In vitro Anthelmintic Activity of Two Aloe-derived Active Principles against Sheep Gastrointestinal Nematodes

Volume 12. Issue 1

2. Pages 1821-1970. 2017

ISSN 1934-578X (printed); ISSN 1555-9475 (online)

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University of Palermo, Viale delle Scienze, Parco d’Orleans II - 90128 Palermo, Italy [email protected]

PROFESSOR VLADIMIR I. KALININ

G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Pr. 100-letya Vladivostoka 159, 690022, Vladivostok, Russian Federation [email protected]

PROFESSOR YOSHIHIRO MIMAKI School of Pharmacy,

Tokyo University of Pharmacy and Life Sciences, Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan [email protected]

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Department of Chemistry, University of Wollongong, Wollongong, New South Wales, 2522, Australia [email protected]

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Department of Chemistry, The University of Alabama in Huntsville, Huntsville, AL 35809, USA

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National Museum of Marine Biology and Aquarium Checheng, Pingtung 944

Taiwan

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Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3 Kanagawa-machi, Kanazawa 920-1181, Japan [email protected]

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HONORARY EDITOR PROFESSOR GERALD BLUNDEN The School of Pharmacy & Biomedical Sciences,

University of Portsmouth, Portsmouth, PO1 2DT U.K.

In vitro Anthelmintic Activity of Two Aloe-derived Active Principles

against Sheep Gastrointestinal Nematodes

Gianluca Fichi

_{, Matteo Mattellini}

_{, Elisa Meloni}

_{, Guido Flamini}

_{and Stefania Perrucci}

_{Dipartimento di Scienze Veterinarie, University of Pisa, Viale delle Piagge n. 2, 56124 Pisa, Italy}

_{Dipartimento di Farmacia, University of Pisa, Via Bonanno Pisano 6, 56126 Pisa, Italy}

[email protected]

Received: May 5

_{, 2017; Accepted: October 22}

_{, 2017}

The in vitro anthelmintic activity on sheep gastrointestinal strongyle (GIS) eggs and larvae of 0.5% aloin and 0.1% aloe-emodin was investigated. From fresh faecal samples collected by ewes naturally infected by Haemonchus, Trichostrongylus and Teladorsagia nematodes, GIS eggs were isolated and cultivated in Petri dishes (100 eggs/dish). For the in vitro evaluation of the anthelmintic activity of tested compounds, the Egg hatch test (EHT), the Larval development test (LDT) and the Larval mortality/paralysis test (LMT) were used. In each assay, the activity of tested compounds was compared to untreated and treated (0.1% thiabendazole, TBZ) controls. Six repetitions were made through the experiment. Obtained data were statistically elaborated using the X2 _{test. In EHT,}

0.5% aloin gave highly significantly different (P<0.01) results from the untreated controls. In LDT, both 0.1% aloe-emodin and 0.5% aloin almost completely prevented the larval development from L1 to L3, showing no significant differences (P<0.01) when compared to TBZ. In LMT, larval mortality observed in

0.5% aloin treated plates was significantly higher (P<0.01) than that observed in TBZ treated controls. These results show the in vitro anthelmintic properties on sheep GIS of the examined plant secondary metabolites. In LDT and/or LMT, the activity of 0.5% aloin and 0.1% aloe-emodin was comparable to or higher than that of the reference drug.

Keywords: Aloin, Aloe-emodin, Anthelmintic activity, Gastrointestinal nematodes, Sheep.

Worldwide, disease caused by gastrointestinal strongyles (GIS) is

considered one of the most important health constraints affecting

productivity in small ruminants [1]. GIS may limit sheep production

by causing retarded growth, weight loss, reduced food consumption,

lower milk production, impaired fertility and even mortality in

heavy parasite burden [2]. Sheep GIS control programs rely mostly

on the use of synthetic anthelminthics [3]. However, the

anthelmintic resistance developed by these nematodes worldwide,

the risk of residues in sheep products and the cost of commercial

anthelmintic drugs, have led to the need to find alternative

treatments and methods for sheep nematode control [1, 4, 5]. In

particular, there is a clear desire to create systems of sheep

production that rely less heavily on chemotherapy [1]. Among

alternative strategies, there has been considerable and expanding

interest in the search for effective and safe herbal dewormers [2]. In

fact, the use of plant-derived compounds with anthelmintic

properties seems to be an effective alternative from the standpoint

of parasite control and their low environmental impact from

residues in relation to commercial anthelmintics. Herbal medicine

can increase profits, by reducing the use of conventional

anthelmintics and extending the useful life of the limited number of

available anthelmintics [1, 6].

Aloin and aloe-emodin are two active principles mainly found in the

leaves of several Aloe species [7, 8]. In ethnoveterinary studies, the

antiparasite activity of Aloe species against some helminths of farm

animals, including the anthelmintic efficacy of Aloe rupestris and

Aloe ferox against the sheep GIS species Haemonchus contortus,

has been shown [9-11]. Moreover, the antiprotozoal activity of aloin

and aloe-emodin has been demonstrated [12-14].

In this context, this study was aimed to evaluate the in vitro

anthelmintic activity on GIS of 0.5% aloin and 0.1% aloe-emodin.

More specifically, the efficacy of tested compounds was evaluated

on GIS eggs obtained by naturally infected ewes and on GIS larvae

obtained from the cultivation of eggs. For the in vitro evaluation of

the anthelmintic activity of tested compounds, the Egg hatch test

(EHT), the Larval development test (LDT) and the Larval

mortality/paralysis test (LMT) were used. Furthermore, GIS larvae

were identified at the genus level. In each test, plates treated with

tested compounds were compared to untreated plates (untreated

controls) and plates treated with 0.1% thiabendazole (TBZ) (treated

controls).

Identified GIS larvae were found to belong mostly to the genus

Haemonchus and to a lesser extent to the genera Trichostrongylus

and Teladorsagia (Table 1).

Table 1: Mean percentage of each gastrointestinal strongyle genus identified in

positive faecal samples from a naturally infested ewe living in an organic sheep flock located in Tuscany (Italy).

Genus Percentage

Haemonchus 93%

Trichostrongylus 4%

Teladorsagia 3%

Haemonchus contortus is the species of the genus Haemonchus

infecting sheep and other livestock and it is considered one of the

main parasites of small ruminants all over the world [15].

From the evaluation of the in vitro anthelmintic activity of tested

compounds, in EHT aloin gave highly significantly different results

(P<0.01) from the untreated controls (Table 2). In particular, in this

test 0.5% aloin showed 39.7% inhibition of egg hatch, while only

13.4% of eggs were found un-hatched in control plates (Table 2).

However, the inhibition of the egg hatch of 0.5% aloin and, mainly,

0.1% aloe-emodin (16.6%) was significantly lower than that

observed in 0.1% TBZ treated plates (94.8%) (P<0.01) (Table 2).

In LDT, the two examined compounds showed significant

differences (P<0.01) from the untreated controls (Table 2).

NPC Natural Product Communications

2017

Vol. 12

No. 12

1897 - 1899

1898 Natural Product Communications Vol. 12 (12) 2017

Fichi et al.

Table 2: In vitro inhibition (in %) of sheep gastrointestinal strongyle egg hatch (EHT),

larval development (LDT) and larval motility/vitality (LMT) by 0.5% aloin and 0.1% aloe-emodin when compared to untreated and treated (0.1% thiabendazole) controls.

Compounds EHT % Inhibition (Confidence Interval) LDT % Inhibition (Confidence Interval) LMT % Inhibition (Confidence Interval) 0.5% Aloin 39.7B (27.6-51.8) 96.9 A (94.6-99.2) 65.4 A (59.3-71.5) 0.1% Aloe-emodin 16.6 Cb (12.1-21.0) 97.7 A (95.8-99.6) - Untreated Controls 13.4Cc (8.8-17.9) 21.3C (17.1-25.5) 4.2C (1.5-6.9) 0.1% Thiabendazole 94.9 A (94.1-100) 100.00 A (100-100) 27.7 B (19.9-35.6) A, B, C P<0.01; a, b, c P<0.05.

Moreover, in this test 0.5% aloin (96.9%) and 0.1% aloe-emodin

(97.7%) almost completely prevented the larval development from

L

to L

, showing no significant differences (P<0.01) when

compared to 0.1% TBZ (Table 2). In LMT, 0.5% aloin (65.4%)

gave the best results, since larval mortality observed in such treated

plates was found significantly higher (P<0.01) than in the untreated

controls (4.2%) but also in 0.1% TBZ treated controls (27.7%)

(Table 2).

The aim of the present study was to evaluate the in vitro

anthelmintic potential efficacy of two aloe-derived active principles,

aloin and aloe-emodin, in comparison to the reference drug

thiabendazole against gastrointestinal strongyles of sheep. Results

obtained in this study revealed that 0.5% aloin and 0.1%

aloe-emodin effectively targeted the larval developmental stages of these

parasites. Although both these compounds were not as effective as

the reference drug in reducing the hatching of the eggs, their effects

on GIS larvae resulted in significantly reduced larval development.

Especially aloe-emodin showed the same efficacy of the reference

drug when used at the same concentration (0.1%). Moreover, 0.5%

aloin also caused increased paralysis and/or death of GIS larvae.

Though aloin was tested at a higher concentration (0.5%) than the

reference drug, the activity of this compound was higher in causing

paralysis and/or death of the larvae. Results here obtained confirm

the anthelmintic efficacy previously reported for some aloe-derived

extracts, as the inhibition of Haemonchus contortus egg hatching

observed for A. rupestris and A. ferox [9, 10]. The anthelmintic

activity of these Aloe species has been related to their content in

amino acids, saponins and sterols [9]. Results from this study show

for the first time that aloin and aloe-emodin are endowed with in

vitro anthelmintic properties against sheep gastrointestinal

strongyles. Results are also indicative that the anthelmintic

properties previously reported for several aloe species may possibly

depend on their content in aloin and aloe-emodin. Therefore, it

would be interesting to proceed with further studies on the

effectiveness and toxicity of these natural active principles,

especially with regard to the evaluation of their efficacy as

de-wormers and as a mean to control the environmental development

of gastrointestinal strongyles when administered to infected animals

as dietary supplement.

Experimental

Plant Material: Two commercial products, aloin (Sigma-Aldrich

S.r.l., Milan, Italy) and aloe-emodin (Sigma-Aldrich S.r.l., Milan,

Italy), were used. Aloin was used at 0.5% and aloe-emodin was

used at the concentration of 0.1% and was not tested in the Larval

Mortality/ Paralysis Test. The needed concentrations of tested

compounds were obtained by dilute them directly in the medium

used for GIS cultivation.

Egg recovery, suspension and cultivation of eggs and larvae:

Individual fresh faecal samples collected from naturally infested

ewes living in an organic sheep flock located in Tuscany (Italy)

found positive for about 2000 GIS nematode eggs per gram of

faeces (EPG) after analysis with a McMaster method [16], were

used in the study.

Recovery, suspension and cultivation of GIS eggs were performed

by using previous reported methods [17, 18]. Briefly, for the egg

concentration and purification about 10 g of selected faecal samples

were mixed and homogenized with distilled water. The mixture was

strained and centrifuged for 2 min at 2300 rpm. The sediment was

collected, suspended in saturated NaCl solution and centrifuged for

2 min at 1000 rpm. The superficial layer of the supernatant was

collected, diluted in distilled water and centrifuged at 800 rpm for 2

min to collect the sediment containing the eggs. GI nematode eggs

were counted and a batch of about 100 eggs was cultivated in Petri

dishes. Each dish (100 eggs/ dish) contained 3ml of a culture

medium composed by 0.54 ml of saline solution, 0.06 ml of Earl

Balanced Salt Solution (Sigma-Aldrich S.r.l., Milan, Italy), 12 μL of

lyophilized Escherichia coli (E. coli strain W, lyophilized cells,

Sigma-Aldrich S.r.l., Milan, Italy), suspended in water and

sterilized for an hour at 100°C, 12 μg of amphotericin B

(Amphotericin B from Streptomyces approx 80% HPLC,

Sigma-Aldrich S.r.l., Milan, Italy), 60 mg of yeast extract (Sigma-Sigma-Aldrich

S.r.l., Milan, Italy) and about 2.4 ml of distilled water. Dishes were

incubated in the dark at 22°C. The same culture medium and

environmental conditions were used to cultivate larvae obtained by

egg cultures, by placing about 100 larvae/dish.

In vitro evaluation of the anthelmintic activity of tested

compounds: For the in vitro evaluation of the anthelmintic activity

of tested compounds, the Egg hatch test (EHT), the Larval

development test (LDT) and the Larval mortality/paralysis test

(LMT), were used [19-21]. In EHT, culture plates containing about

100 GIS nematode eggs/plate were prepared. All plates were

checked after 48 hours. Hatched first stage larvae (L1) and

unhatched eggs were then counted under the microscope and the

percentages of hatched eggs were calculated.

In LDT, live L1 larvae obtained from additional untreated GIS

nematode egg cultures after 48 hours of cultivation were isolated,

counted and about 100 L1/Petri dish were used. Plates were

checked after 7 days in order to evaluate the development from first

stage larvae (L

) to third stage larvae (L

)

L

larvae were counted

under the microscope after adding a drop of Lugol’s iodine solution

in each plate and the percentages of L3 on the total number of

larvae of each plate were calculated.

In LMT, about 100 motile L3/plate, derived from additional

untreated larval cultures, were used. After 24 hours, the percentage

of L3 that was found motionless for at least 6 seconds at

microscopical examination was calculated for each plate. In each

test, plates treated with each tested compound were compared to

untreated plates (untreated controls) and plates treated (treated

controls) with 0.1% thiabendazole (TBZ) (2-(4-Thiazoly)

Benzimidazole minimum 99%, Sigma S.r.l., Milan, Italy). Six

repetitions were made through the experiment.

Identification of GI nematode genera: L

recovered from further

additional plates were identified at the genus level based on their

morphological and metric features [22] and the mean percentage of

each identified genus on the total number of larvae was calculated.

Anthelmintic activity of aloin and aloe-emodin

Natural Product Communications Vol. 12 (12) 2017 1899

Statistical Analysis: Data from EHT, LDT and LMT obtained from

untreated and treated control cultures and from cultures treated with

the tested compounds, were statistically elaborated with X

_with

P<0.01 or P<0.05 significance level [23]. The confidence intervals

were also calculated.

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Natural Product Communications Vol. 12 (12) 2017

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Natural Product Communications

2017

Volume 12, Number 12

Contents

Original Paper

Page

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A New Picrotoxane Sesquiterpene Glucoside from Dendrobium nobile

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Antioxidant Sesquiterpenes from Penicillium citreonigrum

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Sessilifol A and B, Urease Inhibitory Pimarane-type Diterpenes from Hymenocrater sessilifolius

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