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Introduction
Helminths, commonly known as parasitic worms, are multicellular invertebrate organisms characterized by elongated, round, flat or cylindrical bodies (Castro, 1996). Parasitic infections are particularly prevalent in developing and underdeveloped countries with inadequate hygienic conditions. Approximately 1.7 billion people worldwide are infected with Ascaris (common roundworm), Trichuris (whipworm) and hookworm. Schistosomiasis is endemic in 70 countries, with millions suffering due to a lack of clean water and sanitation facilities (Hedley, 2015).
In endemic areas, these infections are treated periodically with anthelmintic medicines. Piperazine hydrochloride, albendazole, mebendazole, ivermectin and levamisole are the approved drugs widely used to treat human and animal parasites (Yadav et al., 2017). For example, albendazole inhibits parasite microtubule polymerization by binding to the β-tubulin. This process disrupts the glucose uptake in nematodes, limiting them only to glycogen stores. As a result, it causes energy loss, consequently leading to the mortality of parasites (Venkatesan, 1998). Frequent treatment of helminths through anti-parasitic drugs has allowed parasites to develop resistance against these medicines. Resistance, as in other microorganisms, stems from the genetic mutations caused by deleting one or more alleles (Martin, 1997). If diseases are part of nature affecting humans, plants and animals, so do cures, as remedies are hidden in the very nature waiting to be explored. Before the industrial and scientific revolution, primeval people used plants, herbs, and shrubs to cure diseases and treat wounds. Therefore, scientists are now focusing on harnessing plants and their compounds to develop alternative medicines to overcome parasitic resistance with less adverse effects compared to pharmaceutical drugs.
Olea europaea L. (Olive) is a plant widely used in the Mediterranean region. Olive leaves, fruit, and oil are extensively consumed due to their tremendous pharmacological potential. Olives are rich in vitamins K, C and other antioxidants that can reduce the risks of cardiovascular diseases, cancer, inflammation caused by reactive oxygen species, diabetes, and high blood pressure (Soler-Rivas et al., 2000). Oleuropein is generally the most prominent phenolic compound found in olives. It belongs to secoiridoids, a specific group of coumarin-like compounds usually glycosidically bound and produced from the secondary metabolism of terpenes. Basically, oleuropein is an ester of hydroxytyrosol and oleosidic skeleton. Hydroxytyrosol is found in olives and olive oil, in the form of its elenolic acid ester, known as oleuropein (Soler-Rivas et al., 2000). Anticancer, antidiabetic, antimicrobial, antioxidant, antihypertensive and cardioprotective, enzyme inhibition, anti-inflammatory and antinociceptive, gastroprotective, neuroprotective, and other predominant biological activities of olives are also associated with oleuropein (Hashmi et al., 2015).
Aspiculuris tetraptera belongs to the Oxyuridae family. It is a natural and common parasite that inhabits the colon and cecum of mice and other rodents (Moulia et al., 1993; Kozan et al., 2006). A. tetraptera has a direct life cycle and infection occurs after ingestion of eggs by the host from environmental contaminants, which takes approximately 23 – 25 days. A. tetraptera eggs can be easily detected in feces (Behnke, 1974). Short embryonic period, direct development and incidence of autoinfection may also contribute to the pervasiveness of pinworms. Nevertheless, pinworm helminths are considered relatively non-pathogenic, and infections are known to be asymptomatic (Plachý et al., 2016). Therefore, A. tetraptera is widely used to determine the efficacies of various chemotherapeutic agents.
Given the above information and published literature, A. tetraptera was established as a model parasite in determining the unexplored anthelmintic potential of OLE and oleuropein.
Materials and Methods
Collection of the O. europaea leaves extract, Oleuropein and Albendazole
Aqueous extract of olive leaves was procured from Kale Natural Herbal Products, Foods Cosmetics, and Agricultural Products LTD. Co, Balıkesir, Turkey. Oleuropein content in the extract was 13 %, as described. Oleuropein, >98 % HPLC of analytical grade, was purchased from Gül Laboratory Equipment and Chemical substances and Bio Medical Scientific Products LTD. Co, Istanbul, Turkey. Albendazole was provided by the Faculty of Veteri nary Sciences, Uludağ University, Bursa, Turkey.
Animals
Male and Female Balb/c albino mice (25 – 30g) were purchased from Uludağ University experimental animals center, Bursa, Turkey. Animals were housed in standard cages, kept at recommended room temperature (20 – 22°C) with 12 hrs artificial light and 12hrs dark cycles for 7 days prior to pharmacological studies with free access to standard laboratory feed and tap water. All animals were deprived of food overnight before dosing, except the tap water was available ad libitum.
Fecal Flotation Method
The saturated NaCl (salt) flotation method was used to diagnose A. tetraptera eggs from stool samples collected from each cage (Sueta et al., 2002).
In vivo experimental assay
Animals were exposed to pinworm-contaminated bedding from an in-house colony of naturally infected mice with A. tetraptera. The stool samples from approximately 200 mice were microscopically examined for endoparasite eggs of A. tetraptera using a fecal flotation technique for detecting infected mice. Infected animals were randomly distributed into 7 groups (n=13). OLE, oleuropein, and ABZ were orally administered for 7 days. Groups 1, 2, and 3 (experimental mice) were treated with 250 mg/kg, 500 mg/kg and 1000 mg/kg concentrations of OLE, and groups 4 and 5 (experimental mice) received 5 mg/kg and 20 mg/kg of % 98.0> oleuropein at body weight. Animals in Group 6 were constituted as untreated control and were given tap water. Group 7 was given a 10 mg/kg dose of a reference drug, ABZ. 21 animals randomly died during oral administrations, leaving 70 animals for autopsy, as mentioned in Table 1.
The number of A. tetraptera collected from autopsied animals in treatment and control groups and descriptive statistical data (n=13).
Groups
The number of animals autopsied post-treatment (n)
The number of minimum and maximum A. tetraptera collected in necropsy (Min.-Max.)
Mean ± SEM
Median
Q1
Q3
Efficacy (%)
Olea europaea 250 mg/kg
11/13
0 – 1055
146.2 ± 92.9
48
1
115
70.03
Olea europaea 500 mg/kg
6/13
1 – 863
329 ± 153
258
2
661
63.18
Olea europaea 1000 mg/kg
6/13
0 – 365
69.8 ± 59.3
9
0
365
92.19
Oleuropein 5 mg/kg
13/13
2 – 959
374.5 ± 88.7
286
92.5
665.5
9.27
Oleuropein 20 mg/kg
11/13
1 – 397
143.6 ± 45.5
56
19
299
70.56
Tap Water (control group)
10/13
0 – 1661
537 ± 180
381
35
950
0
Albendazole 10 mg/kg
13/13
0 – 153
31.2 ± 12.9
4
0
58
92.43
Mice were euthanized by cervical dislocation for necropsy on the 8th-day post-treatment. Gastrointestinal tract was removed and washed with normal saline. The contents were examined under a stereomicroscope for adult A. tetraptera parasites. Syphacia spp. was also found during necropsies. The numbers of these parasites were determined by keeping records. Despite A. tetraptera being a prime target of our study, data about Syphacia spp was also included in Table 2 to increase the robustness of this study.
Efficacy percentages (%) of Olea europaea, Oleuropein, and Albendazole used in different concentrations against Syphacia spp. compared to the control group (water).
Control groups
Efficacy Percentage (%)
Parasite species
Oleuropein 5 mg/kg
Oleuropein 20 mg/kg
Olea europaea 250 mg/kg
Olea europaea 500 mg/kg
Olea europaea 1000 mg/kg
Albendazole 10 mg/kg
Water (Control) Group
Syphacia spp.
−83.70
−218.84
−413.73
−531.30
−110.54
2.23
Efficacy was calculated from the geometric means of A. tetraptera counts as mentioned below:
\% {Efficacy} = {{{\rm{a}} - {\rm{b}}} \over {\rm{a}}} \times 100
“a” is the geometric mean number of A. tetraptera in the control group and “b” is the geometric mean number of A. tetraptera in the treated group.
Statistical analysis of data
Data obtained from experimental groups were expressed as statistical mean ± standard error of the mean (SEM). Statistical differences between the treatments and the control groups were evaluated by Minitab (Version 15.0: Minitab Inc., State College, PA, USA) and Mann-Whitney U tests. P<0.05 was considered to be statistically significant.
Ethical Approval and/or Informed Consent
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional guidelines on the care and use of laboratory animals. Experiments followed the ethical, care and non-unnecessary suffering regulations mandated by the Turkish Animal Ethics Committee of Bursa Uludağ University (Ethical council number: UÜHADYEK-2020-05/02).
Results
During the study, no adverse effects and reactions were observed clinically in any of the mice treated with olive leaves extracts and oleuropein. The number of adult A. tetraptera collected from the gastrointestinal tracts of control and experimental groups, minmax values, geometrical means, and percentages of efficacy are given in Table 1. Total A. tetraptera in animal groups administered with 250 mg/kg, 500 mg/kg and 1000 mg/kg of OLE were; 1,608, 1,976, and 419, while mice treated with 5 mg/kg and 20 mg/kg of oleuropein had 4,869 and 1,580 parasites respectively. Control and ABZ-treated groups contained 5,367 and 406 worm counts. On the other hand, a total of 5,342 Syphacia spp. counts were observed during necropsies in control and all the treated groups. The numbers of these species were much lower as compared to A. tetraptera. Hence, it is most unlikely that Syphacia spp. para sites have interfered with the results. The geometric means of adult A. tetraptera obtained in the control group were significantly higher, followed by 5 mg/kg oleuropein and 500 mg/kg OLE. The remaining groups had comparatively lower geometric mean values. Efficacies of 500 mg/kg and 5 mg/kg oleuropein were low. 1000 mg/kg concentration of OLE and 10 mg/kg ABZ illustrated high efficacy percentage, while 250 mg/kg OLE and 20 mg/kg dose of oleuropein displayed moderate anthelmintic effects. Statistical analysis of ABZ was found to be significant (p<0.05), while that of 1000 mg/kg OLE was equal but not significant (p=0.05). Statistical analysis of remaining concentrations was not significant (p>0.05). In the case of oleuropein, anthelmintic effects increased in a dose-dependent manner. Since oleuropein is the most prominent phenolic compound in olive cultivars, the anthelmintic activity of O. europaea can be attributed to this compound. The results of the in vivo anthelmintic assay and related statistical data are presented in Table 1.
Discussion
Today, it is estimated that approximately one-third of three billion people living in underdeveloped and developing regions of Asia, sub-Saharan Africa, and America are directly or indirectly exposed to co-infestations. Ascariasis, trichuriasis, hookworm, tapeworm, intestinal helminths, and schistosomiasis infections are most prevalent (Hotez et al., 2006; Hotez et al., 2007). Hookworm and schistosomiasis can induce complications during pregnancy, resulting in reduced neonatal birth weight, premature birth, and increased maternal morbidity as well as mortality (Christian et al., 2004).
A plethora of anthelmintic drugs is used to treat helminth infections worldwide. Approved anthelmintic medicines are becoming inadequate in the face of resistance exhibited by the parasites. Kingdom Plantae has always been a tremendous source of natural remedies against infections, inflammation, gastrointestinal and cardiovascular diseases. Gürağaç Dereli et al. (2019) reported that methanolic extract of aerial parts of Polygonum cognatum reduced S. obvelata egg count by 66.8 % and A. tetraptera by 73.4 % as compared to doramectin (100 %). It was deduced that the anthelmintic activity of P. cognatum could be related to phenolic compounds and tannins present in the plant (Gürağaç Dereli et al., 2019). In another study, 250 μL methanolic extract of Plantago major leaves showed 27.62 % and 250 μL aqueous extract illustrated 39.25 % efficacy, much lower than 0.2 mg kg−1 dose of ivermectin, which exhibited 88.57 % anthelmintic efficacy against A. tetraptera respectively (Türel et al., 2013). In another study, aqueous and ethanol extracts of Cucurbita maxima showed lower efficacy than ivermectin (91 %) against A. tetraptera (Ayaz et al., 2015). The studies mentioned above suggest that specific plants and their compounds can possess anti-parasitic activities against helminth infections. Unexpected results have also been reported in another in vivo anthelmintic assay in which 250 mg/kg, 500 mg/kg and 1000 mg/kg of Rosmarinus officinalis (Rosemary) plant extracts showed low anti-parasitic activities; 25.44 %, 46.54 %, and 7.16 %, against A. tetraptera infected mice. On the contrary, rosmarinic acid, a polyphenol constituent of the rosemary plant, increased worm burden by −8.17 % as compared to ABZ, which caused an 89.1 % reduction in A. tetraptera counts (Eylek, 2021). Polyphenols-rich and tanniferous plant extracts or isolated tannins have not been proven to exert anthelmintic activities equal to that of the readily available drugs in the sense of half maximal effective concentration (EC50) values (Spiegler et al., 2017). However, there is still a huge difference between in vitro or low-impact animal studies and evidence regarding the unambiguous efficacy of polyphenols and polyphenols–enriched extracts.
A safety assessment study of ethanolic extract of O. europaea leaves in Winstar rats was conducted to investigate OLE’s acute and subacute oral toxicity. Acute toxicity was assessed using a single dose of 2000 mg/kg of ethanolic extract of olives (EEO) and subacute toxicity was evaluated during 28 days using 100 mg/kg, 200 mg/kg and 400 mg/kg doses of EEO, administered by oral gavage. According to the results, neither single nor three different concentrations of EEO induced mortality or any sign of abnormalities and toxicities among the treated animals (Guexa et al., 2018). This study implied that OLE and oleuropein could be used at higher concentrations to study their anthelmintic potential in animals or humans. Since indigenous people in the Mediterranean region consume olives and related products, no fatal side effects among humans stemming from olive consumption have been reported till today. Bhattacharjee et al. (2016) investigated the anthelmintic effects of crude aqueous, petroleum ether, chloroform, and methanol extract of O. europaea leaves on Pheretima posthuma (earthworm). According to the study, various extracts of O. europaea showed significant worm reduction compared to piperazine citrate (Bhattacharjee et al., 2016). The study and its' result seem ambiguous because anthelmintic effects were analyzed in vitro in free-living and soil-dwelling worms, and no efficacy percentage was evaluated to support the findings. O. europaea leaves contain immense antioxidant properties. Lins et al. (2018) suggested in a recent study that OLE inhibits the action of reactive oxygen species (ROS) and protects human red blood cells against oxidative damage.
Oleuropein has low anthelmintic validity because it has not been studied thoroughly for its anti-parasitic potential (Bisignano et al., 1999). However, oleuropein has several potent pharmacological properties, including antioxidant, anti-inflammatory (Moa et al., 2019), hypotensive (Tsoumani et al., 2021), anti-neuropathic, anticancer, antimicrobial (Gamli, 2016), and hypoglycemic (Cristiano et al., 2021). Oleuropein also decreases low-density lipoprotein (LDL) levels in the blood (Ahamad et al., 2019). In addition, the cardioprotective activity of O.europaea can be attributed to oleuropein (Andreadou et al., 2007). Oleuropein also exhibits anti-ischemic and hypolipidemic activities (Andreadou et al., 2006). Several compounds, like polyphenols, alkaloids, flavonoids and terpenes etc., may be causative agents for the anthelmintic activity of the plants, particularly; polyphenols compounds are known to have anti-parasitic potential. Phenolic compounds may exhibit their anthelmintic activity by uncoupling oxidative phosphorylation or binding to free proteins in the gastrointestinal tract of host animals (Mali & Wadekar, 2008).
Polyphenols or tannins extracted from plants can be used for long-term chronic therapeutics rather than for treating acute parasite infections in animals and humans. Hence further research is required to elucidate the mode of action of polyphenol or tannin-enriched plant extracts, which could possibly lead to the identification of potential drug targets.
Conclusion
In light of the derived results, it can be concluded that the anthelmintic activity of O. europaea has stemmed from oleuropein. Our study illustrates the high anthelmintic potential of OLE against A. tetraptera. Anthelmintic activity of 250 mg/kg, 1000 mg/kg, 5 mg/kg and 20 mg/kg concentrations of OLE and oleuropein increased in a dose-dependent manner except that of 500 mg/kg. This unexpected deviation might have resulted from mishandling and random placement of oral gavage into the trachea of mice instead of the esophagus during the treatment, which led to the suffocation and instant death. Although 1000 mg/kg of OLE reduced a significant amount of parasite counts, it must be kept in mind that the number of animals in this treatment group also declined to 6 compared to ABZ, which remained 13. Since olive is known to be non-toxic, further studies with higher doses of OLE and oleuropein are required to explore different dimensions of our findings. Oleuropein holds potential anthelmintic effects that can be used either as an alternative drug or a supplementary diet to treat parasitic resistance. We also suggest determining Eggs per gram (EPG) values during the formation of the groups in drug efficacy trials against A. tetraptera infections in mice to consolidate the results in future studies. When ingested, oleuropein metabolizes into hydroxytyrosol. Therefore, various pharmacological aspects of olives may be attributed to hydroxytyrosol and more comparative studies are required to test this hypothesis.
