Research Article

Antischistosomal Activity of Trigonella foenum (Fenugreek) Crude Seeds Water Extract on Infected Mice

Gamalat Y. Osman1, Elham M. Hassan1*, Tarek A. Salem2, Azza H. Mohamed1

1Department of Zoology, Faculty of Science, Menoufia University, Shebin El-Kom, Egypt; 2Department of Molecular Biology, Genetic Engineering and Biotechnology Institute, Sadat City University, Sadat City, Egypt.

Received | March 23, 2022; Accepted | May 04, 2022; Published | June 18, 2022

*Correspondence | Elham M. Hassan, Department of Zoology, Faculty of Science, Menoufia University, Shebin El-Kom, Egypt; Email: [email protected]

Citation | Osman GY, Hassan EM, Salem TA, Mohamed AH (2022). Antischistosomal activity of Trigonella foenum (Fenugreek) crude seeds water extract on infected mice. Adv. Anim. Vet. Sci. 10(7):1458-1465.

DOI | https://dx.doi.org/10.17582/journal.aavs/2022/10.7.1458.1465

ISSN (Online) | 2307-8316

Copyright: 2022 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Trigonella foenum (fenugreek) is a plant that belongs to the Fabaceae family and is cultivated worldwide as a semiarid crop (Wani and Kumar, 2018). It’s found in northern Africa and India and famous as a human food with a distinctive flavor, it can also be drunken as a tea. It’s used as whole seeds or powder and was applied firstly in the pharmaceutical fields in ancient Egypt. The majority of the common active constituents of fenugreek seeds are phenolic compounds, in addition to amino acids, protein, vitamin A, starch and minerals such as Calcium, Phosphorus, Magnesium, Iron, Zinc and Manganese (Kumar et al., 2009; Wani and Kumar, 2018). One of the main phenolic constituents of fenugreek seeds is rutin, a bioflavonoid phenolic compound found in a variety of plants including citrus, which is characterized by its distinctive pigment (Patel and Patel, 2019). Coumarin derivatives are also important constitutions that are commonly found in fenugreek seeds (He et al., 2015). They are colorless crystalline solids with a sweet odor found in many plants like cinnamon and cherry (Kruger et al., 2018). Other fenugreek phenolic constituents are ferulic and sinapic acids. They are organic hydroxy-cinnamic acids found naturally in the plant cell wall and represent the precursor of aromatic compounds manufacturing (Bunzel et al., 2003; Pajak et al., 2018). Fenugreek seeds have considerable medical importance. For example, it had been reported to improve body health and digestion functions in rabbits (Abdel-Wareth et al., 2021). Moreover, administration of ethanolic or ether extracts of fenugreek seeds exhibited anti-inflammatory, anti-arthritic activities, and antioxidant effects in arthritic albino rats (Suresh et al., 2012; Pundarikakshudu et al., 2016). Also, water extract of fenugreek was reported to exert an anti-helminthic effect (Swarnakar et al., 2014). The antiparasitic and anti-inflammatory effects of fenugreek seeds are attributed to its active constituents like rutin, coumarin, sinapic and ferulic acids (Chauhan et al., 2018; Yin et al., 2019; Lopes et al., 2020; Quinn et al., 2020; Liseth et al., 2021).

Schistosomiasis is a chronic disease that is caused by the trematode blood fluke Schistosoma and is responsible for the infection of at least 236.6 million people who required preventive treatment, it is second to malaria in tropical countries and is listed as a neglected tropical disease (WHO, 2021) and has a high prevalence in Africa. Schistosomiasis can strike either intestine or urinary tract resulting in pathogenic symptoms like diarrhea, bloody stool or urine, abdominal pain, or even more hazardous cases of hepatic and kidney failure (Abdel Gawad et al., 2018; WHO, 2021).

Different drugs were developed for schistosomiasis treatment and the most efficient one is PZQ. Though it’s the most effective drug with fewer side effects, recent researches still provide proof about Schistosoma-drug resistance. Pinto et al. (2021) found that the worm of S. mansoni that was previously exposed to PZQ developed new protein content which was less sensitive to the drug.

This study aims to estimate the anti-schistosomal effect of fenugreek seeds water extract on Schistosoma mansoni infected mice.

Materials and Methods

Fenugreek plant seeds (Trigonella foenum)

Fenugreek dry seeds were purchased from a local market, Shebin El-Kom, Menoufia. Fenugreek water extract (FWE) was prepared according to Sakr and Abo-El-Yazid (2012) in the laboratory of Faculty of Science, Menoufia University and followed the standards.

Standard drug (Praziquantel, PZQ)

PZQ drug was purchased from Egyptian International Pharmaceutical Industries Company (EIPICO). Each tablet was grinded and freshly suspended in distilled water, administered orally to mice in 3 doses each of 250 mg/kg for three alternative days (Utzinger et al., 2003).

Experimental animals

Swiss male albino mice CD-I strains (weighing 20±2 g) and Biomphalaria alexandrina snails infected with Schistosoma mansoni miracidia were obtained from Theodor Bilharz Research Institute (TBRI) Giza, Egypt. Mice were maintained under standard laboratory conditions and feeding at Parasitological Research Lab in Zoology Department, Faculty of Science, Menoufia University. Egyptian strain of Schistosomamansoni cercariae were shed from laboratory-bred infected B.alexandrinasnails (Ethical number: MUFS /S /Pa /1 /20).

Chemical analysis of FWE using high-performance liquid chromatography (HPLC)

HPLC analysis was carried out using Agilent Technologies 1100 series liquid chromatography according to Kim et al. (2005).

Mice infection

Infected snails were washed with de-chlorinated tap water and left uncovered under white fluorescent light for one hour in a tube for cercarial shedding according to Pellegrino et al. (1962). Infection of mice was performed by subcutaneous injection of 70 ± 5 S. mansoni cercariae to each mouse according to Moore et al. (1977).

Experimental design

Thirty-five mice were divided into 5 groups (7mice/each) as follow:

Group 1: served as normal control; Group 2: infected with 70±5 S. mansoni cercariae and served as an infected group; Group 3: infected and treated with 3 doses of PZQ each of hich 250 mg/kg for three alternative days up to 8 weeks post-infection; Group 4: infected and treated with FEW in a dose of 0.5 ml/mouse for 10 successive days up to 8 weeks post-infection; Group 5: infected and treated with PZQ accompanied to FWE up to 8 weeks post-infection.

Parasitological parameters

Worm load

At the end of the treatment, mice were killed by decapitation. Hepatic and porto-mesenteric vessels were perfused to recover worms for subsequent counting (Cheever, 1969; Duvall and De Witt, 1967).

Scanning electron microscope (SEM)

Adult S. mansoni worms from the experimental groups were fixed in glutaraldehyde buffer solution with 0.1 M sodium cacodylate buffer (pH 7.2) at room temperature for subsequent examination (Matos-Rocha et al., 2016). Joel JSM-5300, Japan scanning electron microscope was applied at Faculty of Science, Alexandria University, Egypt.

Egg count

According to the method of Cheever and Anderson (1971), three samples were collected from the liver and intestine of each mouse and were prepared (0.5 g tissue / 5ml 5% KOH for each sample) and examined under the lower power of the microscope.

Oogram pattern

Three tissue fragments (each of 1 cm) were collected from the liver and intestine of each mouseand placed between a slide and a cover-slip. One hundred eggs were counted microscopically and classified into immature, mature, and dead eggs according to the method of Pellegrino et al. (1962).

Granuloma diameter and liver histopathology

Representative samples from liver tissues were taken from the animal groups and fixed in neutral formalin (10%). Paraffin-embedded sections (5µm thick) were stained using Hematoxylin and Eosin (Romeis, 1989). Granuloma containing a single egg was measured using an ocular micrometer. For each mouse, 40-50 granulomas were measured and the percent reduction in granuloma diameter relative to the infected controls was calculated (Mahmoud and Warren, 1974).

Statistical analysis

Data are presented as Mean ± Standard deviation (M±SD). Student t-test, for normally-distributed data, was used to calculate the significance of differences observed between mean values of experimental and control groups in each experiment at a level of significance of P < 0.05 (Sokal and Rohlf, 1981).

Results

Chemical analysis of FWE applying high-performance liquid chromatography (HPLC)

As shown in Table 1 and Figure 1, FWE was analyzed by the High-performance liquid chromatography (HPLC) technique into different phenolic compounds. Rutin represented the highest concentration (2705.96 mg/g) among phenolic constituents and followed by p-coumaric which recorded 768.59 mg/g. Then, p-hydroxybenzoic recorded 513.36 mg/g, while other phenolic constituents showed lower concentrations (Ferulic, Sinapic, and Apigenin-7-glucoside).

 

Table 1: High-performance liquid chromatography (HPLC) analysis of FWE (Phenolic compounds).

Compounds	µg/g
Rutin	2705.96
p-coumaric	768.59
p-hydroxybenzoic	513.36
Ferulic	355.29
Sinapic	346.85
Apigenin-7-glucoside	214.00

 

.

Table 2: Effect of FWE and /or PZQ on worm burden and total egg count of S. mansoni infected mice.

Effect of FWE and /or PZQ on worm burden in S. mansoni infected mice
Treatment/ Worm	Ic ± Standard deviation		I+FEW ± Standard deviation		Change % (Compared to infected control group)
Male	4.3± 0.9		2.2 ± 0.3		48.8 ↓
Female	3 ± 0.5		3.5± 0.4		16.6 ↑
Couple	5 ± 0.8		3 ± 0.3		40 ↓*
Total	17. ±1.1		11.7± 0.9		32.4↓*
Effect of FWE and /or PZQ on total egg count in S. mansoni infected mice
Egg/ Treatment	Egg count/gm (Liver tissue) ±Standard deviation	Reduction↓% (Compared to infected control group)		Egg count/gm (Intest. tissue) ±Standard deviation		Reduction↓% (Compared to infected control group)
Ic	15333 ±1009	---		25567 ± 1559		---
I+PZQ	5433 ±978	64.6 ↓*		266.7 ± 41		98.9 ↓*
I+FWE	7467 ± 965	51.3 ↓*		4233 ± 609		83.4 ↓*
I+FWE+PZQ	3500 ± 500	77.2 ↓*		1867 ± 340		92.7 ↓*

 

* Significant change compared to infected control group. (P < 0.05). Ic (infected control), I+PZQ (infected and treated with PZQ), I+FWE (infected and treated with fenugreek water extract), I+FWE+PZQ (infected and treated with fenugreek water extract accompanied with PZQ).

 

Effect of FWE and/or PZQ on some parasitological parameters of S. mansoni infected mice

Worm burden

Worm burden was illustrated in Table 2A, S. mansoni infected mice treated with FEW (I+WE) showed a clear reduction in total worm number and number of couples by 32.4 % and 40%, respectively, as compared to the infected control group (IC). There is a significant (P > 0.05) reduction in male worm number in infected mice treated with FWE by 48.8%, while no worms were observed in praziquantel treatments (PZQ) either alone or accompanied with fenugreek as compared to the infected control group.

Scanning electron microscope (SEM) of S. mansoni worms (Figures 2, 3).

 

Total egg count/g tissue

As shown in Table 2B, treatments of infected mice either with PZQ or FWE led to a significant (P > 0.05) reduction in the total hepatic egg count by 64.6% and 51.3%, respectively. While egg percentage was reduced by 77.2% in the infected mice were treated with PZQ accompanied to FWE as compared to that of the infected control group. In the intestinal tissue, treatment of infected mice with FEW significantly (P > 0.05) reduced eggs count by 83.4%, while, treatment of infected mice with PZQ showed the highest reduction in total intestinal egg count by 98.9%, as compared to the infected control group (Table 2).

Oogram pattern

Oogram pattern in liver tissue was illustrated in Table 3A. Immature egg showed a significantly (P > 0.05) increased percentage in the infected group treated with PZQ by (68.8%), while FWE showed extra elevated immature egg percentage by 87%, as compared to the infected control group. The highest increase in the immature percentage was recorded in the infected mice treated with FEW accompanied with PZQ (109.1%) as compared to that of the infected control group. Treatment of infected mice with PZQ showed a significant (P > 0.05) reduction in mature egg percentage (54.2%), and also, but to less extent, FWE reduced mature egg count significantly (P > 0.05) by 33.9%. The highest reduction rate in mature egg percentage was recorded in the infected mice treated with FWE accompanied to PZQ by 73.6 %, as compared to the infected control group.

 

Oogram pattern in intestine tissue was illustrated in Table 3B. The highest immature count was recorded in the infected group treated with FWE accompanied with PZQ (144.3 %). While, the dead egg percentage was reduced closely in either PZQ or FWE by 56.9 % or 50%, respectively. The most reduction in mature egg count was recorded in the infected group treated with PZQ accompanied with FWE by 70.4%, as compared to the infected control group.

Hepatic granuloma diameter (GD)

Granuloma diameter was illustrated in Table 3C. In comparison with infected control mice, all treated groups showed a significant (P > 0.05) reduction in granuloma diameter in closed rates, and the most reduced diameter was exhibited in the infected group was treated with FEW accompanied to PZQ by 18.6% (Figure 4).

 

Table 3: Effect of FWE and /or PZQ on oogram patern and hepatic granuloma diameter of S. mansoni infected mice.

Effect of FWE and /or PZQ on oogram pattern (liver tissue) in S. mansoni infected mice.
Egg stage/ Treatment	Immature % ±Standard deviation	Increase ↑ % (Compared to infected control group)		Mature % ±Standard deviation		Reduction↓% (Compared to infected control group)	Dead % ±Standard deviation		Increase↑% (Compared to infected control group)
Ic	15.4 ± 3.6	---		74.2 ± 4.3		---	11 ± 1.6		---
I+PZQ	26 ± 1.9	68.8 ↑*		34 ± 2.2		54.2 ↓*	40 ± 3.4		263.6 ↑*
I+FWE	28.8 ± 4	87 ↑*		49 ± 4.4		33.9 ↓*	22.2 ± 3		101.8 ↑*
I+FWE+PZ	32.2 ± 4.2	109.1 ↑*		19.6 ± 2		73.6 ↓*	48.2 ± 3.7		338↑*
Effect of FWE and /or PZQ on oogram pattern (intestine tissue) of S. mansoni infected mice.
Egg stage/ Treatment	Immature % ±Standard deviation	Increase ↑ % (Compared to infected control group)		Mature % ±Standard deviation		Reduction ↓ % (Compared to infected control group)	Dead % ±Standard deviation		Increase ↑ % (Compared to infected control group)
I+c	17.6 ± 1.5	---		50.6 ± 4		---	32 ± 3.1		---
I+PZQ	29 ± 3	64.8 ↑*		20.8 ± 2		58.9 ↓*	50.2± 2.6		56.9 ↑*
I+FWE	24.8 ± 3	40.9 ↑		27 ± 2		46.2 ↓*	48 ± 3		50 ↑
I+FWE+PZQ	40 ± 4	144.3 ↑*		15 ± 2		70.4 ↓*	45 ± 3		40.6↑*
Effect of FWE and /or PZQ on hepatic granuloma diameter in S. mansoni infected mice.
Treatment	Ic		I+PZQ		I+FWE			I+FWE+PZQ
Granuloma diameter ±Standard deviation	34.4 ± 0.9		29 ± 1.9		29.3 ± 0.8			28 ± 2
Reduction ↓ % (Compared to infected control group)	----		15.7 ↓*		14.9↓*			18.6 ↓*

 

* Significant change compared to infected control group. (P < 0.05). Ic (infected control), I+PZQ (infected and treated with PZQ), I+FWE (infected and treated with fenugreek water extract), I+FWE+PZQ (infected and treated with fenugreek water extract accompanied with PZQ).

 

Discussion

In the current study, the effect of FWE treatment on some parasitological parameters of mice infected with S. mansoni was investigated. Treatment of S. mansoni infected mice with the standard drug praziquantel (PZQ) alone or combined with (FWE) showed a complete absence of worms. The treatment of infected mice with FWE significantly reduced total and coupled worm numbers, as compared to the infected control group. Meanwhile, the analysis of fenugreek water extract by using HPLC revealed that rutin and P-cumaric phenolic compound were the main constituents of FWE, they were found in high concentrations. Such constituents were reported to have anti-parasitic activities. Chauhan et al. (2018) reported that rutin exhibited a strong anti-leishmanial effect against both sensitive and resistant strains by enhancing cell-mediated immune response. Therefore, in the present work, FEW can be speculated to have a similar attitude against S. mansoni worms. An in vitro study on fenugreek water extract by Kor et al. (2013) found that it was effective against resistant species of Plasmodium. They added that fenugreek seeds exhibited an anti-microbial activity against Helicobacter pylori and restricted the growth of staphylococcus and pseudomonas in vitro. Also, water extract of fenugreek seeds was reported to have a direct anti-helminthic effect on Gastrolthylax carmenifer in cattle (Swarnakar et al., 2014). That was by causing rupture of tegument parenchymal cells, detachment of the tegument surface lead finally to disability of the parasite to be connected to the host.

In the present study, treatment of S. mansoni-infected mice either with PZQ or FEW caused significant reductions in total egg count in liver and intestine, somewhat PZQ reduction rate exceeded that of fenugreek, while accompanied treatment of them got the highest reduction rate of egg count in the liver. Generally, the reduction of egg capacity in each treatment can be in part attributed to their reducing action on total worm burden and also the number of coupled worms, so consequently, egg-laying was clearly reduced. A study by Lopes et al. (2020) found that cumaric acid showed anti-parasitic activity against Leishmania braziliensis and Plasmodium falciparum by blocking enzymes that are responsible for the parasite’s development. A previous study revealed that cumaric acid prevented the growth of Entamoeba histolytica (Muruato et al., 2021). Aqua’s extract of the cinnamon plant was reported to have an inhibitory effect on Plasmodium falciparum development in vitro (Parvazi et al., 2016). Since, cumaric acid is one of the main constituents of cinnamon (Kruger et al., 2018), therefore, FWE also can be assumed to have a resembling effect on Schistosoma. Thus, it can be deduced that cumaric acid in FEW can reduce the total egg count by restricting the worm development and thus preventing egg-laying.

 

The obtained results of oogram pattern of the hepatic and intestinal tissue, PZQ, and FWE significantly reduced mature egg and elevated dead egg percentages, while combined treatment of them caused better effect as compared to that of the infected control group. The water extract of fenugreek seeds was reported to have selective lethal effects on malignant cells in vitro by restricting abnormal cell growth provoking apoptosis (Alsemari et al., 2014), it may have a similar effect on the parasite by restricting egg growth.

Granuloma is an immune-inflammatory response towards S. mansoni egg; it protects the hepatic tissues from egg secreted toxins. But if the immune response around granuloma was not regulated, it will cause a hazardous effect on tissues (Guimaraes et al., 2015). In the current work, PZQ and FWE showed a significant reduction in granuloma diameter, while accompanied treatment got the smallest diameter, as compared to the S. mansoni-infected group. The reducing effect of FWE on granuloma diameter could be due to its active phenolic compounds ferulic and sinapic acids that were confirmed by HPLC. According to Quinn et al. (2020), Sinapic acid-containing extracts were found to have anti-inflammatory properties by reducing pro-inflammatory cytokines in human blood in vitro. Ferulic acid was reported to inhibit the inflammation induced by lipo-polysaccharide by inhibiting reactive oxygen species generation, inhibiting the intracellular malon-dialdehyde formation, and switching the intracellular antioxidant enzyme activities to normal levels (Yin et al., 2019). in addition, fenugreek seeds were reported to have a strong antioxidant activity by raising glutathione (Patil and Jain, 2014). So it can deduce that FWE can neutralize the egg secreted products, restricting extra inflammatory reactions and consequently reducing the granuloma diameter.

Conclusions and recommendations

Fenugreek water extract (FWE) exhibited marked anti-parasitic activities against S. mansoni adult worms and eggs; it also reduced granuloma diameter and tissue damage. Furthermore, the combination of FWE with PZQ ameliorated the antiparasitic efficacy of PZQ against S. mansoni-infected mice as compared to that of the PZQ alone.

Novelty Statement

FWE were used for the treatment of Schistosoma mansoni and showed a direct antihelminthic activity toward the worms and eggs and also anti-inflammatory effect on granuloma. Chemical analysis of FWE was performed to deduce its active properties and support the current data.

Author’s Contribution

GYO, AHM and TAS conceived the study and designed the experiments. EMH performed the experiments. GYO, AHM, TAS and EMH analyzed results. EMH wrote the maniscript. All authors reviewed and approved the final version of the maniscript.

Conflict of interest

The authors have declared no conflict of interest.

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