Effect melittin of bee Apis mellefera venom in pathogenic parasites in vitro

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Jasim , This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7420042/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The results showed that Entamoeba histolytica was killed when the concentration of 0.6 µg/micromole was 280000 amoebae/ml and killed 70%. When the concentration was 0.8 micrograms/micromole, the killing rate was 380000 amoebae/l, and the killing rate was 90.5%. The percentage of Giardia was 3 000 (6000) parasites/mL at the first concentration of 0.6 µg/micromole, and 60% at a concentration of 0.8 micromole/micromole was 7000 par0,6asites/mL. The percentage of killings was 80%. Trichomonas vaginalis . The concentration of 0.6 µg/micromole depended on parasite movement of 85%. The percentage of the last concentration was 90%, and the killing rate was determined by the concentration of venom on the parasite. Biomedical Engineering Apis mellifera Trichomonas vaginalis Concentration Venom Giardia Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Recently, the study of biological antibacterial materials has attracted considerable attention from medical and pharmaceutical researchers, leading us to study new species that have few side effects on human health and the environment. These germs cannot develop immunity against them. These biological compounds are old, sophisticated weapons. Many animal venoms, such as cobra, scorpions, and frogs, have shown antibacterial properties [1]. They were later used as pharmaceutical preparations for their effective role as the primary immune response mechanism [2]. These ancient materials were isolated from the kingdoms and people of living beings [3]. The honey bee Apis mellifera is an economical insect beneficial to humans and the environment it is easy to breed. Its various products are beneficial in terms of food and medicine, especially for honey bees, which were used by the ancient Egyptians, Indians and Greeks to treat various diseases. [4] Bee poison is currently used in complementary medicine for blood clotting, analgesics, anti-inflammation and as a treatment for some chronic diseases, such as tendonitis and scleroderma [5, 6]. Studies on bee venom began in the late 19th century [7], known as antimicrobial peptides (AMPs), a unique and varied group of molecules divided into subgroups based on the composition and structure of amino acids, varying in length and effect., is composed of 12–50 amino acids, with a molecular mass is less than 10 kilo Dalton, whose net-positive charge is as acidic as 2 þ -7 þ because there is a surplus of the essential acids (arginine, lysine and histidine). These compounds contain a high water content of up to 80% and are liquid, transparent, colorless, with pungent odor, water-soluble taste and some acids, resistant to alkaloids, 1.13, pH 5.5 [10, 11]. These aquatic compounds react with bacterial membranes as part of the mechanism of their effects [12]. These substances show some advantages that are encouraged to be used as alternatives to traditional medicines because of their speed of action and not to development resistance against the target organism [13]. These compounds have high toxic against the positive and negative bacterial gout, fungi, metazoans and cancer cells [14]. Melittin is the most representative bio peptide antibiotic in antibacterial compounds [8][9], consisting of 26 aquatic amino acids (arginine, lysine, lysine, arginine), and its molecular weight is 846.462, which represents 40%-50% of the dry weight produced. The honey bee A. mellifera [10] of the acid and alkaline gland in the Venom sac venom sac located at the end of the last ventricular loop of the worker is used to defend itself and the cell from enemies by using a stinging machine. poison in the body of the victim through it [15, 16]. Many human parasites are infected with amoebae, the condition of the tissue Entamoeba histolytica , causing the disease Amebiasis Amebeasis or Amebic dysentery. This parasite is spreads among poor strata of society, malnutrition or possibly unhealthy conditions. The most important places in the tropical and subtropical regions [17]. According to the Health World Organization, amoeba parasites cause more than 100,000 deaths per year, the second most common type of malaria. [18] Amebic liver abscess (amoebic liver abscess) is the condition of the primary parasite. Protozoa require the mucous medium of the intestine to spread to other members of the body, such as the liver. Giardia lamble Giardia is one of the two laryngeal lupus epidemiologists and causes giardia diarrhea. It has two parallel pylori vegetarians [19] [20], containing two nuclei and four lactates, which have a macrophage phase in the external environment [21]. The infectious stage when the is eaten with contaminated food and water is digested by gastric acid in the intestinal cavity, leading to release from the sac into two vegetative stages that multiply bilaterally [22,23]. The Trichomonas vaginalis parasite is one of the parasitic parasites that has a single stage, affecting the vaginal parasite in females and urethral males, and the parasite is infected by approximately 25 m Lyon pregnant women [24]. Because of the lack of studies on this subject, bee venom was selected as a biological compound against the parasites Entamoeba histolytica, Giardia gamble and Trichomonas vaginalis . The study aimed to measure the effect of bee venom by collecting local bee strains and testing its effectiveness on the parasites above and comparing the results with the effect of pure poison imported from the company Sigma. Materials and methods Lyophilized bee venom and fetal bovine serum were obtained from Sigma (Minnesota, USA) and Lichrosol (Merck com., Germany). The local bee venom consists of [ 15 ] A. mellefera bees (the cell consists of two types of Iraqi and Italian bee breeds) from the saplings of the Faculty of Agriculture - University of Baghdad - Jaderia - Baghdad. Form BV0508, early in the morning, put the device in the entrance of the cell and when passing through the bees will be exposed to the electrical current, as it descends the machine sting and falls venom to the glass plate at the base of the device, take the glass board and scrape the venom, the device is used on another cell to get more venom, kept in a poisonous pyramid after being scrapped in special containers at a temperature of 4 M 0 until purification [ 25 ]. Dissolve the raw venom crystals that weigh 100 mg in 4-ml Lichrosol. The solution was placed in a centrifuge for 20 min at a temperature of 4°C at 12,000 cycles/minute. The floating solution was taken and placed in the centrifuge again and under the same conditions, and the precipitate was neglected. The floating liquid was passed through a membrane filter with pores of 0.22 m until use. Preparation of solutions Stock solution (25) mg of each venom imported from Sigma bee venom (SBV) and from the local bee venom (LBV) toxin purified in 1 ml of PBS solution. Both samples are performed under the same conditions, which are the two fundamental solutions. Protein calibration (calibration): The proteins were measured according to method [ 27 ], where a standard series of fetal bovine serum (FBS) was obtained with concentrations of 0.6 '0.8 µg/µm in the same saline solution (PBS) used to dissolve the toxin. The primary solutions of the samples (SBV) and 50-fold (LBV) on gel electrophoresis were measured. The absorbance was measured with a 565 nm wave ELISA for all samples. The concentration of the protein in the SBV and LBV samples was determined by the projection on the standard curve of a series of increasing concentrations of embryonic cow serum (FBS). Parasite Entamoeba histolytica The E. histolytica parasite was obtained from the patients' stool samples of Kadhimiyah Teaching Hospital. After testing for laboratory diagnosis, mucosal mucus samples were selected as an ideal source for the abundance of parasites of the parasite Trophozoites. Contents Except for the protoplasm [ 24 ]. The middle is the center of Lock-Egg Di phase: medium according to Brand et al. [ 28 ]. To control the biomaterial density of the lamina grown in glass, the blood cell count and the 1% Eocene dye were used based on the no permeability of the cosine dye to the living parasite cells. The amoeba was used with a density of 400 000 amps/liter and 95% vitality. It was selected by adding 1 ml from the amoeba farm to the seed medium (LE) to a test tube. The first group was treated with a concentration of 0.6 micrograms/micromole. The second group was treated with a concentration of 0.8 µg/micromole. The third group remained without treatment (control treatment). The tubes were gently pumped to distribute the mixture equally to the plant medium of the ameba. in the incubator at a temperature of 38°C for 2 h, the tubes were taken, and the percentage of the parasite was estimated. Amoeba kill percentage = \(\:\frac{\:the\:numbeof\:Amoeba\:dyed\:byeosin\:}{total\:number\:of\:Amoeba}\) x100 Parasite Giardia gamble Samples were collected from the liquid stools of the patients of the Kadhimiyah Teaching Hospital. After direct examination and confirmation of the infection, the bag was purified based on Sheffield and Bjorvatan [ 30 ]. The cells of the sac were counted by means of a hem cytometer at 105 cell/ml. The stool specimen was planted after the preparation of the giardia parasite medium (HSP-1) as follows: One milliliter of the 105-cell suspension was placed into the center of the plant, and 3-ml serum was added to the medium with two active antibodies, penicillin and streptomycin. The three 3-day-long incubation tubes were incubated at Candle Jar for a period of 3 days. A routine check was performed on the third day. A drop of the implant was placed on a glass slide. The lid was examined on a 400x and 1000x optical microscope. The trophozoites were found in small numbers. The first group was treated with a concentration of 0.6 micrograms/micromole. The second group was treated with a concentration of 0.8 µg/micromel. The third group remained without treatment (control treatment). The tubes were gently pumped to distribute the mixture equally to the embryonic medium of the parasite. in the incubator at a temperature of 38°C for 2 h, the tubes were taken, and the percentage of the parasite was estimated. Percentage of dead parasite = \(\:\frac{\:the\:numbeof\:parasite\:dyed\:by\:eosin}{total\:number\:of\:parasite\:}\) x100 Parasite Trichomonas vaginalis Vaginal parasites were obtained by taking swabs from the Alawiya and Ibn Al-Baladi Hospitals of women with vaginal itching and suffering from vaginal discharge, consultation with a specialist and after laboratory diagnosis. A moist swab was placed in the middle of the sperm called diamond [ 31 ] after the parasite identified 102 organisms/ml [ 32 ], and a swab was taken and planted at 5 ml from the center of the diamond. Fetal bovine serum, a 56 µm dose for 30 min, preserves the serum at 20°C. Add the swab to 5 ml of the center of the plant plus 0.5 ml of F. b. S and incubate at 35°C for 72 h and examine after 4, 6, 7 days after incubation and note vaginal trigeminal movement. The first group was treated with a concentration of 0.6 micrograms/micromole. The second group was treated with a concentration of 0.8 µg/micromole. The third group remained without treatment (control treatment). The tubes were gently pumped to distribute the mixture equally to the implant medium. in the incubator at a temperature of 38°C for 2 h, the tubes were taken, and the percentage of the parasite was estimated. Percentage of dead parasite = \(\:\frac{\:the\:numbeof\:parasite\:deyed\:by\:eosin}{total\:number\:of\:parasite}\) x100 Results and Dissection The results shown in Figure (1) show that E. histolytica was killed in the form of [2] when the concentration of 0.6 µg/micromole was 280000 amoebae/ml and killed 70%. When the concentration was 0.8 micrograms/micromole, the killing rate was 380000 amps/l, and the killing rate was 90.5%. The percentage of Giardia was 3 000 (6000) parasites/mL at the first concentration of 0.6 µg/micromole, and 60% at a concentration of 0.8 micromole/micromole was 7000 parasites/mL. The percentage of killings was 80%. Trichomonas vaginalis. [ 4 ] The concentration of 0.6 µg/micromole depended on parasite movement at 85%. The percentage of the last concentration was 90%, and the killing rate was determined by the concentration of toxin on the parasite. Male Bradford et al. [ 27 ] mentioned that peptide compounds extracted from various organisms have inhibitory activity against microbes and act as a key to disrupting the cell surface. In some cases, their entry affects calcium levels within these microbes, as well as their effect on mitochondrial function, stimulating self-induction and thus necrosis and apoptosis. There is compelling evidence that the melittin compound can penetrate through cell membranes nonselective by stimulating the formation of pores and openings. It also analyses primitive and eukaryotic cells [ 33 ], the method responsible for the degradation of blood cells and macrophages [ 34 ] [ 35 ] and against cancer cells [ 36 ]. Melittin was killed by Leishmania . [ 37 ] The melittin hybrid cecropia recently showed significant inhibitory activity against Leishmania stages with a slight effect on host cells of the parasite [ 38 , 39 ] and even inside the cells of the animals, giving way to various further research and future studies that support the use of this vital compounds, as alternate chemical compounds that are used to treat diseases caused by these microbes and parasites, as well as cancers, with side effects on human health in general and his animals. Declarations CONFLICTS OF INTEREST The authors declare no conflict of interest. Declarations of Generative AI and AI-assisted technologies in the writing process FUNDING STATEMENT This research received no external funding. ACKNOWLEDGMENTS My profound thanks and appreciation to Mustansiriyah University. www.uomustansiriyah.edu.iq)Baghdad-Ira q for its support and assistance in the present work. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7420042","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":503281019,"identity":"796ee0fb-a771-4151-a333-47c29c103e8c","order_by":0,"name":"Assist Prof Dr.FAYHAA ABBOOD MAHDI AL-NADAWI","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYBACPhDBA0T8DAxsxGlhg2mRbCBVC4PBAaK1SDcf/PCm5rCM8Y3kZw8+VDDI84sdIKBF5liy5Jxjh3nMbqSZG844w2A4c3YCAS0SOQbSPGy3gVoSzKR52xgSDG4T1JL/+TfPv9s8xjPSvxGrJYcNqPI2j4FEDtG2pJlZzu37zyNx5k2Z5IwzEoT9wi+R/PjGm29p9vzt6dskPlTYyPNLE9CCAAJglRLEKgfbd4AU1aNgFIyCUTCSAABfzjxbr/3gUwAAAABJRU5ErkJggg==","orcid":"","institution":"Mustansiriyah University","correspondingAuthor":true,"prefix":"","firstName":"Assist","middleName":"Prof Dr.FAYHAA ABBOOD MAHDI","lastName":"AL-NADAWI","suffix":""},{"id":503284789,"identity":"9949d805-81ac-487b-9c94-747d921ddfbb","order_by":1,"name":"Pro.Dr. Aktham N. Jasim ,","email":"","orcid":"","institution":"Mustansiriyah University","correspondingAuthor":false,"prefix":"","firstName":"","middleName":"Pro.Dr. Aktham N.","lastName":"Jasim","suffix":""}],"badges":[],"createdAt":"2025-08-20 18:37:35","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":true,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-7420042/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7420042/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89624670,"identity":"de41c5f8-f8a5-4ca2-8f21-c7ec2f997c52","added_by":"auto","created_at":"2025-08-22 05:35:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":239049,"visible":true,"origin":"","legend":"\u003cp\u003ePercentages of killed \u003cem\u003eEntamoeba histolytica, Giardia gamble\u003c/em\u003e and \u003cem\u003eTrichomonas vaginalis\u003c/em\u003e parasites at concentrations of 0.6 and 0.8 μg/micromole in honey bees in vitro.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7420042/v1/04298088f52e39c87227a98f.png"},{"id":89624669,"identity":"b0c214f1-dd19-4ebf-b01a-34dda8985135","added_by":"auto","created_at":"2025-08-22 05:35:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":588193,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cem\u003eEntamoeba histolytica\u003c/em\u003e) parasite\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7420042/v1/c26d13da9c1157f414b88177.png"},{"id":89624676,"identity":"258c7222-7373-41b7-9327-18a34102c901","added_by":"auto","created_at":"2025-08-22 05:35:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":287935,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eGiardia gamble\u003c/em\u003e parasite\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7420042/v1/05231991e225f2fea4ed1696.png"},{"id":89625442,"identity":"88cfd454-29cd-4d5c-a9ae-6e487d4f02ea","added_by":"auto","created_at":"2025-08-22 05:43:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":286528,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eTrichomonas vaginalis \u003c/em\u003eparasite\u0026nbsp;\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7420042/v1/2ace8b772be0947c80c804d4.png"},{"id":89626662,"identity":"72358107-a9f6-49be-a9e6-e6b44b224ab5","added_by":"auto","created_at":"2025-08-22 05:59:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2225807,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7420042/v1/06abca36-159c-4a09-ba01-eb8c2917f9bb.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eEffect melittin of bee \u003cem\u003eApis mellefera\u003c/em\u003e venom in pathogenic parasites in vitro\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRecently, the study of biological antibacterial materials has attracted considerable attention from medical and pharmaceutical researchers, leading us to study new species\u0026nbsp;that have few side effects on human health and the\u0026nbsp;environment. These germs cannot develop immunity against them.\u003c/p\u003e\n\u003cp\u003eThese biological compounds are old, sophisticated weapons. Many animal venoms,\u0026nbsp;such as cobra, scorpions, and frogs, have shown antibacterial properties [1]. They were later used as pharmaceutical preparations for their effective role as the primary immune response mechanism [2]. These ancient materials were isolated from the kingdoms and people of living beings [3]. The honey bee \u003cem\u003eApis mellifera\u003c/em\u003e is an economical insect beneficial to humans and the environment\u0026nbsp;it is easy to breed. Its various products are beneficial in terms of food and medicine, especially for honey bees, which were used by the ancient Egyptians, Indians and Greeks to treat various diseases. [4] Bee\u0026nbsp;poison is currently used in\u0026nbsp;complementary\u0026nbsp;medicine for blood clotting, analgesics, anti-inflammation and as a treatment for some chronic diseases, such as tendonitis and scleroderma [5, 6].\u003c/p\u003e\n\u003cp\u003eStudies on bee venom began in the late 19th century [7], known as antimicrobial peptides (AMPs), a unique and varied group of molecules divided into subgroups based on the composition and structure of amino acids, varying in length and effect., is composed of 12–50 amino acids, with a molecular mass is less than 10 kilo Dalton, whose net-positive charge is as acidic as 2 þ -7 þ because there is a surplus of the essential acids (arginine, lysine and histidine). These compounds contain a high water content of up to 80% and are liquid, transparent, colorless, with pungent odor, water-soluble taste and some acids, resistant to alkaloids, 1.13, pH 5.5 [10, 11]. These aquatic compounds react with bacterial membranes as part of the mechanism of their effects [12]. These substances show some advantages that are encouraged to be used as alternatives to traditional medicines because of their speed of action and not to development resistance against the target organism [13]. These compounds have high toxic against the\u0026nbsp;positive and negative bacterial gout, fungi, metazoans and cancer cells [14].\u003c/p\u003e\n\u003cp\u003eMelittin is the most representative bio peptide antibiotic in antibacterial compounds [8][9], consisting of 26 aquatic amino acids (arginine, lysine, lysine, arginine), and\u0026nbsp;its molecular weight\u0026nbsp;is 846.462, which represents 40%-50% of the dry weight produced. The honey bee \u003cem\u003eA. mellifera\u003c/em\u003e [10] of the acid and alkaline gland in the Venom sac venom sac located at the end of the last ventricular loop of the worker is used to defend itself and the cell from enemies by using a stinging machine. poison in the body of the victim through it [15, 16].\u003c/p\u003e\n\u003cp\u003eMany human parasites are infected with amoebae, the condition of the tissue \u003cem\u003eEntamoeba histolytica\u003c/em\u003e, causing the disease Amebiasis Amebeasis or Amebic dysentery. This parasite is spreads among poor strata of society, malnutrition or possibly unhealthy conditions. The most important places in the tropical and subtropical regions [17]. According to the\u0026nbsp;Health World Organization, amoeba parasites cause more than 100,000 deaths per year, the second most common type of malaria.\u0026nbsp;[18]\u003c/p\u003e\n\u003cp\u003eAmebic liver abscess (amoebic liver abscess) is the condition of the primary parasite. Protozoa require the mucous medium of the intestine to spread to other members of the body, such as the liver. \u003cem\u003eGiardia lamble\u003c/em\u003e Giardia is one of the two laryngeal lupus epidemiologists and causes giardia diarrhea. It has two parallel pylori vegetarians [19] [20], containing two nuclei and four lactates, which have a macrophage phase in the external environment [21]. The infectious stage when the is eaten with contaminated food and water\u0026nbsp;is digested by gastric acid in the intestinal cavity, leading to release from the sac into two vegetative stages that multiply bilaterally [22,23]. The \u003cem\u003eTrichomonas vaginalis\u003c/em\u003e parasite is one of the parasitic parasites that has a single stage, affecting the vaginal parasite in females and urethral males, and\u0026nbsp;the parasite is infected by approximately 25 m Lyon pregnant women [24].\u003c/p\u003e\n\u003cp\u003eBecause of the lack of studies on this subject, bee venom was selected as a biological compound against the parasites \u003cem\u003eEntamoeba histolytica, Giardia gamble\u003c/em\u003e and \u003cem\u003eTrichomonas vaginalis\u003c/em\u003e. The study aimed to measure the effect of bee venom by collecting local bee strains and testing its effectiveness on the parasites above and comparing the results with the effect of pure poison imported from the company Sigma.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eLyophilized bee venom and fetal bovine serum were obtained from Sigma (Minnesota, USA) and Lichrosol (Merck com., Germany).\u003c/p\u003e\n\u003cp\u003eThe local bee venom consists of [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e] \u003cem\u003eA. mellefera\u003c/em\u003e bees (the cell consists of two types of Iraqi and Italian bee breeds) from the saplings of the Faculty of Agriculture - University of Baghdad - Jaderia - Baghdad. Form BV0508, early in the morning, put the device in the entrance of the cell and when passing through the bees will be exposed to the electrical current, as it descends the machine sting and falls venom to the glass plate at the base of the device, take the glass board and scrape the venom, the device is used on another cell to get more venom, kept in a poisonous pyramid after being scrapped in special containers at a temperature of 4 M 0 until purification [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eDissolve the raw venom crystals that weigh 100 mg in 4-ml Lichrosol. The solution was placed in a centrifuge for 20 min at a temperature of 4\u0026deg;C at 12,000 cycles/minute. The floating solution was taken and placed in the centrifuge again and under the same conditions, and the precipitate was neglected. The floating liquid was passed through a membrane filter with pores of 0.22 m until use.\u003c/p\u003e\n\u003ch3\u003ePreparation of solutions Stock solution\u003c/h3\u003e\n\u003cp\u003e(25) mg of each venom imported from Sigma bee venom (SBV) and from the local bee venom (LBV) toxin purified in 1 ml of PBS solution. Both samples are performed under the same conditions, which are the two fundamental solutions.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eProtein calibration (calibration):\u003c/h2\u003e\n\u003cp\u003eThe proteins were measured according to method [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e], where a standard series of fetal bovine serum (FBS) was obtained with concentrations of 0.6 '0.8 \u0026micro;g/\u0026micro;m in the same saline solution (PBS) used to dissolve the toxin.\u003c/p\u003e\n\u003cp\u003eThe primary solutions of the samples (SBV) and 50-fold (LBV) on gel electrophoresis were measured. The absorbance was measured with a 565 nm wave ELISA for all samples. The concentration of the protein in the SBV and LBV samples was determined by the projection on the standard curve of a series of increasing concentrations of embryonic cow serum (FBS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eParasite\u003c/strong\u003e \u003cstrong\u003eEntamoeba histolytica\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eE. histolytica\u003c/em\u003e parasite was obtained from the patients' stool samples of Kadhimiyah Teaching Hospital. After testing for laboratory diagnosis, mucosal mucus samples were selected as an ideal source for the abundance of parasites of the parasite Trophozoites. Contents Except for the protoplasm [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. The middle is the center of Lock-Egg Di phase: medium according to Brand et al. [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. To control the biomaterial density of the lamina grown in glass, the blood cell count and the 1% Eocene dye were used based on the no permeability of the cosine dye to the living parasite cells. The amoeba was used with a density of 400 000 amps/liter and 95% vitality. It was selected by adding 1 ml from the amoeba farm to the seed medium (LE) to a test tube.\u003c/p\u003e\n\u003cp\u003eThe first group was treated with a concentration of 0.6 micrograms/micromole. The second group was treated with a concentration of 0.8 \u0026micro;g/micromole. The third group remained without treatment (control treatment). The tubes were gently pumped to distribute the mixture equally to the plant medium of the ameba. in the incubator at a temperature of 38\u0026deg;C for 2 h, the tubes were taken, and the percentage of the parasite was estimated.\u003c/p\u003e\n\u003cp\u003eAmoeba kill percentage =\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\:the\\:numbeof\\:Amoeba\\:dyed\\:byeosin\\:}{total\\:number\\:of\\:Amoeba}\\)\u003c/span\u003e\u003c/span\u003e\u003cem\u003ex100\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eParasite\u003c/strong\u003e \u003cstrong\u003eGiardia gamble\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSamples were collected from the liquid stools of the patients of the Kadhimiyah Teaching Hospital. After direct examination and confirmation of the infection, the bag was purified based on Sheffield and Bjorvatan [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. The cells of the sac were counted by means of a hem cytometer at 105 cell/ml. The stool specimen was planted after the preparation of the giardia parasite medium (HSP-1) as follows:\u003c/p\u003e\n\u003cp\u003eOne milliliter of the 105-cell suspension was placed into the center of the plant, and 3-ml serum was added to the medium with two active antibodies, penicillin and streptomycin. The three 3-day-long incubation tubes were incubated at Candle Jar for a period of 3 days. A routine check was performed on the third day. A drop of the implant was placed on a glass slide. The lid was examined on a 400x and 1000x optical microscope. The trophozoites were found in small numbers. The first group was treated with a concentration of 0.6 micrograms/micromole. The second group was treated with a concentration of 0.8 \u0026micro;g/micromel. The third group remained without treatment (control treatment). The tubes were gently pumped to distribute the mixture equally to the embryonic medium of the parasite. in the incubator at a temperature of 38\u0026deg;C for 2 h, the tubes were taken, and the percentage of the parasite was estimated.\u003c/p\u003e\n\u003cp\u003ePercentage of dead parasite =\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\:the\\:numbeof\\:parasite\\:dyed\\:by\\:eosin}{total\\:number\\:of\\:parasite\\:}\\)\u003c/span\u003e\u003c/span\u003e\u003cem\u003ex100\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eParasite\u003c/strong\u003e \u003cstrong\u003eTrichomonas vaginalis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVaginal parasites were obtained by taking swabs from the Alawiya and Ibn Al-Baladi Hospitals of women with vaginal itching and suffering from vaginal discharge, consultation with a specialist and after laboratory diagnosis. A moist swab was placed in the middle of the sperm called diamond [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e] after the parasite identified 102 organisms/ml [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e], and a swab was taken and planted at 5 ml from the center of the diamond. Fetal bovine serum, a 56 \u0026micro;m dose for 30 min, preserves the serum at 20\u0026deg;C. Add the swab to 5 ml of the center of the plant plus 0.5 ml of F. b. S and incubate at 35\u0026deg;C for 72 h and examine after 4, 6, 7 days after incubation and note vaginal trigeminal movement.\u003c/p\u003e\n\u003cp\u003eThe first group was treated with a concentration of 0.6 micrograms/micromole. The second group was treated with a concentration of 0.8 \u0026micro;g/micromole. The third group remained without treatment (control treatment). The tubes were gently pumped to distribute the mixture equally to the implant medium. in the incubator at a temperature of 38\u0026deg;C for 2 h, the tubes were taken, and the percentage of the parasite was estimated.\u003c/p\u003e\n\u003cp\u003ePercentage of dead parasite =\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\:the\\:numbeof\\:parasite\\:deyed\\:by\\:eosin}{total\\:number\\:of\\:parasite}\\)\u003c/span\u003e\u003c/span\u003e\u003cem\u003ex100\u003c/em\u003e\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results and Dissection","content":"\u003cp\u003eThe results shown in Figure (1) show that \u003cem\u003eE. histolytica\u003c/em\u003e was killed in the form of [2] when the concentration of 0.6 \u0026micro;g/micromole was 280000 amoebae/ml and killed 70%. When the concentration was 0.8 micrograms/micromole, the killing rate was 380000 amps/l, and the killing rate was 90.5%. The percentage of \u003cem\u003eGiardia\u003c/em\u003e was 3 000 (6000) parasites/mL at the first concentration of 0.6 \u0026micro;g/micromole, and 60% at a concentration of 0.8 micromole/micromole was 7000 parasites/mL. The percentage of killings was 80%. \u003cem\u003eTrichomonas vaginalis.\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e] The concentration of 0.6 \u0026micro;g/micromole depended on parasite movement at 85%. The percentage of the last concentration was 90%, and the killing rate was determined by the concentration of toxin on the parasite.\u003c/p\u003e\n\u003cp\u003eMale Bradford et al. [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e] mentioned that peptide compounds extracted from various organisms have inhibitory activity against microbes and act as a key to disrupting the cell surface. In some cases, their entry affects calcium levels within these microbes, as well as their effect on mitochondrial function, stimulating self-induction and thus necrosis and apoptosis. There is compelling evidence that the melittin compound can penetrate through cell membranes nonselective by stimulating the formation of pores and openings. It also analyses primitive and eukaryotic cells [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e], the method responsible for the degradation of blood cells and macrophages [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e] [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e] and against cancer cells [\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eMelittin was killed by \u003cem\u003eLeishmania\u003c/em\u003e. [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e] The melittin hybrid cecropia recently showed significant inhibitory activity against \u003cem\u003eLeishmania\u003c/em\u003e stages with a slight effect on host cells of the parasite [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e] and even inside the cells of the animals, giving way to various further research and future studies that support the use of this vital compounds, as alternate chemical compounds that are used to treat diseases caused by these microbes and parasites, as well as cancers, with side effects on human health in general and his animals.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCONFLICTS OF INTEREST\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cdiv class=\"Heading\"\u003eDeclarations\u0026nbsp;of Generative AI and AI-assisted technologies in the writing process\u003c/div\u003e\n\u003ch2\u003eFUNDING STATEMENT\u003c/h2\u003e\n\u003cp\u003eThis research received no external funding.\u003c/p\u003e\n\u003ch2\u003eACKNOWLEDGMENTS\u003c/h2\u003e\n\u003cp\u003eMy profound thanks and appreciation to Mustansiriyah University.\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.uomustansiriyah.edu.iq)Baghdad-Ira\u003c/span\u003e\u003c/span\u003eq for its support and assistance in the present work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChen X, Zhang L, Wu Y, Wang L, Ma C, Xi X, Bininda-Emonds ORP, Shaw C, Chen T, Zhou M. 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Int Arch Allergy Immunol 181:783\u0026ndash;789\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTorres MDT, Silva AF, Andrade GP, Pedron CN, Cerchiaro G, Ribeiro AO, Oliveira VX, de la Fuente-Nunez C (2020) The wasp venom antimicrobial peptide polybia-CP and its synthetic derivatives display antiplasmodial and anticancer properties. Bioeng Transl Med 5:e10167\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu R, Li D, Tang Q, Wang W, Xie G, Dou P A novel peptide from Vespa ducalis induces apoptosis in osteosarcoma cells by activating the p38 mapk and jnk signaling pathways. Biol. Pharm. Bull. 41, 458\u0026ndash;464. [7] Gong, Yuan J, Gao H, Hu Z (2018) F. Wasp venom and acute kidney injury: The mechanisms and therapeutic role of renal replacement therapy. 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Leishmaniasis Antimicrob Agents Chemother 48(2):64\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Mustansiriyah University ","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Apis mellifera, Trichomonas vaginalis, Concentration, Venom, Giardia","lastPublishedDoi":"10.21203/rs.3.rs-7420042/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7420042/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe results showed that \u003cem\u003eEntamoeba histolytica\u003c/em\u003e was killed when the concentration of 0.6 \u0026micro;g/micromole was 280000 amoebae/ml and killed 70%. When the concentration was 0.8 micrograms/micromole, the killing rate was 380000 amoebae/l, and the killing rate was 90.5%. The percentage of \u003cem\u003eGiardia\u003c/em\u003e was 3 000 (6000) parasites/mL at the first concentration of 0.6 \u0026micro;g/micromole, and 60% at a concentration of 0.8 micromole/micromole was 7000 par0,6asites/mL. The percentage of killings was 80%. \u003cem\u003eTrichomonas vaginalis\u003c/em\u003e. The concentration of 0.6 \u0026micro;g/micromole depended on parasite movement of 85%. The percentage of the last concentration was 90%, and the killing rate was determined by the concentration of venom on the parasite.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e","manuscriptTitle":"Effect melittin of bee Apis mellefera venom in pathogenic parasites in vitro","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-22 05:35:20","doi":"10.21203/rs.3.rs-7420042/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ccda45e0-a0bb-4c23-a146-86ed3fa57989","owner":[],"postedDate":"August 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":53544048,"name":"Biomedical Engineering"}],"tags":[],"updatedAt":"2025-08-22T05:35:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-22 05:35:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7420042","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7420042","identity":"rs-7420042","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}