Abstract: Background: Blastocystis is the most common enteric protozoa in humans and animals worldwide. The hazardous side effects and potential development of drug resistance to the standard drug, metronidazole (MTZ), necessitate the search for safer and more efficient alternative therapeutics. Objective: The present study was designed to investigate the in vitro activity and in vivo efficacy of two natural medicinal compounds, Olive Leaf Extract (OLE) and Bee Pollen Compound (BPC) against subtype 3 Blastocystis isolated from symptomatic patients. Materials and Methods: Cultured parasites were challenged with a graded concentration of OLE (500, 1000 μg mL–1), BPC (500, 1000 μg mL–1) and MTZ (150, 250 μg mL–1) to assess its in vitro growth by counting the number of viable cells after one and two h of incubation and morphological ultrastructural changes were evaluated using Transmission Electron Microscope (TEM). The anti-Blastocystis effects of OLE and BPC were determined in 40 laboratory bred Swiss albino mice inoculated with Blastocystis isolates using parasitological, histopathological and immunohistochemical methods. Results: Blastocystis isolated from symptomatic patients were subtype 3. The OLE and BPC showed statistically significant (p<0.05) in vitro growth inhibition of Blastocystis in a concentration dependent manner and a statistically significant (p<0.05) in vivo reduction of the number of Blastocystis in stool samples and intestinal contents of infected treated mice higher than MTN effects with apoptotic-like death and Programmed Cell Death (PCD) in Blastocystis. The BPC normalized villous architecture and increased local IgA secretory villous cells. Conclusion: These findings highlighted the potential therapeutic effects of OLE and BPC as potent safe natural alternatives against blastocystosis. The BPC was superior to OLE in ameliorating the severity of tissue pathology and had an immunostimulatory effect on intestinal cells.
INTRODUCTION
Blastocystis is a unicellular, anaerobic, eukaryotic, extracellular and luminal parasite that lives in the intestinal tract of different hosts including humans. It has a worldwide distribution and it is one of the most frequently identified parasites in human fecal samples in the developing countries in the tropics and subtropics1. Its prevalence might exceed 50% in developing countries2 and reached about 20% in developed countries3. Its prevalence in Egypt reached to 33%4. Human infection is related to poor personal hygiene, absence of sanitation, exposure to animals and consumption of contaminated food or water. Blastocystis transmission is proposed to be by the fecal-oral route5. Blastocystosis can be asymptomatic or cause gastrointestinal symptoms6,7. Blastocystis might act as an opportunistic pathogen in immunocompromised patients8. Symptomatic irritable bowel syndrome and inflammatory bowel disease patients also had increased rates of Blastocystis spp. infection9.
Blastocystis has 4 main forms which have been described in stools and/or in-vitro cultures: vacuolar, granular, amoeboid and cyst forms10. The extensive genetic diversity of Blastocystis spp. has been demonstrated using different molecular assays11,12.
The diagnosis of blastocystosis is currently based on microscopic detection of the protozoa in direct smears carried out before or after cultivation of the fecal sample or molecular identification of the parasite DNA13. The pathogenicity and treatment of Blastocystis infections remain controversial. However, it is generally acceptable that treatment of Blastocystis infection is needed when debilitating symptoms are present with presence of several Blastocystis cysts in stool specimens and with absence of other clear cause of symptoms14.
Although MTZ is the standard therapy for Blastocystis infections, there are several reports of treatment failure, indicating the presence of drug-resistant isolates15. Furthermore, MTZ has many side effects and drawbacks, including: headache, vertigo, nausea and a metallic taste in the mouth with pancreatitis, central nervous system toxicity at higher doses and reversible neutropenia16. It is also mutagenic in bacteria and carcinogenic in mice and rats when given at high doses over long periods of time17. All these reasons motivated the search for alternative therapies of Blastocystis infection, particularly, the study of natural active agents.
Olives and olive oil are considered the cornerstone of the Mediterranean diet and are well-known for their overall health benefits18. Propolis, royal jelly and bee pollen are recognized as valuable therapeutic components and as healthy foods. Several manuscripts discovered in ancient Egypt, China and Greece described the active healing properties of bee products. Many of them are widely used in medicinal purposes for the treatment of bacterial and viral infections, to stimulate the immunity, for treatment of poorly healing wounds, in a variety of tumor diseases, in gastro-intestinal infections, to promote potency and fertility. Bee products have comparable healing properties as established drugs but without side effects19.
The aim of the present study was to evaluate the in vitro and in-vivo therapeutic efficacy of two natural medicinal agents; OLE and BPC on subtype 3 Blastocystis in comparison with MTZ, the standard therapy for Blastocystis infection.
MATERIAL AND METHODS
Blastocystis isolates
Stool sample Collection: Fresh stool samples were obtained from 20 symptomatic patients complaining of intestinal symptoms including diarrhoea, abdominal cramps and bloating attending Theodor Bilharz Research Institute (TBRI) outpatient clinic.
Stool sample processing: Stool specimens were microscopically examined immediately after collection by a wet smear preparation in saline and staining with Lugol’s iodine solution before and after formalin/ethyl acetate concentration and faecal smears stained with modified trichrome and modified Ziehl-Neelsen acid-fast staining20 to exclude the presence of other parasites and to identify cases of Blastocystis infection. Portion of each Blastocytis positive stool sample was cultured on the same day for in vitro assays and another portion was kept at -20°C for molecular assay and genotyping.
Blastocystis molecular characterization: Only three out of the 20 samples were proven to be infected with Blastocystis and were included in this study. At the Lab of Molecular Medical Parasitology (LMMP), Department of Medical Parasitology, Kasr Al-Ainy Faculty of Medicine, Cairo University, Blastocystis isolates were genotyped. Genomic DNA from the positive stool samples was extracted using Favor Prep stool DNA isolation Mini Kit (Favorgen Biotech corporation ping-Tung 908, Taiwan, Cat. No. FASTI001), according to manufacturer’s instructions.
As recommended by Stensvold21, the extracted DNA was amplified by PCR targeting specific SSU rDNA using the primers RD5 (5’-ATCTGGTTGATCCTGCCAGT-3`) and BhRDr (5’-GAGCTTTTTAACTGCAACAACG-3’) to amplify the ~600 bp fragment22 for confirmation of the presence of Blastocystis. The reaction mixture and condition were done in a total volume of 25 μL according to Scicluna et al.22, then seven standardized subtype-specific Sequence-Tagged-Site (STS) primers were used (Table 1) in a PCR reaction mixture and condition according to Yoshikawa et al.23. The amplified products were visualized with 1.5% agarose gel electrophoresis after ethidium bromide staining.
Blastocystis in vitro culturing: Positive Blastocystis stool samples were cultivated in 3 mL of Jones’ medium without rice starch and supplemented with 10% horse serum24, 100 IU mL–1 penicillin and 100 μg mL–1 streptomycin11.
Blastocystis culture was incubated at 37°C with 5% CO2 and monitored daily for 72 h by light microscopy. Culture was regarded as negative when it is failed to detect Blastocystis after 72 h25. Cultures were considered suitable for drug assessment when Blastocystis number mL–1 exceeded 1×103 vacuolar forms.
Drugs and drug preparation
MTN preparation: MTN (Flagyl) was manufactured and purchased from Sanofi-aventis Egypt s. a. e., under licence of Sanofi-aventis/France. It was used as a reference antiprotozoal drug26. Tablet was grinded and dissolved in sterile distilled water to prepare the stock solution of 1 mg mL–1, then stored in a dark bottle at 4°C. Final concentrations of MTZ were adjusted to 150 and 250 μg mL–1.
Preparation of natural products
Olea europaea (OLE): About 430 mg per capsule, containing 20% oleuropein; purchased from Nature’s way, USA and Queen’s Magic BPC: distributed by Eden Pond Labs, USA; each capsule containing propolis (1000 mg), royal jelly (1500 mg) and bee pollen (750 mg). The powder of each capsule from each product was dissolved in sterile distilled water to get a final stock solution of 1 mg mL–1. Final concentrations were adjusted to 500 and 1000 μg mL–1.
In vitro anti-Blastocystis activity of OLE, BPC and MTN
In vitro experiment: Parasite inoculums of size 50×104 parasites mL–1 from cultures in logarithmic growth phase were introduced into each set of culture tubes containing Jones medium at the different concentrations of OLE, BPC or MTN to study parasite growth. Non-treated control cultures of the parasites and negative control (culture media only) were subjected to the same procedure used for the other tested cultures. Culture tubes were used in triplicate for every concentration of each OLE, BPC, MTN and non-treated cultures. Parasites were challenged with a graded concentration of OLE (500, 1000 μg mL–1), BPC (500, 1000 μg mL–1) and MTN (150, 250 μg mL–1). The tested cultures were incubated in humidified CO2 at 37°C for examination after 1 and 2 h.
The treatment period of at least 1 h was used in previous standardized studies and had shown that this time period is necessary to induce the cytotoxic response in these parasites27.
Assessment of in vitro anti-blastocystis activity of OLE, BPC and MTN
Light microscopy for the in vitro study: The activity of the tested drugs against Blastocystis infection was evaluated by counting the number of viable cells after 1 and 2 h using eosin-brilliant cresyl blue dye which stained viable cells with green color and nonviable cells with red color28.
| Table 1: | Blastocystis subtype-specific Sequence-Tagged-Site (STS) primers |
| *ST: Subtype, F: Forward, R: Reverse | |
Enumeration was done in duplication and only intact cells were counted by using a Neubauer cell counting chamber under a Zeiss light microscope (Oberkochen, Germany). The percentage of inhibition of Blastocystis multiplication in the treated cultures in relation to non-treated culture was calculated according to the Eq. 1:
| (1) |
where, "a" is the mean number of parasites in control cultures and "b" is the mean number of parasites in treated cultures.
TEM for the in vitro study: For detection of ultrastructural changes, 1 mL of cultured sediment was taken from untreated culture (control groups) and treated cultures with, 250 μg mL–1 of MTZ, 500 μg mL–1 of OLE and 500 μg mL–1 of BPC after 2 h of incubation, then washed twice in 0.1 M sodium cacodylate buffer (pH 7.2) with 5% sucrose and fixed in 1% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 3 h, then washed twice in buffer; followed by fixation in 2% osmium tetra oxide (OsO4) and in 1% potassium ferrocyanide solution for 1 h then washed twice in 0.5 M sodium cacodylate buffer. The cells were processed29 and tested by JEOL Ltd (JEM-1400 TEM, Japan). These drugs concentrations were selected for TEM according to the least dose of OLE, BPC and MTZ causing significant growth inhibition (p<0.05) in the in vitro study.
In vivo anti-Blastocystis activity of OLE, BPC and MTN
In vivo experiment
Experimental animals: A batch of 40 laboratory bred Swiss albino mice of CD-I strain 3-5 weeks old, weighing 20–25 g were used in this study. Mice were kept in well-ventilated cages of the animal house in the Parasitology Department, TBRI. Throughout the study, the animals were kept on a standard diet containing 24% protein, 4% fat and about 4-5% fiber and water ad libitum. They were kept at room temperature (28±2°C) and away from direct sunlight. The cages were cleaned twice a week to ensure good sanitary condition until required for use. The mice were allowed to adapt to the laboratory environment for 1 week before the experiment and their stools were examined microscopically daily to exclude the presence of parasites.
The OLE and BPC were used in a dose equivalent to the daily recommended dose to maintain good health. The doses were calculated by extrapolation of human therapeutic doses to animal doses according to the table of Paget and Barnes30. The used drugs were given 3 weeks after establishment of infection for seven consecutive days.
Experimental design: The Swiss albino mice used were divided into 5 groups each of which consisted of 8 mice as follows: normal control (uninfected, untreated), infected control (infected, untreated), infected treated with MTN, infected treated with OLE and infected treated with BPC.
Animal infection: Mice were orally infected by intragastric inoculation of 104 vacuolar forms of B. hominis in 4 days old axenic culture per mouse using oesophageal tube.
Three weeks after infection of the mice, faecal pellets were collected and subjected to parasitological examination to detect Blastocystis and to ensure that all mice had been infected.
Scarification: Scarification was done after cessation of treatment by using over dose of ether. Intestinal sections were dissected from individual mice, fixed in 10% formalin, embedded in paraffin for histological examination of 4 mm sections stained with haematoxylin and eosin and for immunohistochemical examination.
Assessment of in vivo anti-Blastocystis activity of OLE, BPC and MTN
Parasitological studies: Quantitative estimation of the intensity of infection in stool samples of Blastocystis infected mice after administration of drugs was determined by microscopic counting of Blastocystis per gram stool. Also, the number of vegetative forms (trophozoites) in intestinal contents was counted in five successive high power fields per animal and then the average was calculated.
Histopathological studies: Intestinal sections from scarified mice were excised, opened longitudinally and embedded in paraffin wax. Transverse sections of paraffin blocks were cut by microtome and mounted on glass slides. Thickness of 5 μm of the deparaffinized sections were performed by dipping slides in 100% xylene and descending grades (100, 95, 80 and 70%) of ethanol for rehydration. Finally, sections were stained with Ehrlich’s hematoxylin and counter stained with eosin31. Five slides/mouse and 3 sections/slide in each group were prepared and examined to detect the histopathological changes occurred due to Blastocystis infection and to assess the cure rates and the degree of healing of intestinal mucosa after drug administration.
Immunohistochemical studies: Immunohistochemical staining was performed on 4 μm, formalin-fixed, paraffin-embedded intestinal sections using IgA antibody at 1:50 dilution (DAKO, Carpinteria, CA). Antigen retrieval was performed in all cases by steam heating the slides in a 1 mmol L–1 solution of EDTA (pH 8.0) for 30 min. After blocking of endogenous biotin, staining was performed using an automated immunostainer followed by detection by using a streptavidin-biotin detection system (DAKO). Positive and negative control sections were used for each assay.
The IgA expression was evaluated according to H score in an attempt to describe the extent of immunohistochemical staining which appeared brown in color. The H score is considered a qualitative scale for the number and intensity of staining, ranging from zero for no staining (scanty), 1+ for mild and 2+ for moderate32.
Statistical analysis: Data were reported as Mean counts±Standard deviation. Statistical analyses were done using computerized statistical software program SPSS 19.0. The independent sample t-test was used to assess the statistical significance of the mean difference between the two study groups. Statistical significance was defined as p<0.05.
Ethical consideration: The protocol of this study was approved by scientific research ethics committee of TBRI. Patients included in the study were informed verbally about the purpose of the study and the collection of stool samples was performed after obtaining their consent. The animal experiments were performed in accordance with the TBRI committee for laboratory animals research guidelines.
RESULTS
Amplification and genotyping of Blastocystis isolates: Genotypic assessment of the isolated Blastocystis samples revealed the subtype 3 with 526 bp band amplified with SB227 primer (Fig. 1, 2).
In vitro study: Effect of different concentrations of OLE, BPC and MTN on the growth of Blastocystis vacuolar form after 1 and 2 h is illustrated in Table 2
In vivo study: Quantitative assessment of the intensity of Blastocystis infection in stool samples and intestinal contents of infected mice after administration of drugs was performed by the conventional diagnostic techniques to count number of Blastocystis per gram stool and results are illustrated in Table 3.
TEM results: TEM micrographs (Fig. 3) showed that untreated cultures had classical Blastocystis morphology (Fig. 3a). MTN-treated cultures showed morphological changes in the form of prominent Mitochondrion-Like Organelle (MLO) at the end of the peripheral cytoplasm with vacuolar compression and a less electron dense central body with presence of electron dense intracellular bodies (Fig. 3b).
| Fig. 1: | DNA analysis of a Blastocystis samples |
Lane L: 50 bp DNA ladder. Lane 1: Positive control, Lane 2: Negative control, Lane 3-5: Isolated samples ~600 bp band amplified with SSU rDNA primer pair |
|
| Fig. 2: | Sub-typing DNA analysis of a Blastocystis samples |
Lane L: 100 bp DNA ladder, Lane 1-3: Isolated samples with 526 bp band amplified with SB227 primer (subtype 3), Lane 4: Negative control, Lane 5: positive control |
|
| Fig. 3(a-d): | TEM photographs of (a) Untreated cultures showing healthy Blastocystis cell, (b) MTN, (c) OLE and (d) BPC-treated cultures showing prominent MLO with vacuolar compression and less electron dense Central Vacuole (CV)
|
| Table 2: | Effect of MTN, BPC and OLE on the in vitro growth of Blastocystis after different incubation periods |
Values are expressed as Mean±SD and Growth Inhibition (%) from control, *Statistically significant compared to infection control group (p<0.05), BPC: Bee Pollen Compound, OLE: Olive Leaf Extract | |
The OLE-treated cultures showed prominent MLO at one end of the peripheral cytoplasm with a less electron dense central body and the presence of membrane-bound electron-dense particles (apoptotic bodies) in the central vacuole which were released into the extracellular space with loss of membrane integrity and presence of electron dense intracellular bodies (Fig. 3c).
| Table 3: | Effect of MTN, BPC and OLE on Blastocystis number in intestinal contents and stool of infected mice 4 weeks post-infection |
Values are expressed as Mean±SD and Reduction (%) from control, *Statistically significant compared to infection control group (p<0.05), BPC: Bee Pollen Compound, OLE: Olive Leaf Extract | |
The BPC-treated cultures showed prominent MLO at one end of the peripheral cytoplasm with vacuolar compression and a less electron dense central body (Fig. 3d).
Histopathological results: Histopathological examination of intestinal sections in infected non-treated mice revealed profound histopathological changes in the morphology of intestinal mucosa in the form of shortening and broadening of villi, loss of villous architecture, crypt hyperplasia, decrease the ratio of villous height and crypt length, mucosal ulceration and infiltration of inflammatory cells (Fig. 4a, b).
Histopathological examination of intestinal sections in treated groups with MTN or OLE revealed partial improvement in the histopathological changes, with persistent moderate villous atrophy and moderate inflammatory reactions (Fig. 4c, d). In contrast, treatment group with BPC revealed marked improvement in the form of returning of the villous like pattern, villous-crypt ratio with presence of few scattered inflammatory cells and focal broad villi (Fig. 4e).
Immunohistochemical results: Immunohistochemical staining of intestinal sections in infected non-treated mice revealed few IgA secretory cells which appeared as brown color in the lamina propria cells of the core of villi (Fig. 5a), while immunohistochemical staining of intestinal sections in treated groups with MTN or OLE revealed occasional IgA secretory cells (Fig. 5b, c), however, with BPC treatment there was a significant increase (p<0.05) in the number group IgA secretory cells (Fig. 5d).
DISCUSSION
In the present study, genotypic assessment of the isolated positive Blastocystis samples revealed infection with subtype 3 Blastocystis. This is agreed with several epidemiological studies which reported that subtype 3 is the most predominant subtype3,33-35. These studies demonstrated the importance of subtype 3 in terms of its prevalence, pathogenic implications and treatment.
Both OLE and BPC showed a statistically significant (p<0.05) growth inhibition of Blastocystis in a concentration dependent manner higher than MTZ effects.
The BPC showed the highest percentage of reduction in the number of Blastocystis in stool samples and intestinal contents of infected treated mice followed by OLE with a statistical significant difference (p<0.05) in comparison with the control group.
Mokhtar et al.36 stated that ST1 and ST3 Blastocystis were susceptible to MTZ, at two concentrations (50 and 100 μg mL–1) without growth after 24 h, while the concentration of 10 μg mL–1 showed a significant reduction (p<0.05) in the parasite counts for all incubation periods (24, 48, 72 h) with a cytocidal efficacy after 72 h.
Similarly, Yakoob et al.37 reported a sensitivity of all Blastocystis isolates to MTZ in comparison to healthy controls at concentrations of 0.01 and 0.1 mg mL–1, these isolates were mostly ST3 and coinfection of ST3 and ST1, however in the same study isolates from IBS patients showed variable efficacy of MTZ and were mostly of ST1. Also, Girish et al.38 demonstrated high growth inhibition of MTZ (0.1 and 1 mg mL–1) against ST1, ST3 and ST5. Moreover, many clinical studies reported effective parasite clearance with MTZ39,40. This controversy about MTZ efficacy may be due to intra-subtype differences in the susceptibility mechanisms of the drug, possibly this may be attributed to the presence of different alleles in each subtype41.
Olive trees and leaves are considered low-cost sources which are rich in phenolic compounds with recognized antioxidant and anti-inflammatory properties42. Oleuropein, which is the main component of olives and olive leaves, have several biological properties: antitumor, antiviral, antibacterial and antiparasitic activity43-49. In the year 2000, oleuropein was pretended in a U.S. patent (Fredrickson, Num: 6,117,844) as having powerful antiviral activity.
Oleuropein demonstrated antiparasitic activity against Toxoplasma gondii, it showed proliferation inhibition of Madin-Darby bovine kidney cells infected with Toxoplasma gondii tachyzoites (in vitro) and decreased the parasite expansion (55.4%) in the peritoneal cavity of Toxoplasma-infected mice48.
| Fig. 4(a-e): | Histopathological examinations of intestinal sections from (a, b) Infected untreated control group showing several Blastocystis granular forms (black arrows) (H&EX1000), severe villous atrophy (red arrows), (c) MTN, (d) Olive Leaf Extract (OLE) treated infected groups showing moderate villous atrophy (red arrows) and (e) BPC treated infected group showing normal villous architecture (H&EX200) |
Also, oleuropein revealed anti-parasitic activity against Leishmania infantum, Leishmania donovani and Leishmania major50. Similarly, Sifaoui et al.51 stated that flavonoids present in five different Tunisian olive extracts, had leishmanicidal activities against the four Leishmania species, an antioxidant efficacy and a protective role against membrane lipoperoxidative damages.
Propolis showed potent antimicrobial properties, it has been used as a chemotherapeutic agent since ancient times and it is considered as an anti-inflammatory drug and wound healing agent. Propolis is one of the most powerful natural antibiotics with a wide spectrum efficacy.
| Fig. 5(a-d): | Immunohistochemical examinations of intestinal sections from (a) Infected untreated control group showing few IgA secretory brownish cells, (b) MTN, (c) OLE treated infected groups showing scanty IgA secretory brownish cells and (d) BPC treated infected group showing mild focal increase of IgA secretory brownish cells (ImmunohistochemistryX200) |
Its therapeutic applications did not stimulate germ resistance and did not damage useful microflora52.
Several studies stated the anti-parasitic activities of propolis against cultured Blastocystis ST3 and ST137, Leishmania infantum and Leishmania tropica strains53, Giardia lamblia, Trichomonas vaginalis, Toxoplasma gondii, Leishmania donovani and Trypanosoma cruzi 54,55.
Similarly, AlGabbani et al.56 demonstrated that methanolic extract of Saudi propolis had antimalarial and antioxidant properties and provided protection against spleen tissue destruction in Plasmodium chabaudi infected mice. In addition, royal jelly and pollen acted as a potent antibiotic, they caused inhibition of all types of bacteria, both Gram-positive and Gram-negative and microbes. They were beneficial for a wide range of health conditions57,58.
In the present study the OLE and BPC caused apoptotic-like death and PCD in treated cultured Blastocystis as was evidenced by ultrastructural morphological changes.
The Programmed Cell Death (PCD) pronounces a biochemical and pathological process of cell removal that theatres a significant role in upholding tissue homeostasis59, 60.
Mishra et al.61 and Mishra62 had provided detailed reviews on the biotechnological approach of apoptosis in living organisms and some of the relative features of PCD in plants and animals.
Similarly, Nasirudeen et al.63 reported apoptosis-like features and PCD in growing cultures of MTN treated axenic B. hominis. Raman et al.64 revealed a raise in the number of MLO in symptomatic Blastocystis subtype 3 treated with MTN.
The presence of MLOs’ implicated that these treated parasites need greater energy for the purposes of internal reorganization in response to the drug effects64 which revealed that there was an increase in the MLO number in symptomatic Blastocystis subtype 3 due to MTN treatment.
In addition to its anti-Blastocystis activity, BPC in the present study reduced Blastocystis induced intestinal tissue pathology, as evidenced by the return of the villous like pattern, villous-crypt ratio and few scattered inflammatory cells present. Also, BPC improved host defense Blastocystis infection as evidenced by increased IgA secretory levels in intestinal mucosa. These IgA secretory cells play a central and predominant role in the host defense against Blastocystis infection. Compared to BPC, both MTZ-treated and OLE-treated mice showed only partial improvement in villous atrophy with moderate inflammation, as well as the presence of few local secretory IgA in the immunohistochemical stained intestinal sections.
Likewise, Dimov et al.65 reported that bee products have a non-specific immunostimulant efficacy and Scheller et al.66 found that propolis ethanolic extract stimulates antibody production by mice spleen cells.
Wang et al.67 reported that polyphenol-rich propolis extracts strengthen the function of intestinal barrier which is attributed to activation of AMP-activated protein kinase and extracellular signal–regulated kinases signaling pathways, which is highlighted the potential application of propolis for human gut health.
CONCLUSION
It is concluded that OLE and BPC had potential therapeutic effect and could be used as safe natural therapeutic alternatives against Blastocystis infections. In addition to its anti-Blastocystis activity, BPC has a restorative effect on the host tissue as it normalized the villous architecture and increased the local IgA secretory villous cells.
SIGNIFICANCE STATEMENT
This study highlighted the possible therapeutic effects of olive leaf extract and bee pollen compound that can be beneficial and potent safe natural alternatives for blastocystosis. This study may open fresh avenues for development of an alternative therapy in order to augment or even replace some of the standard chemotherapeutic agents currently employed in the treatment of blastocystosis.
ACKNOWLEDGMENT
The authors deeply appreciate the support given by Faculty of Agriculture Research Park (FARP), Electron Microscope Department, Cairo University, Egypt to image the electron microscopic pictures.