main compo-

nent, quercetin against Entamoeba histolytica and

Trichomonas vaginalis. Iranian Journal of Parasito-

logy, 16(3), 394–401. https://doi.org/10.18502/ijpa.

v16i3.7092

Elizondo-Luévano, J. H, Castro-Ríos, R., Sánchez-Gar-

cía, E., Hernández-García, M. E., Vargas-Villarreal,

J., Rodríguez-Luis, O. E., & Chávez-Montes, A.

(2018). In Vitro Study of Antiamoebic Activity of

Methanol Extracts of Argemone mexicana on Tro-

phozoites of Entamoeba histolytica HM1-IMSS.

The Canadian Journal of Infectious Diseases &

Medical Microbiology, 2018, 7453787. https://doi.

org/10.1155/2018/7453787

Elizondo-Luévano, J. H., Hernández-García, M. E., Pérez-

Narváez, O. A., Castro-Ríos, R., & Chávez-Montes,

A. (2020b). Berberina, curcumina y quercetina como

potenciales agentes con capacidad antiparasitaria.

Revista de Biología Tropical, 68(4), 1241–1249.

https://doi.org/10.15517/rbt.v68i4.42094

Elizondo-Luévano, J. H., Verde-Star, J., González-Hor-

ta, A., Castro-Ríos, R., Hernández-García, M. E.,

& Chávez-Montes, A. (2020a). In Vitro Effect of

Methanolic Extract of Argemone mexicana against

Trichomonas vaginalis. The Korean Journal of Para-

sitology, 58(2), 135–145. https://doi.org/10.3347/

kjp.2020.58.2.135

Fessi, H., Puisieux, F., Devissaguet, J. P., Ammoury, N., &

Benita, S. (1989). Nanocapsule formation by interfa-

cial polymer deposition following solvent displace-

ment. International Journal of Pharmaceutics, 55(1),

1–4. https://doi.org/10.1016/0378-5173(89)90281-0

Hadaruga, D. I., Hadaruga, N. G., Bandur, G. N., Rivis, A.,

Costescu, C., Ordodp, V. L., & Ardelean, A. (2010).

Berberis vulgaris extract/β-cyclodextrin nanoparti-

cles synthesis and characterization. Revista de Chi-

mie, 61(7), 669–675.

IBM Corp. (2013). IBM SPSS Statistics (Version 22.0,

Software). IBM Corp.

Imenshahidi, M., & Hosseinzadeh, H. (2016). Berbe-

ris Vulgaris and Berberine: An Update Review.

Phytotherapy Research, 30(11), 1745–1764. https://

doi.org/10.1002/ptr.5693

Jarrad, A. M., Debnath, A., Miyamoto, Y., Hansford, K. A.,

Pelingon, R., Butler, M. S., Bains, M. S., Karoli, T.,

330 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 319-331, January-December 2022 (Published May 18, 2022)

Blaskovich, M. A. T., Eckmann, L., & Cooper, M. A.

(2016). Nitroimidazole carboxamides as antiparasitic

agents targeting Giardia lamblia, Entamoeba histo-

lytica and Trichomonas vaginalis. European Journal

of Medicinal Chemistry, 120, 353–362. https://doi.

org/10.1016/j.ejmech.2016.04.064

Kantor, M., Abrantes, A., Estevez, A., Schiller, A.,

Torrent, J., Gascon, J., Hernandez, R., & Och-

ner, C. (2018). Entamoeba Histolytica: Updates in

Clinical Manifestation, Pathogenesis, and Vaccine

Development. Canadian Journal of Gastroentero-

logy and Hepatology, 2018, 4601420. https://doi.

org/10.1155/2018/4601420

Karłowicz-Bodalska, K., Han, S., Freier, J., Smoleński, M.,

& Bodalska, A. (2017). Curcuma longa as medicinal

herb in the treatment of diabetic complications. Acta

Poloniae Pharmaceutica - Drug Research, 74(2),

605–610.

Khachane, P., Date, A. A., & Nagarsenker, M. S. (2011).

Eudragit EPO nanoparticles: Application in impro-

ving therapeutic efficacy and reducing ulcerogenicity

of meloxicam on oral administration. Journal of Bio-

medical Nanotechnology, 7(4), 590–597. https://doi.

org/10.1166/jbn.2011.1322

Khoee, S., & Yaghoobian, M. (2009). An investigation

into the role of surfactants in controlling particle

size of polymeric nanocapsules containing peni-

cillin-G in double emulsion. European Journal of

Medicinal Chemistry, 44(6), 2392–2399. https://doi.

org/10.1016/j.ejmech.2008.09.045

Kocaadam, B., & Şanlier, N. (2017). Curcumin, an active

component of turmeric (Curcuma longa), and its

effects on health. Critical Reviews in Food Science

and Nutrition, 57(13), 2889–2895. https://doi.org/10.

1080/10408398.2015.1077195

Kumar, S. S. D., Houreld, N. N., & Abrahamse, H.

(2018). Therapeutic potential and recent advances

of curcumin in the treatment of aging-associated

diseases. Molecules, 23(4). https://doi.org/10.3390/

molecules23040835

Kuriyama, A., Jackson, J. L., Doi, A., & Kamiya, T. (2011).

Metronidazole-induced central nervous system toxi-

city: A systematic review. Clinical Neuropharma-

cology, 34(6), 241–247. https://doi.org/10.1097/

WNF.0b013e3182334b35

Lancheros, R. J., Beleño, J. Á., Guerrero, C. A., & Godoy-

Silva, R. D. (2014). PLGA nanoparticles produc-

tion by emulsion and evaporation to encapsulate

N-acetylcysteine (NAC). Universitas Scientiarum,

19(2), 161–168. https://doi.org/10.11144/Javeriana.

SC19-2.pnpm

Magalhães, L. G., Machado, C. B., Morais, E. R., Bueno

De Carvalho Moreira, É., Soares, C. S., Da Silva,

S. H., Da Silva Filho, A. A., & Rodrigues, V.

(2009). In vitro schistosomicidal activity of curcumin

against Schistosoma mansoni adult worms. Para-

sitology Research, 104(5), 1197–1201. https://doi.

org/10.1007/s00436-008-1311-y

Mahmoudvand, H., Saedi Dezaki, E., Sharififar, F., Ezat-

pour, B., Jahanbakhsh, S., & Fasihi Harandi, M.

(2014). Protoscolecidal effect of Berberis vulgaris

root extract and its main compound, berberine in cys-

tic echinococcosis. Iranian Journal of Parasitology,

9(4), 503–510.

Majidzadeh, H., Araj-Khodaei, M., Ghaffari, M., Torbati,

M., Ezzati Nazhad Dolatabadi, J., & Hamblin, M. R.

(2020). Nano-based delivery systems for berberine:

A modern anti-cancer herbal medicine. Colloids

and Surfaces B: Biointerfaces, 194(6). https://doi.

org/10.1016/j.colsurfb.2020.11118

Meerasa, A., Huang, J. G., & Gu, F. X. (2012). CH50: A Revi-

sited Hemolytic Complement Consumption Assay for

Evaluation of Nanoparticles and Blood Plasma Pro-

tein Interaction. Current Drug Delivery, 8(3), 290–

298. https://doi.org/10.2174/156720111795256165

Mehra, M., Sheorain, J., & Kumari, S. (2016). Synthesis

of berberine loaded polymeric nanoparticles by cen-

tral composite design. AIP Conference Proceedings,

1724. https://doi.org/10.1063/1.4945180

Molina-Garza, Z. J., Bazaldúa-Rodríguez, A. F., Quintani-

lla-Licea, R., & Galaviz-Silva, L. (2014). Anti-Trypa-

nosoma cruzi activity of 10 medicinal plants used

in northeast Mexico. Acta Tropica, 136(1), 14–18.

https://doi.org/10.1016/j.actatropica.2014.04.006

Ofridam, F., Lebaz, N., Gagnière, É., Mangin, D., & Elais-

sari, A. (2021). Polymethylmethacrylate derivatives

Eudragit E100 and L100: Interactions and comple-

xation with surfactants. Polymers for Advanced Tech-

nologies, 32(1), 379–390. https://doi.org/10.1002/

pat.5093

Panic, G., Duthaler, U., Speich, B., & Keiser, J. (2014).

Repurposing drugs for the treatment and control of

helminth infections. International Journal for Para-

sitology: Drugs and Drug Resistance, 4(3), 185–200.

https://doi.org/10.1016/j.ijpddr.2014.07.002

Pérez, K. C., Galaviz, L., Iracheta, J. M., Lucero, E. A., &

Molina, Z. J. (2017). Actividad contra Trypanosoma

cruzi (Kinetoplastida: Trypanosomatidae) de extrac-

tos metanólicos de plantas de uso medicinal en Méxi-

co. Revista de Biologia Tropical, 65(4), 1459–1469.

https://doi.org/10.15517/rbt.v65i4.27153

Pina-Barrera, A. M., Alvarez-Roman, R., Baez-Gonzalez,

J. G., Amaya-Guerra, C. A., Rivas-Morales, C.,

Gallardo-Rivera, C. T., & Galindo-Rodriguez, S. A.

(2019). Application of a Multisystem Coating Based

on Polymeric Nanocapsules Containing Essential

Oil of Thymus Vulgaris L. To Increase the Shelf Life

of Table Grapes (Vitis Vinifera L.). IEEE Transac-

tions on Nanobioscience, 18(4), 549–557. https://doi.

org/10.1109/TNB.2019.2941931

331

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 70: 319-331, January-December 2022 (Published May 18, 2022)

Prasad, S., Gupta, S. C., Tyagi, A. K., & Aggarwal,

B. B. (2014). Curcumin, a component of golden

spice: From bedside to bench and back. Biote-

chnology Advances, 32(6), 1053–1064. https://doi.

org/10.1016/j.biotechadv.2014.04.004

Raju, M., Kulkarni, Y. A., & Wairkar, S. (2019). The-

rapeutic potential and recent delivery systems of

berberine: A wonder molecule. Journal of Functio-

nal Foods, 61(6), 103517. https://doi.org/10.1016/j.

jff.2019.103517

Rana, A. K., & Misra-Bhattacharya, S. (2013). Current

drug targets for helminthic diseases. Parasitolo-

gy Research, 112(5), 1819–1831. https://doi.

org/10.1007/s00436-013-3383-6

Rosmalena, R., Elya, B., Dewi, B. E., Fithriyah, F., Desti,

H., Angelina, M., Hanafi, M., Lotulung, P. D., Pra-

sasty, V., & Seto, D. (2019). The antiviral effect of

indonesian medicinal plant extracts against dengue

virus in vitro and in silico. Pathogens, 8(2), 1–11.

https://doi.org/10.3390/pathogens8020085

Sasidharan, S., & Saudagar, P. (2020). Encapsulation and

delivery of antiparasitic drugs: a review. In Encapsu-

lation of Active Molecules and Their Delivery System

(pp. 323–342). Elsevier Inc. https://doi.org/10.1016/

b978-0-12-819363-1.00017-x

Shabanzadeh, P., Senu N., Shameli, K., Ismail, F., &

Mohagheghtabar, M. (2013). Application of artificial

neural network (ANN) for the prediction of size

of silver nanoparticles prepared by green method.

Digest Journal of Nanomaterials and Biostructures,

8(2), 541–549.

Sun, Y., Chen, D., Pan, Y., Qu, W., Hao, H., Wang, X., Liu,

Z., & Xie, S. (2019). Nanoparticles for antiparasitic

drug delivery. Drug Delivery, 26(1), 1206–1221.

https://doi.org/10.1080/10717544.2019.1692968

Theel, E. S., & Pritt, B. S. (2016). Parasites. Microbiology

Spectrum, 4(4). https://doi.org/10.1128/microbiols-

pec.DMIH2-0013-2015

Turkeltaub, J. A., McCarty, T. R. III., & Hotez, P. J.

(2015). The intestinal protozoa: emerging impact on

global health and development. Current Opinion in

Gastroenterology, 31(1). https://journals.lww.com/

co-gastroenterology/Fulltext/2015/01000/The_intes-

tinal_protozoa__emerging_impact_on_global.7.aspx

Vaughn, A. R., Branum, A., & Sivamani, R. K. (2016).

Effects of Turmeric (Curcuma longa) on Skin Health:

A Systematic Review of the Clinical Evidence.

Phytotherapy Research, 1264(10), 1243–1264.

https://doi.org/10.1002/ptr.5640

Verma, S., & Sharma, D. (2018). Berberine: A pioneer

remedy for various ailments. Pharma Innovation,

7(10), 194–200. http://www.thepharmajournal.com/

archives/2018/vol7issue10/PartD/7-9-47-287.pdf

Wendel, K. A., & Workowski, K. A. (2007). Tricho-

moniasis: Challenges to appropriate management.

Clinical Infectious Diseases: An official publication

of the Infectious Diseases Society of America, 44(3),

123–129. https://doi.org/10.1086/511425

WHO (World Health Organization). (2018). Infection Sur-

veillance. Southern Medical Journal (Vol. 70).

Wright, C. W., Marshall, S. J., Russell, P. F., Anderson,

M. M., Phillipson, J. D., Kirby, G. C., Warhurst, D.

C., & Schiff, P. L. (2000). In vitro antiplasmodial,

antiamoebic, and cytotoxic activities of some mono-

meric isoquinoline alkaloids. Journal of Natural Pro-

ducts, 63(12), 1638–1640. https://doi.org/10.1021/

np000144r