Waste and Biomass Valorization (2022) 13:3523–3533

https://doi.org/10.1007/s12649-022-01732-x

ORIGINAL PAPER

In vitro anthelmintic activity of extracts from coffee pulp waste,

maize comb waste and Digitaria eriantha S. hay alone or mixed,
against Haemonchus contortus
G. S. Castañeda‑Ramírez1 · I. Y. Lara‑Vergara2 · J. F. J. Torres‑Acosta3 · C. A. Sandoval‑Castro3 · J. E. Sánchez4 ·
J. Ventura‑Cordero5 · V. G. García‑Rubio4 · L. Aguilar‑Marcelino1

Received: 30 September 2021 / Accepted: 13 February 2022 / Published online: 3 March 2022
© The Author(s), under exclusive licence to Springer Nature B.V. 2022

Abstract
Purpose This study evaluated the in vitro anthelmintic (AH) activity against Haemonchus contortus of ten extracts obtained
from coffee pulp waste (Coffea canephora (Co)), maize comb waste (Zea mays (Zm)), pangola grass hay (Digitaria eriantha
Steud (Di) and different mixtures of those materials.
Methods Three batches prepared with individual feedstuffs (T1, T2 and T3), 3 batches formed with 2 feedstuffs (50:50
proportion; T4, T5 and T6), a batch combining 3 feedstuffs (T7) and 3 batches combining 3 feedstuffs (T8, T9 and T10).
The batches of individual feedstuffs and mixtures were used to determine their chemical composition as well as preparing 10
methanol–water (70–30%) extracts. The in vitro tests used against H. contortus were egg hatch test (EHT), larval mortality
test (LMT) and larval exsheathment inhibition test (LEIT).
Results Chemical composition suggested that the nutritional value of Co and the batches including Co (T1, T4, T6 to T10)
could be used for ruminant nutrition, but the Di and Zm showed very poor nutritional potential unless they are combined
with Co. Extracts showing activity against eggs were T4 and T8 (P < 0.05). Significant ­L3 mortality was reported for extracts
T1, T3, T4 and T8 (P < 0.05). Extracts of T3, T4, T7, T8 and T10 showed an EC50 < 1000 μg/mL for the L ­ 3 exsheathment
inhibition (P < 0.05). Chemical analyses showed the presence of coumarins and flavonoids in all the extracts.
Conclusion Extracts obtained from T4 and T8 showed the best overall activity in the three in vitro tests against H. contortus
and a good nutritional quality that could be suited for ruminant nutrition.

* L. Aguilar‑Marcelino
aguilar.liliana@inifap.gob.mx
1
Centro Nacional de Investigación Disciplinaria en Salud
Animal e Inocuidad, INIFAP, Km 11 Carretera Federal
Cuernavaca‑Cuautla, No. 8534, Col. Progreso, Jiutepec,
Morelos C.P. 62550, México
2
Centro Universitario Amecameca, Universidad Autónoma del
Estado de México, Carretera Amecameca‑Ayapango km 2.5,
C.P. 56900 Amecameca, México
3
Facultad de Medicina Veterinaria y Zootecnia, Universidad
Autónoma de Yucatán, Km 15.5 Carretera Mérida‑Xmatkuil,
C.P. 97100 Mérida, Yucatán, México
4
El Colegio de La Frontera Sur, Apdo. Postal 36,
C.P. 30700 Tapachula, Chiapas, México
5
School of Biological Sciences, Queen’s University Belfast,
Chlorine Gardens, Belfast BT9 5BL, UK

13
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3524
Graphical Abstract
Keywords Larval mortality test · Egg hatching test · Anthelmintic activity · Coffea canephora, Zea mays and Digitaria
eriantha Steud
Statement of Novelty
The purpose of this research was to find out if the follow‑
ing by-products; coffee pulp, maize and pangola grass, and
their mixtures, had in vitro anthelmintic activity against
the haematophagous nematode Haemochus contortus. The
use of alternative methods of control against sheep para‑
sites is necessary to avoid damage to the environment and
to decrease anthelmintic resistance. In addition, the use
of by-products from another process is beneficial to the
industry because it makes use of by-products that may not
have had a purpose before.
Introduction
The irrational use of commercial anthelmintic (AH) drugs
for the control of gastrointestinal nematodes (GIN) in
ruminant production systems has generated AH resistant
worm populations and environmental damage [1]. As a
result, alternative methods of GIN control could help to
decrease the use of commercial AH drugs. The search for
novel control methods includes the use of agro-industrial
by-products, some of which have been tested against GIN
with positive results [2]. Recent studies [3] suggested the
potential AH activity of macromycetes (Pleurotus spp.)
13
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Waste and Biomass Valorization (2022) 13:3523–3533
mycelia against Rhabditoid larvae. More recently, the AH
activity against Haemonchus contortus, an important GIN
affecting the abomasum of ruminants, has been reported
for extracts obtained from macromycetes under in vitro
conditions [4, 5]. Further studies suggested that extracts
obtained from basidiomas, mycelia and degraded materi‑
als of Pleurotus spp. also have in vitro AH activity against
H. contortus [6–8]. The latter showed the potential AH
activity of the degraded substrate, which is a mixture of
substrates that provide macro and micronutrients neces‑
sary for the production of basidiomas. These substrates
are mainly of lignocellulosic nature, which is appropri‑
ate for the development of edible mushrooms. For exam‑
ple, the production of 1 kg of edible mushrooms requires
approximately 60 kg of substrate. Hence, a large quantity
of waste materials must be discarded after mushrooms are
harvested, representing a great ecological problem. How‑
ever, those waste products of the production of basidiomas
of macromycetes could represent an important resource
for ruminants in the form of an alternative method of GIN
control.
The search for AH activity in the macromycete pro‑
duction should also consider the potential activity of the
substrate before it is used for the production of the edible
mushrooms. To the best of our knowledge, there is no infor‑
mation on the potential AH activity of substrates used for
the production of the fungus. The substrates used for the
production of macromycetes include materials obtained after
Waste and Biomass Valorization (2022) 13:3523–3533 3525

harvesting different crops [9]. The use of byproducts with Table 1  Proportions of feedstuffs Coffea canephora, Zea mays and
AH activity began with the use of husks and leaves of plants Digitaria eriantha Steud included in batches used for the chemical
analyses and the production of extracts tested against Haemonchus
with importance in agriculture. Some of these examples are contortus eggs or L
­3
the evaluation of the AH ovicidal effect of the acetone–water
extracts of Theobroma cacao (shell and seed), as well as Identifica‑ Proportion of substrate included Yield (%)
tion of
percolated residue of Coffea arabica. An ovicidal effect was extract Corn residue— Digitaria Coffea
observed in the extracts of T. cacao (shell and seed) [2]. Zea mays (Zm) eriantha canephora
(Di) (Co)
Later, another study evaluated the shell of T. cacao, and it
showed AH activity against larvae and eggs of H. contortus T1 0 0 100 7.98
[10]. In 2016 the residues of C. arabica were evaluated, T2 0 100 0 5.28
where the percolated residue of coffee from the coffee indus‑ T3 100 0 0 1.1
try was evaluated and an in vitro AH effect was observed T4 0 50 50 7.70
in this product [11]. Recently, the essential oil from orange T5 50 50 0 3.35
peels was evaluated in vivo on sheep against H. contortus. T6 50 0 50 4.53
The essential oil was administered in the food concentrate T7 33.33 33.33 33.33 3.52
for 10 days, the number of eggs per feces was counted in T8 66.66 16.66 16.66 3.52
grams among other variables and a deworming activity of T9 16.66 16.66 66.66 7.26
the treatments with orange peel oil was observed. The search T10 16.66 66.66 16.66 5.98
of AH activity of by-products is an emerging activity due
to its potential ecological and economic implications. Cur‑
rently, plant materials that could help fight and control gas‑ but were not seeded with the fungi. The batches of indi‑
trointestinal parasites are discarded in most industries. For vidual substrates and different mixtures were used to obtain
this reason, the aim of the study was to evaluate in vitro extracts and the chemical analyses.
ten hydroalcoholic extracts from different mixtures of three
materials to determine their AH value against H. contortus
and to learn about some of their chemical components. Chemical analysis of different feedstuffs
and mixtures

Materials and methods
Preparation of the mixtures used
For this experiment, maize comb waste (Zea mays (Zm)),
coffee pulp waste (Coffea canephora (Co)), and pangola
grass hay (Digitaria eriantha Steud (Di) were acquired
locally in the Soconusco region, Chiapas, Mexico. The Zm
residues were obtained from local maize growers, the Di
hay from local farmers who sell it as livestock feed and the
Co pulp came from a mill dedicated to the processing of C.
canephora. The individual substrates were used to prepare
different mixtures as shown in Table 1. The inclusion levels
of substrates were based on the conventional materials and
mixtures used for the commercial production of Pleurotus
spp. It included 3 batches with the individual feedstuffs (T1,
T2 and T3), 3 batches formed with 2 feedstuffs in a 50:50
proportion (T4, T5 and T6), a batch combining 3 feedstuffs
in equal proportion (33.3% each; T7), and 3 batches com‑
bining 3 feedstuffs with 2/3 of one ingredient and 1/6 of the
other two ingredients as described in Table 1. Batches of
one kilogram (at 65% humidity) of the individual substrates
and the different mixtures were sterilized at 1.05 kg/cm 2
(120 °C) for 1 h. These materials were prepared in the same
manner as those used for the production of Pleurotus spp.
Each of the 10 mixtures was dried in an oven at 40 °C until
reaching constant weights. Once dry, the samples were
ground into a mesh with an opening of 1 mm. The analy‑
ses were: dry matter (DM#7.007), crude protein (CP) (no.
2057), ash (#7,009) [12], neutral detergent fiber (NDF) [13],
and acid detergent (ADF) [9]. Also, total phenols (TP) and
total tannins (TT) were determined by the Folin-Ciocalteu
method as described [14]. The content of condensed tannins
(CT) was quantified by the vanillin method using catechin
as standard [15].
Hydroalcoholic extracts
Hydroalcoholic extracts were made from the mixtures and
substrates, using a 70–30% v/v methanol–water solution.
Each of the mixtures and substrates (150 g each) were depos‑
ited in different 4 L Erlenmeyer flasks, adding the hydroal‑
coholic solution and left to macerate for 24 h. After that,
the separation was carried out by filtration, obtaining the
extract without solid residues. These extracts were concen‑
trated using a rotavapor to eliminate the methanol solvent.
Finally, the extracts were lyophilized and stored at − 20 °C
until use [5]. The yield of each extract was determined, and
the formula is presented in the data analysis section.

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3526
Production of Haemonchus contortus eggs
and larvae
The H. contortus eggs were obtained from an artificially
infected 4-month-old male donor lamb (25 ± 1 kg). The
donor was kept indoors under pen conditions and was fed
a full diet including chopped Medicago sativa hay a con‑
centrate feed and water ad libitum. This lamb was experi‑
mentally infected with H. contortus larvae which have been
characterized as resistant to ivermectin and benzimidazole.
After a period of 21 days, faecal samples of the donor lamb
were used to confirm the presence of H. contortus eggs using
the McMaster technique [16]. Once the H. contortus eggs
were present in the faeces, the donor animal was housed in a
metabolic cage. The faeces of the donor animal were used to
obtain eggs and were also collected for coproculture, incu‑
bated for 7 days at 28 °C, then the larvae (­ L3) were recovered
using the Baermann technique [17].
Recovery of Haemonchus contortus Eggs
The faeces of the donor animal were collected and macer‑
ated until a homogeneous mixture was obtained, this mix‑
ture was sieved (using successive sieves # 40, 100, 200 and
400). The sieved sample was recovered in a 250 mL beaker.
Afterward, washing was carried out, part of the sample was
placed in 15 mL tubes and sucrose was added at 20% (1:4),
a band was formed by density where the eggs were found,
which were collected. This sample of eggs was washed with
water and quantified to know the concentration of eggs [17].
Egg Hatch Test (EHT)
A first evaluation of the ten extracts was performed in 96
well plates at 3600 µg/mL following the methodology
described [18, 19]. The extracts were evaluated at different
concentrations (150, 300, 600, 1200, 2400 and 3600 µg/mL)
using a final volume of 100 µL, including the egg suspension
(200 eggs), with their respective positive controls (thiaben‑
dazole 50 µg/mL) and negative controls (PBS/pH 7.4), each
with four replicates. Incubations were left at 28 °C for 48 h
to hatch the eggs. Finally, each well was read in the opti‑
cal microscope (10X and 40X) observing and counting the
hatched larvae and unhatched eggs as described by Vargas-
Magaña [2].
Mortality Bioassay Against Larvae
This test consisted of exposing exsheathed larvae to the
respective extracts and observing the induced mortality.
This bioassay is based on the larval motility test [20], with
some modifications (i.e., no screen inserts are used, and the
exposure time is 72 h). Also, ­L3 are used without sheath
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Waste and Biomass Valorization (2022) 13:3523–3533
since the susceptibility of larvae without sheath is higher
[21]. The exsheathment protocol consisted in incubating
H. contortus ­L3 in 0.18% sodium hypochlorite for 5 min.
The absence of the sheath was verified in 10 aliquots of
10µL. Subsequently, three washes were made with distilled
water and the ­L3 suspension was centrifuged at 168 G for
three minutes. After the last washing, the supernatant was
discarded, and the larvae were recovered in the sedimented
pellet [22]. The 96 well plates were used for the test. Each
extract from individual feedstuffs or the different mixtures
were used at different concentrations (150, 300, 600, 1200
and 2400, 3600 µg/mL) with their respective positive con‑
trols (Levamisole-2000 µM/mL) and negative controls (PBS/
pH 7.4), together with 200 exsheathed L ­ 3 in each well. All
wells had a final volume of 100 µL. The larvae were left
in contact with the extract for 72 h. The final reading was
made using the compound microscope, where live and dead
larvae were observed and quantified. This quantification was
done on slides (10 µL aliquot-drop), observing the larvae,
counting the degraded larvae and those that did not move
after manual stimulation are considered dead. The larvae
were counted as alive when they actively moved after the
incubation time [23].
Larval Exsheathment Inhibition Test (LEIT)
The respective LEIT were conducted following the proce‑
dure described by Jackson and Hoste [19]. A stock solution
was prepared using the respective methanol:water extracts
at 10,000 µg/mL PBS. Levamisole (3000 µg/mL) was used
for the positive controls and the negative controls consisted
of larvae exposed to PBS only. The extract concentrations
used were 2500, 1200, 600, 300 and 150 µg/mL PBS. Each
concentration was added with 1000 μL of a solution contain‑
ing 1000 L­ 3 to obtain the final concentrations of the extracts.
The ­L3 were incubated with the plant extract for 3 h at 24 °C.
After that time, the L­ 3 were centrifuged for 3 min at 168 G
and washed 3 times with the PBS solution. Then, aliquots
of each larval solution were placed in Eppendorf tubes (200
μL each). Four replicates were made for each concentration
and PBS control. The tubes were kept overnight at 8 ºC.
The exsheathment process was artificially induced by con‑
tact with a solution of sodium hypochlorite (2%) and sodium
chloride (16.5%) diluted in 6 mL of PBS (Final chlorine con‑
centration 2.5% = 25 μL). The exsheathment kinetics were
evaluated under a microscope using the 10X objective, and
were recorded at minutes 0, 20, 40 and 60 [19].
Qualitative Phytochemistry
The 10 extracts produced from the individual materials and
mixtures were used for qualitative phytochemistry analy‑
ses. Different methodologies were carried out, in each case
Waste and Biomass Valorization (2022) 13:3523–3533 3527

the reagents were added to 5 mg of extract and were read
after 5 min. A change in colour indicated a positive result.
The Liebermann-Burchard reaction suggested the presence
of triterpenes when changing to purple. The methodology
consisted in adding 1 mL of the acetic anhydride reagent,
100 µL ­H2SO4 and 10 drops of HCL. The Shinoda reac‑
tion suggested the presence of flavonoids when changing to
red. The methodology consisted in adding 1 mL ­CH3OH,
2–3 Mg chips and 2–3 drops HCL. Molisch’s test (carbohy‑
drate-violet): 1 mL of water, 2 drops of alpha-naphthol (1%)
and 7 drops of ­H2SO4. The reaction to identify coumarins
suggested the presence of those substances when the yellow
colour of the extract disappears. The methodology consisted
in adding 1 mL of C ­ H3OH, 1 mL of alcoholic NaOH and 8
drops of HCL. The saponins were confirmed by foam forma‑
tion when adding the extract to 1 mL of water. The Wagner
reaction indicated the presence of alkaloids when the colour
change to brown. The methodology consisted in adding 1
chip of Iodine, KI and 1 mL of distilled water [24].
with the different extracts were analyzed using respective
generalized linear model (GLM) to assess differences
between PBS control and the concentrations evaluated. The
EHT/mortality results obtained for the different extracts
at different concentration levels were compared with their
respective PBS negative controls. For each analysis, a post-
hoc test was performed using Fisher's LSD. The GLM and
post-hoc analyses were performed with Statgraphics Centu‑
rion XV (Statpoint Technologies Inc. 2005).
Respective effective concentration 50% (­ EC50) were cal‑
culated for each in vitro test with the respective extract using
the Polo Plus Program, and the 95% confidence intervals
were also calculated.
Results
Yields of the hydroalcoholic extracts produced with the dif‑
ferent mixtures were included in Table 1.

Data Analysis Chemical Analysis of Different feedstuffs

and Mixtures
The yields of different extracts were calculated as follows:

(Final weight of the freeze dried extract) × (100)

% =
(Initial weight of the material subjected to the preparation of the extract)

The formula used to calculate the percentage inhibition The results obtained from the chemical analyses of the mix‑
of egg hatching obtained for each extract was previously tures evaluated are presented in Table 2. The CP content
described by Vargas-Magaña [2], shown below: fluctuated from 3.66% for Z. maize residue (T3) to 14.94%

Number of larvae
Percentage of hatching (PHT) = × 100
Number of eggs + Number of larvae

Percentage of egg hatching inhibition = PHT − 100 for C. canephora residue (T1). Similarly, the minimum con‑
tent of lignin was recorded for T3 (8.89%) and the maximum
The formula used to estimate the larvae mortality for each for T1 (20.32%). Meanwhile, the NDF content varied from
extract was as follows [5]: 47.84% (T1) to 84.84% in T3. The EE, TT and TP values

Dead larvae
Mortality = × 100
Number of live larvae + Number of dead larvae

The exsheathment inhibition percentage (EI) obtained for were low for all the materials and mixtures ranging from
each extract with the LEIT, was determined as [25]: 0.06 to 0.83% for T3 and T2. No CT were detected in the

Larvae L3 without sheath

Exsheathment % = × 100
Larvae with sheath + Larvae without sheath

individual materials and mixtures.

EI % = 100 − Exsheathment %
The results of egg hatching inhibition, larvae mortality
and larvae exsheathment inhibition of H. contortus obtained

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3528 Waste and Biomass Valorization (2022) 13:3523–3533

Table 2  Chemical analysis (%) Mixtures CP NDF ADF Lignin EE TP TT CT*

of the individual feedstuffs and
mixtures of feedstuffs produced T1 14.94 47.84 46.33 20.32 0.57 0.36 0.17 ND
with different proportions of
T2 5.86 59.2 41.21 11.23 0.76 0.41 0.12 ND
Coffea canephora (Co), Zea
mays (Zm) and Digitaria T3 3.66 84.84 46.59 8.89 0.06 0.37 0.24 ND
eriantha Steud (Di) T4 11.05 48.62 39.52 14.04 0.93 0.45 0.18 ND
T5 4.92 72.51 44.55 9.58 0.42 0.41 0.21 ND
T6 9.95 63.61 47.69 15.66 0.29 0.37 0.17 ND
T7 9.01 60.78 42.95 12.15 0.34 0.38 0.15 ND
T8 6.20 72.59 44.38 9.71 0.26 0.41 0.24 ND
T9 12.13 54.71 45.38 16.65 0.83 0.45 0.23 ND
T10 7.12 60.31 41.32 11.5 0.72 0.43 0.15 ND

CP crude protein, NDF neutral detergent fibre, ADF acid detergent fibre, TP total phenols, TT total tannins,
CT condensed tannins, EE ether extract, ND not detected

Table 3  Effective concentrations 50% ­(EC50) and confidence intervals Steud (Di), obtained when using the egg hatching inhibition test, lar‑
(CI) of hydroalcoholic extracts produced with different proportions vae mortality tests and the larval exsheathment inhibition test against
of Coffea canephora(Co), Zea mays (Zm) and Digitaria eriantha Haemonchus contortus

Test materials Egg hatch inhibition Larvae mortality Larvae exsheathment inhibition
CE50 (µg/mL) 95% Confidence interval CE50 95% Confidence interval CE50 95% Confidence interval
(µg/mL) (µg/mL)

T1 – – 1189.7ª 810.3–1879.6 – –
T2 – – – – 1018.1e 806.2–1344.3
T3 – – 2376.0a 1420.4–6200.4 600.1d 529.7–681.8
T4 2874.6a 1907.2–5799.2 1576.4ª 1330.5–1856.7 520.0 cd 392.8–713.7
T5 – – – – 2618.4f 1617.0–9083.7
T6 – – – – 1766.4f 1322.2–2784.4
T7 – – – – 306.3ab 231.4–384.4
T8 1755.2a 1542.9–2009.9 1193.4ª 737.9–1694.7 214.7a 151.9–273.5
T9 – – – – 2079.9ef 1269.4–4063.4
T10 – – – – 385.9bc 335.3–438.7
a,b
Different letters in the same column indicate a significant difference (P < 0.05)
“–” absence of value, it can not be calculated due to the low AH activity

Effective Concentration ­(EC50) of Extracts
The extracts’ ­EC50 values, and respective 95% CI, found for
the EHT, larval mortality tests and the LEIT are shown in
Table 3. The T4 and T8 extracts showed significant activ‑
ity against H. contortus eggs (P < 0.05). The main effect
observed was the ­L1 trapping, which results in a hatching
inhibition effect. In addition, the larvae that emerged from
eggs showed structural damage (Fig. 1).
Subsequently, the extracts showing significant E ­ C50 val‑
ues for the larval mortality test were T1, T3, T4 and T8
(P < 0.05).
Meanwhile, all extracts showed AH activity against
exsheathment H. contortus, with the sole exception of
T1. However, the most effective results for the LEIT test
were the extracts T8, T7 and T10 ­( EC 50 = 214.7, 306.3
and 385.9 μg/mL).
Qualitative Phytochemistry
The presence or absence of secondary compounds tested
in the ten extracts is shown in Table 4. Triterpenes and
alkaloids were not present in the 10 extracts evaluated.
Flavonoids and coumarins were present in all the extracts
while saponins was only observed for extracts T3, T6 and
T7.

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Waste and Biomass Valorization (2022) 13:3523–3533 3529

Fig. 1  Eggs and Larvae of Haemonchus contortus incubated with PBS and extracts showing the anthelmintic effects caused by extracts: a, d
normal egg and larvae, b, c morulated egg, e larvae that emerged whit structural damage and f egg with a larva failing eclosion

Table 4  Presence of Triterpenes Test materials Secondary compounds

(Trit), Flavonoids (Flav),
Carbohydrates (Carb), Trit Flav Carb Cum Sap Alk
Coumarins (Cum), Saponins
(Sap) and Alkaloids (Alk) in Control (without extract) − − − − − −
methanol:water extracts (70:30) T1 − + − +  +  + − −
T2 − +  +  + − +  +  + − −
T3 − +  + − +  + + −
T4 − +  + − +  +  + − −
T5 − + − +  + − −
T6 − + − +  +  + + −
T7 − +  + − + + −
T8 − + − + − −
T9 − + − +  +  + − −
T10 − +  + − + − −

( −) Absence
( +) Presence

Discussion of conventional drugs to beneficial microorganisms in the

environment. The use of lignocellulosic residues from the
The search for new sustainable control alternatives is nec‑ production of edible mushrooms represents a potential
essary to limit the negative impact of AH resistant GIN low-cost feed source for ruminants. Some of these agroin‑
populations in ruminants and to limit the potential damage dustrial by-products and waste materials are currently

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3530
explored in the search for alternatives that can be used
as nutraceuticals against GIN of ruminants [2, 11, 26].
For this reason, this protocol also evaluated three waste
materials and their mixtures which are commonly used for
the production of Pleurotus spp. to identify their potential
nutraceutical value before they are used for the production
of edible mushrooms.
Bromatological Composition
This study evaluated the nutritional characteristics of rumi‑
nant feeds such as CP, ash, NDF, ADF, CT, TT, and TP
(Table 2). The mixtures and single materials presented CP
values < 15%. It was evident that Co (T1) had the best nutri‑
tional potential as it had the highest CP content, and the
lowest ADF and NDF values, which should result in a better
ruminal degradability, only limited by its considerably high
lignin content. The latter implies that all the mixture batches
containing Co (T4, T6 to T10) had CP values > 6.2%, and
limited ADF and NDF values. Those mixtures containing
Co had nutritional values comparable to plants consumed
by sheep and goats in the tropical forest of Yucatán [27].
The evaluation of D. eriantha hay (Di) showed a low CP
content (T2 = 5.86%) but the latter was expected as most
tropical grasses show low CP content (< 5% CP) [28]. Simi‑
larly, the low CP content of Z. mays cob residue was also
expected. This poor-quality waste product is not considered
suitable for ruminant feeding due to its low CP content as
well as high fibre content [29]. The present study showed
that all the materials and mixtures had low TT and TP val‑
ues. Meanwhile, the three different independent feedstuffs
and their respective mixtures showed no CT content, which
could be due to the high temperatures used to sterilize all
the batches (120 ºC for 1 h) for its use in the production
of edible mushrooms. The CT can be denaturalized with
high temperatures [30]. The T1 and mixtures containing
Co (T4 and T6-T10) can be considered potentially useful as
ruminant feeds. It is important to mention that, for in vivo
nutraceutical evaluations, more factors such as digestibility,
availability, absence of any toxic or harmful effects should
also be explored before these materials can be considered
good for animal nutrition [31]. Those aspects still need to
be explored.
In vitro Evaluation of Extracts Against Eggs of H.
contortus
Extracts T4 and T8 showed the best activity against the egg
hatching of H. contortus (Table 3). The T4 extract was pre‑
pared with Co and Di (50% of each ingredient), while the
T8 extract was prepared with 16% Co, 16% Di and 66% Zm.
Thus, the two extracts with the best activity had two ingredi‑
ents in common: Co and Di. To the best of our knowledge,
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Waste and Biomass Valorization (2022) 13:3523–3533
this is the first study to show evidence of AH activity for the
C. canephora pulp. Previous studies explored the AH activ‑
ity of the spent coffee ground of Coffea arabica against H.
contortus eggs [2, 11].
It is evident that the extract of Co (T1) was not as active
as the combinations T4 and T8. Thus, it seems that the com‑
pounds of these mixtures of ingredients show synergistic
activity against H. contortus eggs as already reported [32,
33]. The effect against eggs observed with these extracts was
associated with larvae trapped inside the eggs, which is the
most common effect reported for hydroalcoholic extracts of
different tropical plants when confronted to H. contortus [2].
It is also important to mention that the few larvae that were
able to hatch from eggs showed structural damage (Fig. 1),
which is consistent with previous reports obtained with leaf
extracts from tropical trees [27, 34], and also with extracts
obtained from Theobroma cacao leaves and husks [10].
In vitro Evaluation of Extracts Against H. contortus
Larvae
The materials tested in the present study had never been
tested using the mortality test against H. contortus ­L3. A sig‑
nificant effect was observed for T1, T3, T4 and T8 extracts
(Table 3). The activity of those extracts was present only
when using large quantities of extract (> 1189.7 µg/mL),
which may suggest that the mortality test possesses a low
sensitivity. This can be confirmed with other in vitro studies
using the same method with different plant extracts, where
the authors used very large quantities of extracts to find AH
activity, sometimes > 10 times more than in the present study
[7, 8]. However, in the present study we only attempted to
evaluate those extracts at concentrations < 3600 µg/mL. The
latter is based on the low possibility of finding larger con‑
centrations of secondary compounds in the ruminal liquor of
ruminants when these animals consume the plant materials
in a nutraceutical approach. It is possible that other authors
looking for medicinal extracts could be interested in testing
higher extract concentrations.
The evaluation of larval exsheathment inhibition pro‑
duced interesting results. The LEIT is a very sensitive test,
in which AH activity can be detected at very low E ­ C50 val‑
ues. However, the activity of most plants against exsheath‑
ment has been mainly associated with polyphenols, par‑
ticularly CT. In the present study, the plant extracts had
no CT content, hence it was thought that the AH activity
against exsheathment would be limited or non-existing.
Surprisingly, most extracts and combinations showed activ‑
ity against exsheathment of L ­ 3, with the exception of T1
(Table 3). Such activity suggests that other plant secondary
compounds are responsible for that activity. The activity of
T7, T8 and T10 were remarkable and could be interesting
to explore which secondary compounds were associated
Waste and Biomass Valorization (2022) 13:3523–3533
with their AH effect. Most of the other extracts had mild to
low AH activity when compared to previous results using
acetone:water plant extracts from tropical materials [34].
Previous studies never tested tropical grasses, such as the
pangola grass, for its in vitro AH activity, since it is normally
considered a material without any AH activity. Several studies
have used these tropical grasses in the control diets of stud‑
ies involving GIN infected animals [28, 35]. As expected, the
activity of the pangola grass extract (T2) against eggs and lar‑
vae mortality was absent. However, we report here the activity
of the T2 extract against L3 exsheathment. It is possible that
the thermic manipulation of the grass material used for T2
extract could have modified some of its compounds, allowing
them to present AH activity under this special circumstance.
This is again another aspect deserving further research. Simi‑
larly, the results of the AH activity obtained from the corn
residues (T3), i.e. egg hatching inhibition and larvae mortal‑
ity, could also be associated with changes in the secondary
compounds, which would normally be inactive without the
thermic treatment. The latter emphasizes the importance of
manipulation/handling of plant materials before or after pre‑
paring extracts.
The in vitro tests represent a screening tool to explore fur‑
ther in vivo activity [35]. However, it is unknown whether a
material showing activity to any of those tests could result in
a clear in vivo response. Some tested materials have shown
in vivo AH activity [28, 36, 37], but other materials did not
[11]. Considering that complexity of possible outcomes, we
suggest starting bio-guided evaluations of this type of materi‑
als to find out what are the compounds responsible for the
respective activities as performed in previous studies [34] with
the following two approaches:
• To explore extracts with significant activity in all three
in vitro tests. With that idea, we could suggest exploring
T4 (combination of Di and Co) or the T8 (triple combina‑
tion of Zm, Di and Co).
• To explore those with the best exsheathment inhibition
activity: This will consider exploring those materials with
an ­EC50 < 300 µg/mL, which has been used for other plant
materials using this same LEIT. In that case, we can con‑
centrate on the T8 with the triple combination of ingredi‑
ents.
Qualitative Phytochemistry
Flavonoids were found in most of the extracts and it has been
reported that different groups of polyphenols, among them
flavonoids, can have AH activity [32, 38]. So, this group of
compounds may be responsible for some of the AH activity
found in the present study. Coumarins were also found in all
the extracts evaluated, hence we do not consider that the activ‑
ity was associated with those metabolites. However, there are
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
3531
reports that some coumarins have shown AH activity against
a phytoparasite and a free-living nematode (Bursaphelen-
chus xylophilus and Panagrellus redivivus respectively) [39].
More studies are still needed to understand the synergy and
antagonism of the secondary compounds upon the AH activ‑
ity against eggs and ­L3 of GIN. Saponins were found in T3,
T6 and T7, but it was not possible to associate these types of
compounds with any in vitro AH activity.
Conclusion
The T1 and mixtures containing Co (T4 and T6-T10) can be
considered potentially useful as ruminant feeds. The T4 and
T8 extracts showed low AH activity against eggs. Similarly,
T1, T3, T4 and T8 extracts caused ­L3 mortality. Although,
no CT were detected in the extracts under evaluation we
identified inhibition of exsheathment in most tested extracts
(except for T1). Extracts T4 and T8 showed AH activity in
the three in vitro tests, which included eggs and larvae of H.
contortus. Thus, the T4 and T8 materials could be consid‑
ered with potential as nutraceutical materials for ruminants
against H. contortus. Further bio-guided studies should be
implemented to explore the active secondary compounds
contained in those extracts.
Author Contributions C-RGS: Experimental design carried out the
experiment, data analysis, critical review, manuscript approval for
publication, agreement to be accountable for all aspects of the research
paper. L-VIY Carried out the experiment, data analysis, critical review,
manuscript approval for publication. T-AJFJ Bromatological analysis,
critical review, manuscript approval for publication. S-CCA Bromato‑
logical analysis, critical review, manuscript approval for publication.
SJE Production of materials, critical review, manuscript approval for
publication. V-CJ critical review, manuscript´s drafting, manuscript
approval for publication, agreement to be accountable for all aspects
of the work. GRVG Critical review, manuscript approval for publica‑
tion. A-ML Experimental design, critical review, manuscript´s drafting,
manuscript approval for publication, agreement to be accountable for
all aspects of the manuscript.
Funding The present study was financed by the CONACYT's National
Problems Project with Number: 9342634372.
Data Availability The datasets used and/or analyzed during the current
study available from the corresponding author on reasonable request.
Declarations
Conflict of interest All authors declare that they have conflict of inter‑
est.
Ethical Approval For the handling of the animals, the good practices
of CENID-SAI, INIFAP and the MEXICAN OFFICIAL STANDARD
NOM-051-ZOO-199, humane treatment in the mobilization of the ani‑
mals, as well as the FEDERAL LAW ON ANIMAL HEALTH, were
fully adhered to.
13
3532 Waste and Biomass Valorization (2022) 13:3523–3533

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