Evaluation of Amygdalin (B17) and Cucurbita pepo (Pumpkin seed) Activity Against Blastocystis from Diarrheic Patients in Baghdad, Iraq: in Vitro Study

Blastocystis is a ubiquitous human and animal protozoa that inhabit the gastrointestinal tract. Metronidazole is considered the standard drug for the treatment of Blastocystis infection; however, there is growing evidence of treatment failure, hazardous side effects, and appearance of strains resistant to metronidazole. In the last era, many studies have been implicated in the quest for new treatments for Blastocystis infection, especially natural products. Attention has been focused on the effect of Amygdalin (B17) and pumpkin seed on eradicating parasitic infections. The current work was built up to explore the in vitro efficacy of two natural compounds, Amygdalin (B17) and pumpkin seeds against Blastocystis isolated from symptomatic patients. In vitro incubation of the parasite with B17 (200, 400μgmL ̄1), pumpkin seed (200, 400 μgmL ̄1) and metronidazole (100, 150μgmL ̄1) was counted at different periods (one, two, twentyfour and forty-eight hours) and morphological changes were evaluated using Light Microscope. Blastocystis detected from patients with symptoms was subtype 1. The B 17 and pumpkin seed demonstrated statistically significant (p<0.05) growth reduction of Blastocystis in culture. Such results showed the possible therapeutic effects of B 17 and pumpkin seed against blastocystosis as effective safe natural alternatives.


Introduction:
Blastocystis is one of the world's most widely distributed intestinal protists that infect humans and animals 1 . The prevalence of Blastocystis in industrialized and developing countries varies between 0.5-30% and 30-76%, respectively 2. The Blastocystis pathogenicity remains a controversial issue. Originally, it was thought to be a commensal protozoan, but recent studies have supported its pathogenicity 3 . Blastocystis causes enteritis, which results in diarrhea, bloating, abdominal discomfort, and/or vomiting 4 . Clinical studies also connect the parasite with other inflammatory conditions of intestine and skin, like irritable bowel syndrome and urticarial disorders. Immunocompromised people (patients of HIV/acquired immunodeficiency syndrome or cancer) are especially susceptible to infection, indicating that Blastocystis can work as an opportunistic pathogen 5 .The morphological forms recorded from stool samples and/or culture media are vacuolated, granular, trophozoite, and cyst forms 4 . The vacuolated form is the most commonly observed in feces and is responsible for the fecaloral transmission of infection.
Blastocystis sp. is diagnosed by microscopic detection in direct smears performed before or after fecal sample cultivation or molecular parasite DNA identification 6 . Small-subunit (SSU) rRNA gene molecular studies categorized Blastocystis into 17 subtypes (ST1-ST17). From these, the subtypes 1-9 were reported in humans, and ST1 to ST4 being the prevalent subtypes in humans found in >90% of examinations 2 . In a recent study from South America, subtype 12 has also been reported found in humans 7 . Due to the uncertainty surrounding the potential pathogenicity of Blastocystis and the self-limiting nature of the symptoms, this disease is treated equivocally 8  have referred to treatt insufficiency indicating the development of isolates not responding to therapy 9 . Also, metronidazole has many drawbacks and risks, like migraine, dizziness, queasiness a metallic taste in the mouth, reversible neutropenia with rare major adverse reactions including pancreatitis, peripheral neuropathy and CNS toxicity consisting of seizures, encephalopathy, cerebellar dysfunction, paresthesia, mental confusion, and depression. These neurologic reactions generally occur only with high, prolonged, cumulative doses. As a result, a new therapy for Blastocystis especially the analysis of natural active agents has been quested 10 .
In the developing countries, medicinal plants have been widely used for centuries due to their abundance, inexpensiveness, and conventional uses 10 . Amygdalin is a natural plant-derived from pebbles of rosaceous fruit such as apricots, almonds, cherries, peaches, and plums 11 . It is made up of two glucose molecules, a benzaldehyde molecule and a hydrocyanide molecule, it has been shown that benzaldehyde of amygdalin is capable of inducing an analgesic effect and hydrocyanide of amygdalin is capable of inducing an anticancer effect 12. It is also known as vitamin (B17), it possesses several benefits and used to treat many disorders such as asthma, nausea, leprosy, bronchitis, and leukoderma. 13 . It also benefits the digestive system, where it has a calming and defensive effect; as well as the urinary system, where human renal fibroblast apoptosis is encouraged and kidney function improved 14 .
One of the well-known edible plants is the pumpkin. Since some special natural edible substances are present, where it has important medicinal properties. Many Phyto-constituents of alkaloids, flavonoids, palmitic acid, oleic and linoleic groups are present in the pumpkin. Thus, various essential medical characteristics are well known, in particular anti-diabetes, antioxidants, anti-carcinogens, anti-inflammatories, etc. 15 Pumpkin seed has been used as a traditional medicine in various parts of the world treating gastrointestinal parasites with antihelmintic properties especially against tapeworm 16

Materials and Methods:
Collection of fecal samples: Fresh fecal samples were taken from 16 patients complaining of gastrointestinal symptoms like diarrhea, abdominal pain, and distention from the Central Pediatric Teaching Hospital in Baghdad, Iraq. The stool samples were collected in sterile clean stool cups and transferred to Parasitology Laboratory at the College of Sciences for women. Processing of fecal samples: The microscopic examination of fecal samples were done immediately using direct wet smear in saline solution and colored with Lugol's iodine then the concentration technique was done using formalinethyl acetate, stool smears were stained using modified Ziehl-Neelsen acid-fast staining to eliminate the possibility of multiple parasitic infections and to detect Blastocystis 10 .Stool samples contain Blastocystis sp. were divided into two parts; part was used for culture on the same day and the other part for molecular assays and genotyping was stored at -20 ° C. Blastocystis culturing In Vitro: 25µg/ml taken from fecal samples with Blastocystis were cultured in Jones' medium (3ml) not containing rice starch and enhanced with 10% horse serum 17 , and synergistic antibiotics were added 100 IU\ml penicillin, 100 µg\ml streptomycins and 1.25 µg \ml amphotericin B to prevent contamination 18. The culture media were incubated with 5% CO 2 at 37°C for 72 h and light microscopic examination was done daily 19 . We studied to consider the culture media negative when there is no Blastocystis growth after 72h 8,10 . Cultures were used for drug testing when vacuolated forms of Blastocystis were more than 1×10 4 / µL. Blastocystis molecular characterization: sixteen stool samples were infected with Blastocystis and were used in this work. DNA extraction: 1-Separated genomic DNA of the isolated samples was used by the maker 's instructions (Geneaid biotech, Taiwan), using a Geneaid Kit (STLD004, STLD050, STLD100), as indicated. 2-DNA density and purity was measured by nanodrop, with a higher concentration of ethidium bromide (0.7 μg / ml) pre-colored in the buffer of TAE (40 mM tris-acetate, 2 mM EDTA (pH 8.3), and with a 1 kb of the ladder as molecular weight marker (Cat # D-1040, Bioneer, Daejeon, South Korea), the integrity of the DNA was assessed by electrophoresis in standard agarose gel at 0, 8% (w / v).3-The PCR has been used as disengaged DNA. Polymerase chain reaction(PCR): 20 recommended that one PCR fragment was selected for amplification, which is supposed to partially cover 607 bp fragment indented to partially amplify the small subunit ribosomal RNA (SSU) rRNA locus within Blastocystis sp. genomic DNA sequences (Table 1).  The freeze-dried primers were purchased from (Bioneer, Daejeon, South Korea). 1-The PCR reaction was carried out utilizing the AccuPower PCR premix (Cat # K , the accompanying program has been applied. 5-Amplification started with an initial denaturation at 94 ° C for 5 min, followed by 30 denaturation cycles at 94 ° C for 30 seconds, annealing at 59 ° C for 30 seconds and extension at 72 ° C for 30 seconds, and ending with a final extension at 72 ° C for 5 minutes. 6-The amplification was verified by electrophoresis on an ethidium bromide (0.5 mg/ml) 1.5% (w / v) precolored agarose gel in 1 × TBE buffer (2 mM EDTA, 90 mM Tris-Borate, pH 8.3), utilizing a Ladder of 100 bp (Cat # D-1010, Bioneer, Daejeon, South Korea) as a molecular weight marker. All PCR bands were assured to be explicit and consist of only one spotless and sharp band to be effectively submitted for sequencing. DNA Sequencing of PCR products: The settled PCR amplicons were commercially sequenced from termini, forward, and reverse, as indicated by guidance manuals of the sequencing organization (Macrogen Inc. Geumchen, Seoul, South Korea). Additionally, Specific chromatographic information obtained from the ABI sequence documentation was analyzed to ensure that the analysis and classes did not result from PCR or artifact sequence. By looking at the DNA sequences observed for live samples with the DNA sequences obtained from Blastocystis sp., the virtual locations, and other details of the recovered PCR pieces were differentiated. Interpretation of sequencing data: The consequences of sequencing the PCR results of various samples were different and adjusted, then examined along with the individual arrangements of the reference database utilizing Bio Edit Sequence Alignment Editor Version 7.1 (DNASTAR, Madison, WI, USA). The varieties observed in each sequenced test were numbered in PCR amplicons as well as in their relative position within the alluding genome. Drugs: Metronidazole: It has been used as an antiprotozoal reference drug, supplied by Ajanta pharma limited 21 . To set up the stock solution of 1 mg mL-1, the tablet was granulated and disintegrated in sterile refined water, and at that point stored in a dim container at 4 ° C. The last levels of Metronidazole have been standardized to100 and 150μg mL -1 . Pumpkin seed powder: About 650 mg per capsule manufactured and provided by Earth Natural Supplements, Florida, and USA. The capsule's powder was broken down in sterile refined water to achieve a 1 mg mL -1 final stock solution. Final concentrations of 200 and 400 μg mL -1 were adjusted. Amygdalin (B17) Powder: About 500 mg per capsule manufactured in the USA for Zildek Nutrition, New York. The capsule's powder was broken down in sterile refined water to achieve a 1 mg mL -1 final stock solution. Final concentrations of 200 and 400 μg mLwere adjusted. The activity of Metronidazole, Pumpkinseed, and Amygdalin B17 as anti-Blastocystis in vitro In vitro experiment: Inoculums size of 8×10 4 parasites taken from cultures were brought into each group of culture tubes which have Jones medium at the various concentrations of Metronidazole, Pumpkinseed, and Amygdalin B17 to detect the growth of the parasite. Untreated cultures of positive control (only parasites) and negative control (only culture media) were exposed to a similar condition as those utilized for the rest of the studied cultures. Triplicate tubes containing culture media were utilized for each concentration of Metronidazole, Pumpkinseed, Amygdalin B17, and untreated cultures. Blastocystis was tested with a graduated concentration of Metronidazole (100 and 150μg mL -1 ), Pumpkinseed (200 and 400 μg mL -1 ), and Amygdalin B17 (200 and 400 μg mL -1 ). After 1, 2, 24, and 48 h, the cultures tested were incubated at 37 ° C, then the culture was examination. In previous standardized studies, the treatment time of at least 1 h was utilized and indicated that this timeframe is important to stimulate the cytotoxic reaction in these parasites 22 .
Where, in control cultures, "a" is the mean number of organisms and "b" is the mean number of organisms in treated cultures (10). Statistical analysis: Data as Mean counts± Standard deviation were recorded. Statistical analyzes were conducted using computerized SPSS statistical program version 15 the independent sample t-test was used for assessing the statistical significance of the mean difference between the two study groups. Statistical value determination as p<0.05. Ethics Committee approval: The proposal of this study was endorsed by the research ethics committee at the College of Sciences for women. The patients involved in this study verbally on the motivation behind the study and the collection of fecal samples were carried out after obtaining their verbal approval. Results: Amplification and genotyping of Blastocystis isolates: Five samples were positive for Blastocystis by PCR from the 16 positive specimens. Subtype 1was the genotype that was detected using genotypic assessment (Fig. 1). Sequencing results: About the investigated samples of S1 -S5, the sequencing reactions indicated the exact positions after performing the National Center of Biotechnology Information (NCBI) BLASTn for these PCR amplicons. NCBI BLASTn engine has shown about 99% sequences of similarities between the sequenced samples and this target. NCBI BLASTn engine has indicated the presence of remarkable homology with the expected target that covered a portion of the SSU rRNA within Blastocystis sp. genomic DNA sequences. By looking at the DNA sequences observed from these nearby samples with the DNA sequences recovered (GenBank acc.) MK719675.1, the specific positions and different subtleties of the recovered PCR fragment were recognized (Fig. 2).  (Table 2). The sequencing chromatogram of DNA sequences in addition to its particular interpretation was recorded and these variants pattern within the amplified sequences are shown in (Fig.3). In the position 399 th of the PCR amplicon, a nucleic acid substitution of Adenine (A) to Thymine (T) was detected in both S1 and S2 samples, while S3 -S5 samples kept an entire homology with the referring sequences. To summarize all the results obtained from the sequenced 607 bp fragments, the specific situation of the watched mutation was described in (Table 3).  (Table 4). Culture sample of Blastocystis was stained with iodine after 2 h without treatment (Fig.4).  (Table 4).

Discussion:
Inhuman, Blastocystis is the most common enteric protozoan. Though Metronidazole is the treatment of choice, doctors are still skeptical about an antibiotic prescription for Blastocystis as there is still some controversy about its pathogenicity and frequent reports of failure to respond to chemotherapy 23 . Variation from strain to strain in Blastocystis of sensitivity to metronidazole, overprescription as well as abuse of antimicrobial drugs might be some explanations for the failure of the treatment 24 .
Considering the side effects and resistance of many antiparasitic medications, attention has been focused on plant extracts and herbal compounds used in traditional medicine that have therapeutic potential as sources for new treatments 25 . Also, WHO has also recommended the start of studies to identify and characterize new herbal preparations from traditionally known plants and the development of new effective therapeutic agents, particularly in areas where many individuals lack access to needed preventive and treatment care. Natural sources were considered the best option in this regard to isolating new and novel anti-microbial components 26 .
In this study, genotypic evaluation of the isolated positive samples of Blastocystis showed infection with subtype I. This finding is following many epidemiological studies worldwide and, also in Iraq that reported STI as the mostly detected subtype 27,28 .In agreement with our results, several studies stated that ST1 is the prevalent subtype that showed an important role in pathogenic consequences in humans 29,30 . In the present study, Amygdalin (B17), pumpkin seed, and metronidazole demonstrated a statistically valuable (p<0.05) reduction of Blastocystis development according to the used concentration. Amygdalin B17 showed the highest reduction in Blastocystis numbers for all incubation times with statistical valuable change (p<0.05) in comparison with the control untreated group. Amygdalin B17 at concentrations of 200μg/ml showed significant inhibition (p<0.05) in Blastocystis numbers for all incubation times (1, 2, 24, 48 h). It is shown that the highest concentration of Amygdalin B17 (400μg/ml) caused the parasite to die quickly after 48 h and triggered considerably more grounded impacts with mean growth inhibition of 100% similarity with 150 µg mL¯¹ MTZ after 48h.
During the late 1970s, amygdalin got one of the most mainstream natural compounds utilized by tumor patients. Notwithstanding alerts of the potential danger of cyanide harmfulness, amygdalin has encountered a solid renaissance as another alternative for treating cancer 31 . Still, controlled clinical investigations have not been completed and inquiries concerning amygdalin's effectiveness have not yet been agreeably replied 32 .
In a study done by 33 , amygdalin possessed a significant protective effect for chronic liver injury in rats. Furthermore, amygdalin intake abolished the majority of the pathological alterations of the LPSinduced liver damage of rats, the useful effects of amygdalin can be attributed to its action against inflammation and enhancement of liver impairment by inhibition of PI3K/AK JAK2/STAT3 and NF-κB signaling pathways. In another study done by 34 when rats were treated orally with 500 mg/kg of pepsin hydrolysate of almond water-solution on carbon tetrachloride, the action of pepsin was inhibited by the benzaldehyde resulted from the disintegration of amygdalin. Additionally, the level of AST and ALT decreased, hydroxyproline content raised and the extension of euglobulin lysis time was lowered. Also, it could diminish the proliferation of connective tissue of rat liver. Moreover, amygdalin was found to be an effective medication for chronic gastritis and atrophic gastritis in rats 34 .
Pumpkin seed at concentrations of 200μg/ml showed significant inhibition (p<0.05) in Blastocystis numbers for all incubation times (1, 2, 24, 48 h). On the other hand, the most elevated concentration of pumpkin seed which produced the highest reduction of parasite counts was 400μg/ml after 48 h.
There are different varieties of pumpkin, the most noticeable species being Cucurbita Maxima, Cucurbita pepo, and Cucurbita moschata, which are discovered around the world 35 . Pumpkin is one of the most notable palatable plants and has significant medical properties due to the presence of important natural consumable substances; it includes a few Phyto-constituents having a place amnog the classifications of alkaloids, fatty acid and different amino acid which are biologically active specific nurturance 35 .
Separating different broad spectrum of antimicrobial compounds from pumpkins. In a study done by 36 37 . In 1991 38 stated that the crude ethanolic extract of C. maxima is viewed as valuable in vivo inhibition and prevention of the development of parasitemia in vivo. The utilization of C. maxima unrefined ethanolic extract and pyrimethamine expanded invulnerability against malaria. Leaf concentrates of C. maxima display larvicidal and ovicidal properties and can be utilized as a defensive boundary against mosquito chomp 39. The antihelmintic effect of C. moschata was assessed in vitro from aquatic, methanolic, and dichloromethane seed separates against Haemonchus contortus, the parasitic nematode of little ruminants, and it was affirmed that C. moschata has larval growth reduction at all concentrations 40 . Extracts of pumpkin seeds and pomegranate peels against Ascaridia galli have been reported In vitro and In vivo anthelmintic activity by 41 . Pumpkin (Cucurbita pepo L) seeds are a valuable source of protein and bioactive compounds when mathematical models have been used to study the effect of pumpkin extracts on the weight gain of chicken 42 .
In this study, MTZ displayed inhibitory effects on Blastocystis with two concentrations (100 &150 µg mL¯¹). The 100µg mL¯¹ concentration produced highly significant inhibition in Blastocystis numbers within 24 h, whereas the 150µg mL¯¹concentration produced no growth after (48 h). 43 reported that Blastocystis ST1 and ST3 were vulnerable to metronidazole at 50 and 100 μg mL -1 concentrations with no growth within 24 hours, whereas the10 μg mL -1 concentration produced significant inhibition (p<0.05) in Blastocystis numbers for all incubation times (24,48, 72 h) with toxic effect within 72 hours.
So, in correlation with healthy controls at 0.01 and 0.1 mg mL -1 concentrations, 44 recorded vulnerability of all Blastocystis isolates to metronidazole at 0.01 and 0.1 mg mL -1 concentrations, these isolates were, for the most part, ST3 and coinfection of ST3 and ST1, yet a similar report indicated variable adequacy of metronidazole and was for the most part ST1. This dispute over the efficacy of metronidazole can be attributed to variations within subtype in the vulnerability mechanisms of the drugs, likely due to the presence of specific alleles in each subtype 45

Conclusion:
We conclude that Pumpkin seed and Amygdalin (B17) possessed potential beneficial activity and can be used as safe agents derived from nature for Blastocystis STI infections treatment.