Ferreira 

et al. Parasites Vectors (2020) 13:527

https://doi.org/10.1186/s13071-020-04402-w
Parasites & Vectors

RESEARCH Open Access

Ketamine can be produced by Pochonia

chlamydosporia: an old molecule and a new
anthelmintic?
Sebastiao Rodrigo Ferreira1,2, Alan Rodrigues T. Machado3,4, Luís Fernando Furtado1,5, Jose Hugo de S. Gomes6,
Raquel M. de Almeida1, Thiago de Oliveira Mendes7, Valentina N. Maciel3, Fernando Sergio Barbosa1,
Lorendane M. Carvalho8, Lilian Lacerda Bueno1, Daniella Castanheira Bartholomeu1, Jackson Victor de Araújo8,
Elida M. L. Rabelo1, Rodrigo Maia de Pádua6, Lucia Pinheiro Santos Pimenta3 and Ricardo Toshio Fujiwara1*

Abstract 
Background:  Infection by nematodes is a problem for human health, livestock, and agriculture, as it causes deficits
in host health, increases production costs, and incurs a reduced food supply. The control of these parasites is usually
done using anthelmintics, which, in most cases, have not been fully effective. Therefore, the search for new molecules
with anthelmintic potential is necessary.
Methods:  In the present study, we isolated and characterized molecules from the nematophagous fungus Pochonia
chlamydosporia and tested these compounds on three nematodes: Caenorhabditis elegans; Ancylostoma ceylanicum;
and Ascaris suum.
Results:  The ethyl acetate extract showed nematicidal activity on the nematode model C. elegans. We identified
the major substance present in two sub-fractions of this extract as ketamine. Then, we tested this compound on C.
elegans and the parasites A. ceylanicum and A. suum using hamsters and mice as hosts, respectively. We did not find a
difference between the animal groups when considering the number of worms recovered from the intestines of ani-
mals treated with ketamine (6 mg) and albendazole (P > 0.05). The parasite burden of larvae recovered from the lungs
of mice treated with ketamine was similar to those treated with ivermectin.
Conclusions:  The results presented here demonstrate the nematicidal activity of ketamine in vitro and in vivo, thus
confirming the nematicidal potential of the molecule present in the fungus P. chlamydosporia may consist of a new
method of controlling parasites.
Keywords:  Pochonia chlamydosporia, New drugs, Nematicidal molecule, Ketamine

Background go beyond organic deficits because they incur increased

Nematode infection is a problem for human health, as production costs and consequently a reduced food supply
it contributes negatively to growth and cognitive devel- [1–3]. The control of these parasites is usually carried out
opment. In animal production and agriculture, damages using anthelmintics that, in most cases, have not been
totally effective. In addition, resistance to the main group
of drugs used in the treatment and control is already a
*Correspondence: [email protected] consolidated problem [4]. Thus, the search for new drugs
1
Departamento de Parasitologia, Instituto de Ciências Biológicas,
Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, Belo and control alternatives is necessary.
Horizonte, MG 31270‑901, Brazil
Full list of author information is available at the end of the article

© The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and
the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material
in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material
is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the
permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​
mmons​.org/licen​ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/publi​cdoma​in/
zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Ferreira et al. Parasites Vectors (2020) 13:527 Page 2 of 9

Several studies in the literature have described the direct Laboratory of the Federal University of Viçosa, Viçosa,
or indirect nematicidal action of some organisms, such as Brazil. Culture discs with an approximate diameter of 4
fungi, featuring one of the alternatives of biological con- mm were extracted from the tube containing the fun-
trol. The administration of nematophagous fungi is consid- gal isolate and then transferred to a 250 ml Erlenmeyer
ered a promising alternative in the prophylaxis of parasitic flask containing the potato dextrose liquid medium
gastrointestinal helminthiasis [5]. Willcox & Tribe [6] (Difco, Detroit, USA). These were incubated in a hori-
reported the association between the fungus Pochonia zontal shaker (Tecnal, Piracicaba, Brazil) at 28 °C under
chlamydosporia and the nematode Heterodera schachtii, as agitation at 120× rpm, and, after 20 days, this material
the primary and secondary metabolites of nematophagous was filtered to separate the extract from the mycelial
fungi have been known to be involved in this interaction mass. The separated mass was used in the extraction
[5, 7, 8]. In a review by Li et  al. [9], 179 substances from process by maceration.
fungi, including nematophagous, were reported as poten- To obtain the methanolic extract, 400 g of the myce-
tial nematicidal drugs; therefore, these fungi can be con- lium (a product of 30 l of fungal culture) had been sub-
sidered an important source of new drugs. mitted to cold triple maceration for 7 days in methanol.
In the history of drug production and discovery, often The methanolic extract (MeOH) was concentrated
a molecule that is characterized with a well-defined under reduced pressure and lyophilized, obtaining 4.1
function can, with new studies, be employed for a new g of methanolic extract. 3 g of this extract were solu-
purpose. This practice is called drug repositioning bilized in MeOH, and then this solution was parti-
[10]. In helminthology, some drugs that were not origi- tioned with dichloromethane, followed by ethyl acetate,
nally used against helminths have shown high efficacy affording the fractions C
­ H2Cl2 (0.61 g) and EtOAc (0.50
against some worms, such as mefloquine for Echino- g). 0.4 g of the ethyl acetate fraction (EtOAc) was fur-
coccus multilocularis [11]. Sertraline, paroxetine, and ther fractionated by column chromatography in sub-
chlorpromazine have long histories of clinical use as fractions using silica gel as the stationary phase and
antidepressant or antipsychotic medicines. However, then eluted with hexane: ethyl acetate gradients of
according to Weeks et al. [12], they may represent new increasing polarity. These sub-fractions were concen-
classes of anthelmintic drugs. In this perspective, other trated under reduced pressure in a rotary evaporator
drugs that act via ion channels or neurotransmitter and lyophilized (Table 1).
receptors may have promising functions for helminth
control. Here, we extracted, characterized, and evalu- Nuclear magnetic resonance (NMR) spectroscopy
ated P. chlamydosporia metabolites against nematodes. The NMR experiments were obtained on a Bruker
In these studies, it is important to have good models AVANCE DRX400 spectrometer, at a High-Resolution
to test drug effectiveness. In this context, Caenorhab- Magnetic Resonance Laboratory (LAREMAR), of the
ditis elegans is the main model for a drug-screening Chemistry Department-ICEx-UFMG. The fractions were
study, since it presents characteristics that facilitate the analysed by 1H NMR. EtOAc and C ­ H2Cl2 were solu-
assays, such as the feasibility of laboratory maintenance bilized in ­CDCl3 (deuterated chloroform), and MeOH
[13] and the taxonomic and functional proximity to were solubilized in a solution of methanol-d4/buffered
important parasitic nematodes [14]. However, valida- ­KH2PO4 in ­D2O, pH 6. The spectra were acquired at 300
tion of the experiments in an animal model is necessary. K with a spectral window of 16 ppm, a total of 32 k points,
It is important to consider that we also carried out tests 16 intermediate points, acquisition times (AQ), and a
on two parasitic nematodes, Ancylostoma ceylanicum recovery (d1) of 2.6 s and 2.0 s, respectively. For the pro-
and Ascaris suum, using the infection model, hamsters cessing, a line widening of 0.3 Hz was used prior to the
and mice, respectively. Therefore, in the present work, Fourier transform. The phases and baselines were auto-
we have extracted and characterized a new metabolite matically corrected using the TopSpin 1.3 programme,
of P. chlamydosporia: the molecule ketamine. We eval- and, finally, the spectra were calibrated by the TMS sig-
uated this molecule on nematodes, C. elegans, A. suum nal (Trimethylsilane) at 0.00 ppm, with the exception of
and A. ceylanicum, and here, we present unprecedented the sample spectrum MeOH, which was calibrated by
to our knowledge, results of its nematicide activity. the signal of (3-(trimethylsilyl)propionic-2,2,3,3-d4 acid
sodium salt) TSP-d4 at 0.00 ppm. 2D NMR 1H-1H COSY
(correlated spectroscopy), 1H-13C HSQC (Heteronuclear
Methods Single Quantum Correlation), and 1H-13C HMBC (Het-
Fungus cultivation eronuclear Multiple Bond Correlation) experiments were
The P. chlamydosporia used in this study was kept in performed for the F5 sample.
the fungal collection of the Veterinary Parasitology
 Ferreira et al. Parasites Vectors (2020) 13:527 Page 3 of 9

GC‑MS analysis Table 1  Fractions from EtOAc column fractionation

The ethyl acetate fraction and its 7 sub-fractions obtained Mobile phase Mass (g)a Coding name
from the purification by column chromatography (F1-F9)
were analysed by gas chromatography coupled with mass Hex:EtOAC (9:1) 0.01 F1
spectrometry (GC/MS). The analyses were performed Hex:EtOAC (8:2) 0.03 F2
using a Shimadzu GCMSQP2010 Plus, equipped with an Hex:EtOAC (7:3) – F3
AOC-10 automatic injection system (Shimadzu, Tokyo, Hex:EtOAC (6:4) 0.01 F4
Japan). In the experiments, a 30-m-long capillary col- Hex:EtOAC (5:5) 0.04 F5
umn Rxi-1 (100% polydimethylsiloxane), with an inter- Hex:EtOAC (4:6) – F6
nal diameter of 0.25 mm and a film thickness of 0.25 μm, Hex:EtOAC (3:7) 0.06 F7
was used. The injector temperature was 250 °C, and the Hex:EtOAC (2:8) 0.04 F8
temperature programme ranged from 150 °C to 300 °C at Hex:EtOAC (1:9) 0.04 F9
3 °C/min. The injected volume was 1 μl in split-mode at a
  The sub-fractions F3 and F6 of EtOAc were not obtained in sufficient biomass
a ratio of 10:1. MS analysis was carried out in a quadru-
(Holliday-Scott®, Buenos Aires, Argentine) was employed
pole MS system (QP-2010plus) operating at 70 eV under
the same conditions described above. Compounds were
identified through comparison with mass spectral data in as the reference compound. The UPLC-ESI-MS/MS anal-
NIST 08 libraries. ysis was performed twice with the fungus grown at differ-
ent times for confirmation of ketamine’s presence.
UPLC‑ESI‑MS/MS
Culturing Caenorhabditis elegans larvae and motility test
4.0 mg of the ethyl acetate fraction and the standard
molecule (ketamine) were weighed directly in a micro- The strain of C. elegans used in the experiment was kindly
tube and 1 ml of methanol/water (1:1). The HPLC grade provided by Professor Carlos Winter of Universidade de
was added until reaching complete solubilization. After São Paulo (USP). L3 larvae of C. elegans were grown on
that, the ethyl acetate sample was diluted 4× and keta- 8P NGM plates according to the methodology previously
mine was diluted 20×, and then they were automatically described [16]. After 7 days of culturing in a bio-oxygen
injected onto the system. demand (BOD) incubator at 20 °C, the plates were washed
The chemical composition of ethyl acetate fractions was with M9 medium [16] and filtered through three sieves
investigated by ultra-performance liquid chromatography with 40-, 30- and 20-μm pores. L3 larvae retained in the 20
coupled with mass spectrometry in a Waters ACQUITY μm strainer were collected by backwashing. The obtained
UPLC system (Waters, Milford, MA, USA) composed larvae were washed by centrifugation at 700×g for 4 min,
of a binary pump, auto sampler, in-line degasser, and followed by two washes with M9 medium. For the nemati-
photodiode array detector (190–500 nm) (Waters). The cidal assay against C. elegans, the L3 was resuspended in
analyses were performed on an Acquity UPLC BEH C18 M9, and approximately 1000 larvae in 100 μl of suspen-
column (2.1 × 50 mm i.d., 1.7 µm; Waters) at 40 °C, elut- sion were added to each well in a 96-well microplate. The
ing with a linear gradient of water (A) and acetonitrile ethyl acetate extract was added in concentrations of 1000,
(B), both containing 0.1% v/v formic acid, at a flow rate 100, 10, 1, 0.1 and 0.01 μg/ml. For methanol and dichlo-
of 0.3 ml/min (e.g. 5 to 95% of B in 10 min) before return- romethane, concentrations of 5000 and 3000 μg/ml were
ing to the initial conditions in 1 min. A re-equilibration added, and ketamine was used up until the concentration
time of 2 min was maintained between runs. As previ- of 20,000 μg/ml. In the negative control, the M9 medium
ously described, our research group employed the spec- was used with 0.05% dimethyl sulfoxide, and, for the posi-
troscopic conditions [15]. tive control, ivermectin was applied in the same concen-
A mass spectrometer ­XecoTM Triple Quadrupole MS trations used for the tested extracts (1000–0.01 μg/ml).
(Waters) equipped with an electrospray ionization (ESI) Plates containing compounds and larvae were stored in a
source, operating in a negative and positive ionization BOD incubator at 20 °C. After 48 h, 10 μl of a solution con-
mode, was used in the analysis. The cone gas flow was set taining approximately 100 larvae were removed from each
to 90 L/h, and desolvation gas flow was set to 900 L/h at well for analysis, and quantification of the total number of
350 °C. The capillary voltage was set to 3.54 kV, cone gas paralyzed larvae was carried out using an optical micro-
voltage was set to 27 V, and source temperature was set scope at 100× magnification. Larvae were considered par-
to 120 °C. The data were accomplished in the scan mode, alyzed when presenting a straight body and absence of any
with mass range adjusted to m/z 100–700 Da. Ketamine mobility.
Ferreira et al. Parasites Vectors (2020) 13:527 Page 4 of 9

Activity of ketamine on Ancylostoma ceylanicum Statistical analyses

For this experiment, 50 female hamsters (Mesocrice- The data of the recovered nematodes were submitted to
tus auratus), 4 to 6 weeks-old, were divided into five the normality test (Kolmogorov-Smirnov), followed by
experimental groups containing 10 animals each. All the analysis of variance (ANOVA) and Tukey’s post-hoc
animals were maintained in a controlled environment test (P ≤ 0.05). The EPG data were analyzed using two-
and infected with 75 third-stage larvae (L3) of A. cey- way ANOVA and Bonferroni correction (P ≤ 0.01). Non-
lanicum at day 0 of the experiment. After 20 days of linear regression analysis was used to calculate the E
­ D50
infection, when all animals were excreting eggs in the value utilized of a four-parameter sigmoid curve. All the
faeces, they were treated for 5 days, orally, according statistical analyses were carried out in GraphPad Prism
to the following experimental design: Group 1 PBS 7.0.

Química®,Campinas, Brazil) 0.60 mg; Group 3 keta-

(phosphate buffer saline); Group 2 albendazole (Nova
Results

5 ketamine 6 mg (Holliday-Scott®). The eggs per gram

mine 0.06 mg; Group 4 ketamine 0.60 mg; and Group Anthelmintic activity
To evaluate the nematicidal activity of P. chlamydospo-
of faeces (EPG) was performed to monitor the quan- ria, we tested the extracts obtained from the fungus on
tification of eggs throughout the experiment. This C. elegans larvae. Table 2 shows the results of the activ-
quantification was performed every 2 days using the ity of three extracts, and it can be seen that EtOAc was
McMaster chamber [17]. After the treatment period, presented as the most promising of them. Although
the animals were euthanized using an overdose of ­CH2Cl2 and MeOH also showed a larval paralysis per-
anaesthetic (45 mg/kg of xylazine associated with 240 centage above 50%, they had high effective dose (­ED50)
mg/kg of ketamine, intraperitoneally). Then, the small values when compared to the value observed for EtOAc.
intestine was removed and placed in a Petri dish con- We observed that EtOAc was able to paralyze 84% of the
taining PBS, and it was opened longitudinally for the larvae at a concentration of 1000 µg/ml, whereas ­CH2Cl2
recovery of adult worms. and MeOH did not show relevant activity at this same
concentration, as it was necessary to perform the test at
Activity of ketamine on infection of Ascaris suum larvae 5000 µg/ml for these two extracts.
In this experiment, 60 mice of the C57Bl6 lineage were As EtOAc was the most promising, we fractioned it
used, being divided into two groups of 30 animals: a further, obtaining 7 sub-fractions (Table 1). In the motil-
group of 30 animals with treatment administered sub- ity test, we observed that only F4 and F5 were active, and
cutaneously and the other group by oral route (gavage). it presented the following ­ED50 values: 50.9 μg/ml and
The animals had been infected with 2000 eggs of A. suum 210.5 μg/ml, respectively (Fig. 1). The EtOAc and F4 and
larvae and divided into groups, with 6 animals each, that F5 reached the following percentages of immobility: 84,
received the following treatments: Group 1 PBS (phos- 85.5, and 75.7%, respectively, using the concentration of

(Ouro ­Fino® Cravinhos, Brazil); Group 3 ketamine 0.05

phate buffered saline); Group 2 ivermectin 0.05 mg 1000 µg/ml. After the fractionation of EtOAc, the anthel-
mintic activity remained in F4 and F5.

0.50 mg (Holliday-Scott®). The treatment was carried out

mg; group 4 ketamine 0.25 mg; and Group 5 ketamine Unfortunately, the amount of biomass produced by
the fungus was very small, not being possible to isolate
for 7 days, except for the subcutaneous ivermectin group ketamine in sufficient quantity to perform an in vivo test
that received a single dose. using animal models. Therefore, we decided to perform
On the eighth day after infection, the animals were this test using only commercial ketamine on C. elegans
euthanized using an anaesthetic overdose (45 mg/kg of larvae. Figure  1 shows the activity of the drug on C.
xylazine associated with 240 mg/kg of ketamine, intra- elegans. As the commercial ketamine was active on the
peritoneally). To recover and quantify the number of nematode model, we tested it in vivo on the animal mod-
larvae present in the lungs, the lung tissue collected els infected with A. ceylanicum, and A. suum. Figure  2
was triturated in the Petri dish and placed in the Baer- shows that ketamine exhibited activity on A. ceylanicum
mann apparatus for 4 h at 37 °C with PBS. After incu- at the concentrations of 0.6 and 6 mg, and the concen-
bation, the sediment was collected and centrifuged at tration 6mg reduced worm burden parasites to approxi-
600× g for 10 min at 20 °C, and the larvae were fixed mately zero; thus, there was no difference (P ˃ 0.05)

(Leica ­Microsystems®, Wetzlar, Germany) with a 100×

in 10% formalin and counted in the optical microscope between the groups treated with albendazole (0.6 mg)
and ketamine (6 mg). The EPG of the groups treated with
magnification. albendazole (0.6 mg) and ketamine (6 mg) was zero after
4 days of treatment (Fig. 2).
 Ferreira et al. Parasites Vectors (2020) 13:527 Page 5 of 9

Figure  3 shows the ability of ketamine to impair the some CG/MS analyses revealed greater purity in F5, we
infection of A. suum larvae in C57Bl/6 mice. The mean chose to perform two-dimensional NMR analyses for
number of larvae recovered in the lungs of these animals this sample. We confirmed the presence of ketamine
are shown in this figure. Ketamine showed nematicidal F5 by HSQC, HMBC, and COSY (Additional file  1:
activity in the two routes of the drug’s administration, Table S2, Additional file 1: Figures S3–S6 and by UPLC-
oral and subcutaneous, with no difference (P ˃ 0.05) ESI-MS/MS techniques (Additional file  1: Figures  S7,
between the administration routes. In addition, when S8), and we compared it to the standard, commercially
ketamine was administered orally or subcutaneously, available ketamine (Table  4 and Additional file  1: Fig-
there was no difference in the number of larvae recovered ure S7). We identified ketamine by UPLC-MS analyses,
(P ˃ 0.05) between ketamine (0.5 mg) and ivermectin. using ESI in the positive mode, through the ions at m/z
= 238.17 Da [M + ­H]+ and with m/z = 240.10 Da [M
Chemical characterization of P. chlamydosporia extracts + 37Cl] +. In addition, we observed the spectral ions
1
H NMR analysis of these samples (Additional file  1: daughters profile generated from the fragmentation of
Figure S1) revealed characteristic signals of fatty acids ions at m/z 238.17 Da and 240 Da (Table 4).
and aromatic compounds in EtOAc. Using the GC/MS
technique, we could identify the main substances pre- Discussion
sent in EtOAc (Table 3). In sub-fraction 4, we identified There are few investments in the discovery of new drugs
ketamine and the acids cis-7-hexadecenoic, palmitic, for the treatment of parasites. Considering that there
and linoleic acid; however, there were no identified sub- are reports in the literature relating nemathophagous
stances other than ketamine in F5 (Additional file  1: fungi extracts with nematicidal activity and commercial
Figure S2). The sub-fractions F1, F2, F7, F8, and F9 products using nemathophagous fungi [7, 18, 19], we
presented the same fatty acids but not ketamine (Addi- performed extraction of the P. chlamydosporia metabo-
tional file 1: Table S1). lites with an organic solvent; the extract was fractionated
Ketamine was present in the EtOAc fraction and in with solvents of different polarity scale and evaluated the
sub-fractions F4 and F5 only, and these sub-fractions compounds for nematicidal capacity. We observed that
showed paralyzing activity on C. elegans larvae. As EtOAc was able to paralyze 84% of the C. elegans larvae
at a concentration of 1000 μg/ml, whereas C ­ H2Cl2 and
MeOH did not show any relevant activity at the same
concentration, which was probably because of the profile
Table 2 Activity of P. chlamydosporia extracts on C. elegans
larvae and quantity of substances present in EtOAc. The analy-
ses by 1H NMR and GC/MS showed the presence of fatty
Extract Immobility (%) [concentration of 1000 and acids and aromatic compounds in EtOAc and C ­ H2Cl2;
5000 µg/ml]
however, the GC/MS analysis showed that, in addition to
Time: 48 h ED50 (µg/ml) these compounds, ketamine was present in EtOAc. The
EtOAC 84.0 325.3 other substances present in this fraction were the fol-
CH2Cl2 55.5 1400 lowing fatty acids: hexadecenoic; palmitic; linolelaidic;
MeOH 66.9 3365 oleic; linoleic; and vaccenic. The palmitic, oleic, and lin-
oleic acids have the previously described nematicidal
Notes: ­CH2Cl2 and MeOH were tested at concentrations of 5000 µg/ml because
we did not find an effective dose ­(ED50) in the test with a concentration of 1000 activity [19, 20]. The other fatty acids are most likely the
µg/ml. We evaluated the motility of larvae 48 h after contact with the extract

Fig. 1  Activity of ethyl acetate sub-fractions 4 (a) and 5 (b), and ketamine (c) on the motility of C. elegans larvae. The ketamine was tested until
reaching a concentration of 20,000 µg/ml. The motility of larvae was evaluated 48 h after contact with the drug
Ferreira et al. Parasites Vectors (2020) 13:527 Page 6 of 9

Fig. 2  Activity of ketamine on the reduction of A. ceylanicum worm burden in hamsters (Mesocricetus auratus). a Recovered worm burden from
hamster intestines. Bars indicated by the same letter signify that there was no statistically significant difference, according to the Tukey’s test (P
˃ 0.05). Results were plotted as the mean ± standard deviation. b Mean EPG values of the animals in their treatment groups, before (18th day)
and after (24th day) the treatment had begun, significant differences, according to Bonferroni correlation (P < 0.01), were only present in the
albendazole 0.6 mg and ketamine 6 mg

constituents of the fungal structures, such as the cell adhesive traps in predatory fungi, with the example of
membrane. In F4, we identified ketamine and palmitic linoleic acid [21]. However, the role of such metabolites
and linoleic acids, as these fatty acids have nematicidal may go beyond the formation of adhesive traps, since P.
activity that has already been described in the literature chlamydosporia does not form such structures.
[19, 20], and this can explain the fact that sub-fraction 4 The GC/MS analysis of F5 suggested the presence
had the lowest E­ D50 compared to sub-fraction 5 and com- of ketamine with a high degree of purity, since it pre-
mercial ketamine; thus, the substances could be acting in sented a single peak with a retention time of 25.5 min
synergy. On the other hand, F5 indicated only the pres- (Additional file  1: Figure S2). The main peak observed
ence of ketamine. Although the role of fatty acids in nem- by UPLC-ESI-MS/MS analyses displayed a reten-
atode fungus interaction is not totally understood, some tion time of 2.1 min, with a mass/charge ratio at m/z
of these compounds can be present in the formation of 238.17 Da and 240.10 Da. These ions are the adducts

Fig. 3  Activity of ketamine on the reduction of the A suum larvae burden in lungs of mice (C57Bl6). a Administration of drugs by one subcutaneous
route. b Administration of drugs by oral route. Bars indicated by the same letter signify that there was no statistically significant difference,
according to the Tukey’s test (P ˃ 0.05). Results are plotted as the mean ± standard deviation
 Ferreira et al. Parasites Vectors (2020) 13:527 Page 7 of 9

protonated: the ion 238,17 with 35Cl isotope and 240.10 have been a good alternative model for studying early
with the 37Cl isotope. The MS/MS spectra corroborate Ascaris spp., and initial events of infection and immu-
the presence of ketamine in the samples due to the nology are similar for the hosts of A. suum and A. lum-
profile of the ions observed (Table  4, Additional file  1: bricoides [24]. Ketamine was effective in reducing the
Figures S7, S8), and its structure was confirmed by 2D burden parasites recovered from the hamster intes-
NMR analysis (Additional file 1: Figures S3–S6). tines. At a concentration of 6 mg, the worm burden was
Due to the fact that nematicidal activity was observed close to zero, showing results similar to albendazole. In
in EtOAc fraction and F4/F5 sub-fractions, all possess- the experiment with A. suum, we observed reduction in
ing ketamine, and this molecule were commercially the number of larvae that reached the mice lungs, and
available, we confirm the anthelmintic activity of keta- the drug activity was not dependent on the route of
mine on C. elegans; thus, we demonstrated that besides administration, since there was no difference in results
the fatty acids with anthelmintic activity known there, when the drug was administered subcutaneously or
ketamine is responsible for the nematode death/paraly- orally. This information is important because the oral
sis process. Although, the fungi paralysis mechanism route of administration is cheaper, easy to administer,
on the nematode can occur due to a possible synergy and painless. Possibly, the activity of this drug is related
among the substances present. According to Olthof to its ability to bind to GABA receptors and may conse-
& Estey [22], fungal metabolites capable of paralyzing quently lead the nematode to paralysis [25, 26]. There is
and/or killing nematodes are involved in the first con- no report in the literature on the anthelmintic activity
tact between the fungus and the nematode, so it can be of ketamine. It is worth noting that this drug is already
the first method of immobilization, which explains the used as a dissociative anaesthetic. Therefore, for its use
production of ketamine by the fungus. as an anthelmintic, more studies are needed to avoid
The anthelmintic activity of ketamine was confirmed or minimize adverse reactions. Other studies are nec-
in vivo using hamsters and BALB/c mice. Hamsters essary to verify a better form of administration and/or
are permissive hosts for A. ceylanicum infection, and association with another drug or vehicle once this drug
the pathogeny caused by these worms in hamsters is is rapidly metabolized, having a half-life of approxi-
similarly caused in humans [23]. The BALB/c mice mately 15 minutes [27].

Table 3 Substances present in the ethyl acetate fraction Conclusions

identified by GC/MS The unprecedented results presented here confirm the
Substance Retention time Percentage
anthelmintic activity of ketamine in the tested models,
(min) thus demonstrating the possibility of repositioning keta-
mine and corroborating the potential of P. chlamydo-
Ketamine 25.47 15.0
sporia in the discovery of new drug candidates that can
Cis-7-hexadecenoic acid 25.96 0.6
be used in the treatment of several neglected diseases
Palmitic acid methyl ester 26.30 26.3
caused by nematode infections.
Palmitic acid 27.20 3.6
Linolelaidic acid methyl ester 29.55 26.3
Oleic acid, methyl ester 29.65 23.8
Stearic acid, methyl ester 30.11 3.0
Linoleic acid 30.37 0.9
cis-vaccenic acid 30.47 1.9

Table 4  Identification of ketamine in EtOAc fraction by UPLC-ESI-MS/MS

Substance Retention time (min) Retention time (min) [M + ­H]+ m/z (Da) m/z of fragments of [M + ­H]+ (Da)
UV ­lighta ­TIC+

Reference ketamine 2.1 2.3 238.17 179.09, 207.22, 220.13, 238.16

240.15 127.15, 153.94, 208.98, 222.25, 239.48, 240.05
EtOAc fraction 2.2 2.3 238.17 179.22, 192.28, 207.03, 220.26, 238,03
240.10 126.83, 154.13, 209.23, 222.06, 239.92, 240.11
a
  Obtained at 220 nm
Ferreira et al. Parasites Vectors (2020) 13:527 Page 8 of 9

Supplementary information Brazil. 8 Departamento de Medicina Veterinária, Universidade Federal de

Viçosa, Av. Peter Henry Rolfs, s/n, Viçosa, MG 36.570‑000, Brazil.
Supplementary information accompanies this paper at https​://doi.
org/10.1186/s1307​1-020-04402​-w.
Received: 9 July 2020 Accepted: 9 October 2020

Additional file 1: Figure S1. 1H NMR spectrum of MeOH, EtOAc and

­CH2Cl2. Figure S2. GC-MS chromatogram of F5. Table S1. Substances
present in the sub-fractions F1, F2, F7, F8 and F9. Table S2. One and two
dimensional NMR spectral data of F5 and its correlations to structure keta- References
mine. Figure S3. Key HMBC and COSY correlations of Ketamine. Spectrum 1. Hotez PJ, Brindley PJ, Bethony JM, King CH, Pearce EJ, Jacobson J.
of F5 in 2D MNR. Figure S4. 1H-1H COSY, Figure S5. 1H-13C HSQC. Figure Helminth infections: the great neglected tropical diseases. J Clin Investig.
S6. 1H-13C HMBC. Figure S7. UPLC-ESI-MS/MS of F5. Figure S8. Proposal 2008;118:1311–21.
of fragmentation the ion m/z 238.17 Da. 2. Nicol JM, Turner SJ, Coyne DL, den Nijs L, Hockland S, Maafi ZT. Current
nematode threats to world agriculture. In: Jones J, Gheysen G, Fenoll C,
editors. Genomics and molecular genetics of plant-nematode interac-
Abbreviation tions. London: Springer Nature; 2011.
GABA: Gamma-AminoButyric Acid. 3. Vande Velde F, Charlier J, Claerebout E. Farmer behavior and gastroin-
testinal nematodes in ruminant livestock-uptake of sustainable control
Acknowledgements approaches. Front Vet Sci. 2018;5:255.
We thank Dr. Almudena Aranda Martinez for allowing the use the photograph 4. Zajíčková M, Nguyen LT, Skálová L, Raisová Stuchlíková L, Matoušková P.
in graphical abstract image. Anthelmintics in the future: current trends in the discovery and develop-
ment of new drugs against gastrointestinal nematodes. Drug Discov
Authors’ contributions Today. 2020;25:430–7.
RTF and SRF were responsible for the conception of the study. LLB and DCB 5. Braga FR, Araújo JV. Nematophagous fungi for biological control of gas-
assisted in obtaining financial resources. LMC and JVA provided and cultivated trointestinal nematodes in domestic animals. Appl Microbiol Biotechnol.
the fungus. RMA, TAOM, FSB, LFF and EMLR participated in the experimental 2014;98:71–82.
biological assays. ATM, JHSG, LPSP, VNM and RMP participated the chemical 6. Willcox J, Tribe HT. Fungal parasitism in cysts of Heterodera: I. Preliminary
extraction and analyses experiments. All co-authors contributed to the inter- investigations. Trans Br Mycol Soc. 1974;62:585–94.
pretation of the results and revision of the manuscript. All authors read and 7. Degenkolb T, Vilcinskas A. Metabolites from nematophagous fungi and
approved the final manuscript. nematicidal natural products from fungi as alternatives for biological con-
trol. Part II: metabolites from nematophagous basidiomycetes and non-
Funding nematophagous fungi. Appl Microbiol Biotechnol. 2016;100:3813–24.
This research was supported by Brazilian National Research Council (CNPq), 8. Wang YL, Li LF, Li DX, Wang B, Zhang K, Niu X. Yellow pigment aurovertins
the Fundação de Amparo à Pesquisa do Estado de Minas Gerais/FAPEMIG, mediate interactions between the pathogenic fungus Pochonia chla-
Pro-Reitoria de Pesquisa of Universidade Federal de Minas Gerais, and CAPES. mydosporia and its nematode host. J Agric Food Chem. 2015;63:6577–87.
SRF was supported by a doctoral degree fellowship from the CAPES. RTF, DCB, 9. Li G, Zhang K, Xu J, Dong J, Liu Y. Nematicidal substances from fungi.
LLB and JVA are supported by CNPq fellowships. Recent Pat Biotechnol. 2008;1:212–33.
10. Pillaiyar T, Meenakshisundaram S, Manickam M, Sankaranarayanan M. A
Availability of data and materials medicinal chemistry perspective of drug repositioning: recent advances
Data supporting the conclusions of this article are included within the article and challenges in drug discovery. Eur J Med Chem. 2020;165:112275.
and its additional file. 11. Rufener R, Ritler D, Zielinski J, Dick L, da Silva ET, da Silva Araujo A, et al.
Activity of mefloquine and mefloquine derivatives against Echinococcus
Ethics approval and consent to participate multilocularis. Int J Parasitol Drugs Drug Resist. 2018;8:331–40.
The experiments were carried out according to animal care standards, defined 12. Weeks JC, Roberts WM, Leasure C, Suzuki BM, Robinson KJ, Currey H, et al.
by the rules and guidelines of the Brazilian College of Animal Experimentation Sertraline, paroxetine, and chlorpromazine are rapidly acting anthelmin-
(COBEA). The experimental protocols used here were approved by the Animal tic drugs capable of clinical repurposing. Sci Rep. 2018;8:975.
Use Ethics Committee (CEUA) of the Federal University of Minas Gerais (UFMG) 13. Geary TG, Thompson DP. Caenorhabditis elegans: how good a model for
under the protocol No. 45/2012. veterinary parasites? Vet Parasitol. 2001;101:371–86.
14. Coghlan A, Tyagi R, Cotton JA, Holroyd N, Rosa BA, Tsai IJ, et al. Compara-
Consent for publication tive genomics of the major parasitic worms. Nat Genet. 2019;51:163–74.
Not applicable. 15. Pereira AC, Pereira ABD, Moreira CCL, Botion LM, Lemos VS, Braga FC, et al.
Hancornia speciosa Gomes (Apocynaceae) as a potential anti-diabetic
Competing interests drug. J Ethnopharmacol. 2015;161:30–5.
The authors declare that they have no competing interests. 16. Stiernagle T. Maintenance of C. elegans. 2006. In: WormBook: the online
review of C. elegans Biology. Pasadena (CA): WormBook; 2005–2018.
Author details https://www.ncbi.nlm.nih.gov/books/NBK19649/. Accessed 2 Jan 2018.
1 17. Gordon HM, Whitlock HV. A new technique for counting nematode eggs
 Departamento de Parasitologia, Instituto de Ciências Biológicas, Universi-

18. Braga FR, Ferraz CM, da Silva EN, de Araújo JV. Efficiency of the ­Bioverm®
dade Federal de Minas Gerais, Av. Antônio Carlos 6627, Belo Horizonte, MG in sheep faeces. J Counc Sci Ind Res. 1939;12:50–2.
31270‑901, Brazil. 2 Centro de Formação em Ciências da Saúde, Universidade
Federal do Sul da Bahia, Praça Joana Angélica, 250, Teixeira de Freitas, BA (Duddingtonia flagrans) fungal formulation to control in vivo and in vitro
45988‑058, Brazil. 3 Departamento de Química, Instituto de Ciências Exatas, of Haemonchus contortus and Strongyloides papillosus in sheep. 3 Biotech.
Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, Belo Horizonte, 2020;10:62.
MG 31270‑901, Brazil. 4 Departamento de Ciências Exatas, Universidade 19. Degenkolb T, Vilcinskas A. Metabolites from nematophagous fungi and
do Estado de Minas Gerais, Unidade João Monlevade, João Monlevade, MG nematicidal natural products from fungi as an alternative for biological
35930‑314, Brazil. 5 Universidade do Estado de Minas Gerais, Unidade Passos, control. Part I: metabolites from nematophagous ascomycetes. Appl
Avenida Juca Stockler, Nossa Sra. das Gracas, 1130, Passos, MG 37900‑106, Microbiol Biotechnol. 2016;100:3799–812.
Brazil. 6 Departamento de Produtos Naturais, Faculdade de Farmácia, Univer- 20. Li GH, Zhang KQ. Nematode-toxic fungi and their nematicidal metabo-
sidade Federal de Minas Gerais, Av. Antônio Carlos 6627, Belo Horizonte, MG lites. In: Zhang KQ, Hyde K, editors. Nematode-trapping fungi. Fungal
31270‑901, Brazil. 7 Departamento de Bioquímica e Biologia Molecular, Uni- diversity research series. Berlin: Springer; 2014.
versidade Federal de Viçosa, Av. Peter Henry Rolfs, s/n, Viçosa, MG 36.570‑000,
 Ferreira et al. Parasites Vectors (2020) 13:527 Page 9 of 9

21. Anke H, Stadler M, Mayer A, Sterner O. Secondary metabolites with receptor blockade to dopaminergic and cognitive disruptions associated
nematicidal and antimicrobial activity from nematophagous fungi and with the prefrontal cortex. J Neurosci. 1997;17:2921–7.
Ascomycetes. Can J Bot. 1995;73(Suppl. 1):932–9. 26. Ribeiro FAQ, Pereira CSB, Alves AA, Marcon MA. Treatment of human
22. Olthof THA, Estey RH. A nematotoxin produced by the nematophagous cavitary myiasis with oral ivermectin. Rev Bras Otorrinolaringol.
fungus Arthrobotrys oligospora fresenius. Nature. 1963;197:514–5. 2001;67:755–61.
23. Serafim LR, da Silva JPG, de Paiva NCN, dos Santos HA, Quintão Silva 27. Rowland LM. Subanesthetic ketamine: how it alters physiology and
MDG, Carneiro CM, et al. The crowding effect in Ancylostoma ceylanicum: behavior in humans. Aviat Sp Environ Med. 2005;76(Suppl. 7):52–8.
density-dependent effects on an experimental model of infection. Parasi-
tol Res. 2014;113:4611–21.
24. Gazzinelli-Guimarães PH, Gazzinelli-Guimarães AC, Silva FN, Mati VLT, Publisher’s Note
Dhom-Lemos LDC, Barbosa FS, et al. Parasitological and immuno- Springer Nature remains neutral with regard to jurisdictional claims in pub-
logical aspects of early Ascaris spp. infection in mice. Int J Parasitol. lished maps and institutional affiliations.
2013;43:697–706.
25. Moghaddam B, Adams B, Verma A, Daly D. Activation of glutamatergic
neurotransmission by ketamine: a novel step in the pathway from NMDA

Ready to submit your research ? Choose BMC and benefit from:

• fast, convenient online submission

• thorough peer review by experienced researchers in your field
• rapid publication on acceptance
• support for research data, including large and complex data types
• gold Open Access which fosters wider collaboration and increased citations
• maximum visibility for your research: over 100M website views per year

At BMC, research is always in progress.

Learn more biomedcentral.com/submissions