Nematicidal Effect of Shiitake ( Lentinula edodes ) Extracts Against Haemonchus contortus

Abstract

During the previous decades, the indiscriminate use of anthelmintics for the control of gastrointestinal nematodes has generated anthelmintic resistance in different parts of the world. It is necessary to search for new sustainable control alternatives, such as the use of extracts from plants and edible mushrooms. Therefore, the aim of the present study was to evaluate the nematicidal activity of extracts and fractions of the edible mushroom Lentinula edodes against Haemonchus contortus eggs and infective larvae. The basidiomata of L. edodes ECS-401 were provided by the Tropical Fungi Laboratory of El Colegio de la Frontera Sur and were extracted with ethyl acetate, methanol, and water. Fractionation of the most active extract was carried out by open column chromatography. The bioassays were performed in 96-well microtiter plates using 100 eggs/larvae, a final volume of 100 μL, and different concentrations of extracts/fractions (n = 4). Bioassay readings were taken at 48 h for egg hatching inhibition (EHI) and at 24, 48 and 72 h for larval mortality (LM). The ethyl acetate extract exhibited the highest percentage of EHI (100%). For the LM bioassay, the aqueous extract was the most active (69%), but its fractions did not show larvicidal activity. The chemical profile of the aqueous extract was analyzed by high-performance liquid chromatography, which showed the presence of phenols, flavonoids, and terpenes. L. edodes extracts showed ovicidal and/or larvicidal activity.

Introduction

One of the most prevalent gastrointestinal nematodes (GINs) in the world is Haemonchus contortus, considered to be the most important cause of diseases in small ruminants.¹ Among the effects they cause in animals, there is a decrease in zootechnical potential, weight loss, low milk and meat production, and even death.² One study reported that economic losses from GINs in the livestock sector in Brazil are about $7.1 billion per year.³ Anthelmintics have been used as the main method to control GINs in production animals. These drugs are characterized by high efficacy and the short time required to achieve results, although this outcome depends on the susceptibility of the nematode to the utilized anthelmintic class.⁴ However, recent studies have shown that H. contortus has resistance to different anthelmintic types, such as albendazole, levamisole, and ivermectin.^5
–7

Given that the number of small ruminant herds containing anthelmintics-resistant GINs is constantly increasing, producers require other tools to control nematodes and reduce the use of these chemicals.⁸ Edible mushrooms have been considered a great source of active substances due to their ability to degrade different types of substrates, which has led to their classification as functional foods and sources of nutraceuticals.^9,10 It has been reported in the literature that different extracts or metabolites isolated from mushrooms have shown nematicidal activity.^11,12 Among the edible mushroom is Lentinula edodes, which is a source of nutrients and biological compounds with antiviral, immunomodulatory, and antimicrobial properties, among others.¹³ It ranks second in production for commercial purposes in Asia. Also, in different parts of the world, the cultivation of shiitake has been modified to reduce the crop cycle and lower production costs, using agroindustrial waste such as shavings and sawdust mainly from oak.^14,15 In addition, Comans-Pérez et al. ¹⁶ reported a reduction of H. contortus larvae (93.9%) when exposed to the mycelium of L. edodes ECS-401. Therefore, due to this previous work, its different properties and the sustainability in its cultivation, the present work evaluated the nematicidal activity of different extracts made from basidiomata of L. edodes ECS-401 against H. contortus eggs and larvae.

Materials and Methods

Preparation of L. edodes extracts

L. edodes basidiomata were produced according to the methodology reported by Royse and Sánchez,¹⁷ and provided by the Tropical Mushroom Laboratory of El Colegio de la Frontera Sur, Tapachula, Chiapas, Mexico. The material was dried at room temperature and in the shade for 5 days. Subsequently, they were cut into small, 1-cm pieces and macerated with ethyl acetate in a 3:1 ratio (v/w) for 24 h. The obtained extract was filtered (Whatman No. 4) and concentrated in a rotary evaporator (Heidolph G3) at 45°C and dried under a high vacuum with a lyophilizer (Heto Dpywinner DW3) to give the ethyl acetate extract (LeAceOt). This extraction was repeated three times. The basidiomata were dried and placed with methanol and later with water for maceration, repeating the same process as described above. Both extracts were concentrated and dried, giving the methanolic extract (LeMeOH) and the aqueous extract (LeAcu). These three extracts were stored at −20°C until their evaluation.

Obtaining H. contortus eggs and L₃ larvae

A 3-month-old male sheep of the Pelibuey breed was used to obtain H. contortus eggs. To ensure that the sheep were free of nematodes, the sheep was dewormed with 2.5% albendazole (Valbazen^® Lab, 10 mg/kg body weight) and a count of eggs per gram of feces was carried out before infection. The sheep was infected with 350 H. contortus L₃ larvae per kg live weight. After the prepatent period, fecal samples were collected directly from the sheep's rectum. Fresh samples were macerated with water and sieved through a mesh system (No. 35, 100, 200, and 400). The sample was then recovered and added to 15 mL plastic tubes containing 9 mL of 40% sucrose. The samples were centrifuged at 261.9 rad/s for 5 min, thus generating a density gradient where the eggs were left in a ring-shaped phase. Subsequently, the eggs were washed four times with distilled water to remove the remaining sucrose and fecal matter.¹⁸ Finally, a count was carried out: aliquots were placed on slides and observed under the microscope (5 × magnification).

To obtain H. contortus L3 larvae, feces were collected in a basin and a culture medium was made with feces macerated with water and foam rubber, so that it had ∼70% humidity. The basin was covered with aluminum foil and left at room temperature. After 7 days of incubation, the larvae were recovered using the Baermann funnel technique.¹⁹ Finally, the larvae were stored at 4°C until they were used. For the bioassays, the larvae were washed with distilled water and unsheathed with 0.187% sodium hypochlorite.¹⁸ Likewise, a count was made before the assembly of the bioassay. The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant Mexican guidelines regarding animal welfare and unnecessary animal suffering. The Norma Oficial Mexicana (Official Rule Number) NOM-051-ZOO-1995 as well as the Ley Federal de Sanidad Animal (Federal Law for Animal Health) DOF 07-06-2012. These are Good Management Practices policies well established at our institution.

Mortality of H. contortus larvae due to L. edodes extracts

The evaluation of larval mortality (LM) due to L. edodes extracts was carried out in 96-well microtiter plates. Each extract (LeAceOt, LeMeOH, and LeAcu) was evaluated at different concentrations (6.25, 12.5, 25, 50, and 100 mg/mL). Methanol (4%) was used as a negative control (solvent), while a commercial anthelmintic (5 mg/mL ivermectin) served as a positive control. Four replicates of each treatment were performed. Each repetition was performed in a single well, in which the amount of each extract necessary for such concentrations was placed, along with ∼100 H. contortus L₃ exsheathed larvae and 100 μL 4% methanol. The plates were covered with aluminum foil and placed in an incubator at 28°C ± 1°C until reading. Three readings were taken for each extract: 24, 48, and 72 h postconfrontation.

Hatching inhibition of H. contortus eggs by L. edodes extracts

For the evaluation of egg hatching inhibition (EHI), the procedure was the same as described in section 2.3; however, the concentrations for each extract were 1.25, 2.5, 5, 10, and 20 mg/mL. The positive control was 5 mg/mL fenbendazole. Approximately 100 nematode eggs were placed in each well, and a reading was taken at 48 h postconfrontation.

Chemical fractionation of L. edodes extract

LeAcu was separated by column chromatography: 60 g of LeAcu was added to silica gel (60 g, RP-18, Merck) and placed in a glass column (40 × 10 cm), packed with 300 g of silica gel (60–50, RP18, Merck). Four solvent mixtures (water:acetonitrile) were used for the elution system: 100:0, 90:10, 80:20, 50:50, and 0:100%. Volumes of 500 mL (three for each mixture) were collected to obtain 18 fractions. The separation was monitored by thin layer chromatography (TLC), using aluminum plates coated with silica gel (60 F254) and silica gel (RP-18 F254S). The chromatography plates were observed using an ultraviolet lamp at 254 and 360 nm and developed with 4-hydroxybenzaldehyde, serum sulfate, and boron derivative. According to the TLC analysis, the fractions were grouped into six and stored in 15 mL vials (Table 1). Finally, they were dried under a high vacuum with a freeze dryer and stored at −20°C until evaluation.

Table 1.

Summary of the Fractionation of the Lentinula edodes Aqueous Extract

Elution system	Obtained fractions	Assembled fractions	Resulting fractions
100% water	1–4	1	LeR1
100% water	1–4	2–5	LeR2
90:10 water:acetonitrile	5–8	2–5	LeR2
		6–7	LeR3
		8–11	LeR4
80:20 water:acetonitrile	9–10
70:30 water:acetonitrile	11–12
70:30 water:acetonitrile	11–12	12–15	LeR5
50:50 water:acetonitrile	13–15	12–15	LeR5
30:70 water:acetonitrile	16	16–18	LeR6
100% acetonitrile	17
100% methanol	18

Mortality of H. contortus larvae due to the effect of L. edodes

All L. edodes fractions (20 mg/mL) were evaluated against H. contortus L₃ exsheathed larvae (three replicates). The bioassays were performed in 96-well microtiter plates. Methanol (4%) was used as a negative control and a commercial anthelmintic (5 mg/mL ivermectin) served as a positive control. The plates were covered with aluminum foil and placed in an incubator at 28°C ± 1°C until reading. The bioassays were read at 72 h postconfrontation.

Identification of compound groups by high-performance liquid chromatography resolution

The chemical profile of the L. edodes extracts was performed using high-performance liquid chromatography (HPLC) equipment consisting of a separation module (Waters 2695) with a photodiode array detector (Waters 2996) and a LiChrospher^® 100 RP-18 column of 250 × 4 nm (for a particle size of 5 μm). The samples were analyzed at a concentration of 2 mg/mL with a flow rate of 0.9 mL/min and sample injection of 10 μL. Compounds were detected with a wavelength scan from 196 to 600 nm. Water with 0.5% trifluoroacetic acid and acetonitrile were used for the elution system. The analysis lasted 30 min. This analysis was carried out following the method described by Gutiérrez-Román et al. ²⁰

Statistical analysis

The percentages of LM and EHI were square-root and then sine-arc transformed to homogenize the variance and obtain an approximation of a normal distribution. Analysis of variance (ANOVA) was carried out using the GLM procedure of the SAS 2009 statistical package. In addition, the Tukey mean comparison test was performed (P < .05 was considered significant).

Results

Nematicidal activity of L. edodes extracts against H. contortus eggs and L₃ larvae

In the LM bioassay, the LeAcu extract caused the highest mortality (68.84%) at 100 mg/mL and at 72 postconfrontation; this same concentration caused 52.68% mortality at 48 h. The LeMeOH and LeAceOt extracts showed no significant mortality (Table 2). In the case of H. contortus EHI, the lowest LeAceOt extract concentration (2.5 mg/mL) resulted in 96% hatching inhibition. The highest concentrations showed 100% inhibition at 48 h postconfrontation. On the contrary, 20 mg/mL LeAcu caused 36% inhibition (the maximum for this extract), and 10 mg/mL LeMeOH caused less than 20% inhibition (Table 3).

Table 2.

Effect of Different Organic Extracts of Lentinula edodes Basidiomata on Haemonchus contortus Infective Larvae at Different Postconfrontation Times

Treatment (mg/mL)	Mortality (%) ± SD
	LeAceOt			LeMeOH			LeAcu
	24 h	48 h	72 h	24 h	48 h	72 h	24 h	48 h	72 h
6.25	0^b	0^b	13.45 ± 6.45^b	0^b	3.50 ± 1.45^b	11.88 ± 5.04^c	1.27 ± 1.24^d,e	8.13 ± 2.42^d	4.28 ± 5.226^e
12.5	0^b	0^b	5.43 ± 4.09^b,c	0^b	1.46 ± 1.02^b,c	25.09 ± 10.32^b,c	2.81 ± 1.40^c,d	10.67 ± 1.64^d	23.36 ± 9.23^c,d
25	0^b	0.15 ± 0.30^b	5.38 ± 4.02^b,c	0^b	0^c	17.14 ± 7.94^c	6.01 ± 2.00^b,c	9.13 ± 3.56^d	16.38 ± 6.39^d
50	0.19 ± 0.24^b	0^b	4.20 ± 4.86^b,c	0^b	0^c	14.96 ± 6.32^c	9.73 ± 0.84^b	30.13 ± 7.15^c	35.58 ± 4.42^c
100	0^b	0.11 ± 0.22^b	6.53 ± 5.56^b,c	0^b	0.86 ± 1.070^c,d	41.82 ± 5.56^b	10.82 ± 5.16^b	52.68 ± 3.36^b	68.84 ± 11.09^b
Ivermectin (5)	100 ± 0^a	100 ± 0^a	100 ± 0^a	100 ± 0^a	100 ± 0^a	100 ± 0^a	100 ± 0^a	100 ± 0^a	100 ± 0^a
4% Methanol	0^b	0^b	0^c	0^b	0^c	0^d	0^e	0^e	0^e

Data are presented as mean ± SD, n = 4. The same letters in the same column indicate that the values do not differ statistically, according to Tukey's test (P < .05).

LeAceOt, ethyl acetate extract; LeAcu, aqueous extract; LeMeOH, methanolic extract; SD, standard deviation.

Table 3.

Hatching Inhibition of Haemonchus contortus Eggs by Different Organic Extracts of Lentinula edodes at 48 h Postconfrontation

Treatment mg/mL	Egg hatching inhibition (%) ± SD 48h
Treatment mg/mL	LeAceOt	LeMeOH	LeAcu
2.5	96.36 ± 5.24^a	4.68 ± 3.17^c,d	15.67 ± 6.55^c
5	100 ± 0^a	14.21 ± 2.31^b,c	21.59 ± 8.22^c
10	100 ± 0^a	17.71 ± 5.81^b	33.91 ± 15.54^b,c
20	100 ± 0^a	9.95 ± 6.10^c	36.10 ± 5.67^b
40	100 ± 0^a	16.11 ± 11.39^b	35.14 ± 0^b
Fenbendazole (5)	100 ± 0^a	100 ± 0^a	100 ± 0^a
4% Methanol	0^e	0^d	0^d

Data are presented as mean ± SD, n = 4. The same letters in the same column indicate that the values do not differ statistically, according to Tukey's test (P < .05).

Mortality of H. contortus larvae due to the effect of L. edodes fractions

The LeAcu extract fractions had no activity against H. contortus larvae, as all of them presented <13% mortality at 20 mg/mL and 72 h postconfrontation (Table 4).

Table 4.

Mortality of Haemonchus contortus L₃ Larvae Due to the Effect of Different Fractions of the Lentinula edodes Aqueous Extract

Treatment (20 mg/mL)	Mortality (%) ± SD
Treatment (20 mg/mL)	72 h
LeR1	1.15 ± 1.99^b
LeR2	7.64 ± 2.89^b
LeR3	13.27 ± 11.85^b
LeR4	0.62 ± 1.01^b
LeR5	6.25 ± 3.81^b
LeR6	5.40 ± 6.22^b
Ivermectin (5 mg/mL)	100^a
Methanol 4%	0^b

Data are presented as the mean ± SD, n = 3. The same letters in the same column indicate that the values do not differ statistically, according to Tukey's test (P < .05). LeR1–6 indicate the L. edodes aqueous fractions (see Table 3 for additional details).

Identification of compound groups by HPLC

HPLC analysis (reversed-phase column conditions) showed that the aqueous extract (LeAcu) contains at least five main compounds with retention times of 4.6, 6.4, 7.0, 7.2, and 8.0 min, and according to their UV light spectra (λ _max = 200–280 nm) and comparison with data described in the literature, these compounds could correspond to phenols (Fig. 1).²¹ In the case of LeAceOt, which is an extract of medium polarity, the chromatogram showed polar compounds (at least 8) but at low concentrations (determined by the area under the curve of each peak), and the main compounds of this extract appear at higher retention times (less polar compounds) at 27.9, 28.8, and 29.9 min, with UV absorptions from 230 to 280 nm. According to the literature, these compounds may correspond to phenols, coumarins, or flavonoids (Fig. 2).²²

FIG. 1.

Chromatogram of Lentinula edodes aqueous extract.

FIG. 2.

Chromatogram of ethyl acetate extract of Lentinula edodes.

Discussion

The results showed that the LeAcu extract was the most active against H. contortus L₃ exsheathed larvae at the highest concentration (100 mg/mL), whereas the LeAceOt extract showed higher activity in the EHI bioassay (even at a minimum concentration of 2.5 mg/mL). These data suggest that the LeAcu extract contains metabolites with larvicidal activity and the LeAceOt extract comprises ovicidal metabolites. The differences between the extracts are most likely due to the polarity of the compounds present in each extract: more polar compounds are found in the LeAcu extract, while there are less polar compounds in the LeAceOt extract. The response in this study is similar to what González-Cortazar et al. ²³ reported. They evaluated an aqueous extract, an aqueous fraction, and an ethyl acetate fraction of Lysiloma acapulcensis against H. contortus eggs and L₄ larvae. In the EHI bioassay, they reported that the aqueous extract caused 19% inhibition, the aqueous fraction caused 7.7% inhibition, and the ethyl acetate fraction caused 100% inhibition. In the L₄ LM test, the aqueous extract caused 47% mortality, the aqueous fraction was not evaluated, and the ethyl acetate fraction showed 100% mortality. Another work evaluated the aqueous extract and ethanolic extract of Moringa oleifera seeds against H. contortus eggs and L₃ larvae. In the egg inhibition bioassay, the aqueous extract presented 81% inhibition, while the ethanolic extract presented 96% inhibition. On the contrary, in the LM bioassay, the aqueous extract presented 92.5% and the ethanolic extract 57% mortality. It is worth mentioning that the larval activity was measured based on the motility of the larvae.²⁴

Works have also reported ovicidal activity with aqueous extracts. For example, Belemlilga et al. ²⁵ evaluated the aqueous extract of Saba senegalensis leaves against H. contortus adult larvae and eggs; they reported 98% LM and 93% egg inhibition. It should be noted that H. contortus larvae in adult stage already feed; therefore, the effect of these extracts could be related to the consumption of the same.

In the present work, the LeAcu extract fractions did not show activity against H. contortus L₃ exsheathed larvae, contrary to what has been observed in bioassays with extracts. Based on this finding, it can be said that when the extracts are chemically fractionated, they lose their activity, and that the activity may require the interaction of two or more metabolite groups present in the raw extracts. This finding is contrary to what has been reported by other works where the fractions presented greater nematicidal activity compared with the crude extracts.^12,26,27

The larvicidal activity of the LeAcu extract fractions was low compared with what was expected; however, it should be noted that for the continuation of the present study we intend to carry fractionate the LeAceOt extract and evaluate its ovicidal activity. Given that the LeAceOt extract showed 96% egg inhibition at the concentration of 2.5 mg/mL, this endeavor should identify the metabolite groups responsible for such activity, or if something similar occurs as observed with the LeAcu extract fractions against L₃ larvae (i.e., the interaction of several compounds is required for the ovicidal activity).

HPLC analysis showed that the major compounds in L. edodes extracts and fractions were phenols, flavonoids, coumarins, and terpenes. Some of the described metabolite groups resemble those reported by Rivera et al. ²⁸ This is consistent with some extracts and fractions obtained from plants and fungi reported as nematicides against H. contortus. Such is the case of phenols and flavonoids reported by some studies.^23,29,30 Likewise, Artemisia absinthium extracts, rich in phenols and flavonoids, present nematicidal activity against Meloidogyne incognita larvae.³¹ In addition, terpenes are also nematicidal.^12,25,32

Currently there are no studies of the nematicidal activity of basidiomata of the edible mushroom L. edodes. Therefore, this work is the first to report this activity and may serve as a background for future research.

The use of edible mushrooms for the control of GINs is a research topic that has gained interest in recent years, since few studies have been reported of edible mushrooms with nematicidal activity of livestock interest, particularly in sheep farming, compared with that reported in the agricultural area with plants infected with phytoparasites.³³ However, it is important to continue researching the edible mushrooms, since they are an important source of nutrients and bioactive compounds, and so, in addition to helping to control GINs in ruminants, they would also contribute to the diet of sheep through nutraceutical products.¹⁰ In addition, edible mushrooms are easy and inexpensive to obtain, since their cultivation has been adapted using different substrates of agroindustrial waste.¹⁵ Thus, both basidiomes and bioactive compounds can be produced cost effectively.

Conclusions

L. edodes basidiomata have in vitro nematicidal activity against H. contortus eggs and larvae. The LeAcu extract has higher larvicidal activity, while the LeAceOt extract has higher ovicidal activity. The LeAcu extract fractions did not show larvicidal activity, and so, the use of the crude extract is seemingly more efficacious compared wiht the fractions. The major compounds in L. edodes extracts may correspond to the phenolic, coumarin, flavonoid, and terpene types; these may be responsible for the nematicidal activity.

Although more study is needed, L. edodes could be used as a supplement in the sheep diet through nutraceutical products to reduce GINs. This would help reduce the use of anthelmintics and consequently resistance.

Footnotes

Acknowledgment

We thank the Consejo Nacional de Ciencia y Tecnología (CONACYT), Mexico, for the scholarship granted to J.A.P.-A (211375).

Author Disclosure Statement

No competing financial interests exist.

Funding Information

The present article was financed by the National Problems project, Consejo Nacional de Ciencia y Tecnología, Mexico (CONACYT), project number 9342634372.

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Nematicidal Effect of Shiitake ( Lentinula edodes ) Extracts Against Haemonchus contortus

Abstract

Introduction

Materials and Methods

Preparation of L. edodes extracts

Obtaining H. contortus eggs and L3 larvae

Mortality of H. contortus larvae due to L. edodes extracts

Hatching inhibition of H. contortus eggs by L. edodes extracts

Chemical fractionation of L. edodes extract

Mortality of H. contortus larvae due to the effect of L. edodes

Identification of compound groups by high-performance liquid chromatography resolution

Statistical analysis

Results

Nematicidal activity of L. edodes extracts against H. contortus eggs and L3 larvae

Mortality of H. contortus larvae due to the effect of L. edodes fractions

Identification of compound groups by HPLC

Discussion

Conclusions

Footnotes

Acknowledgment

Author Disclosure Statement

Funding Information

References

Obtaining H. contortus eggs and L₃ larvae

Nematicidal activity of L. edodes extracts against H. contortus eggs and L₃ larvae