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ahmed@ahmedhegazi com ahmedhegazi128@gmail ahmedhegazi

MANAGEMENT OF MULTIPLE SCLEROSIS: APITHERAPY MANAGEMENT OF MULTIPLE SCLEROSIS: APITHERAPY. IN THE NAME OF GOD IN THE NAME OF GOD. Is bee venom having antibacterial activity?. Ahmed G. Hegazi and Samira Shawky. ahmed@ahmedhegazi com ahmedhegazi128@gmail.com www.ahmedhegazi.com

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  1. MANAGEMENT OF MULTIPLE SCLEROSIS: APITHERAPY MANAGEMENT OF MULTIPLE SCLEROSIS: APITHERAPY IN THE NAME OF GOD IN THE NAME OF GOD Is bee venom having antibacterial activity? Ahmed G. Hegazi and Samira Shawky ahmed@ahmedhegazi com ahmedhegazi128@gmail.com www.ahmedhegazi.com National Research Center, Dokki, Giza, Egypt

  2. The bilogy of honey bee(apis mellifera) The bilogy of honey bee(apis mellifera) Kingdom: Animalia Phylum: arthropoda Class: insecta Family: apidae Genus: apis Species: apis mellifera Apis mellifera (honey bee)

  3. Historic Record • Humans eventually began providing cavities for honey bees to nest in. • Earliest records are Egyptian

  4. Egyptian beekeeping Harvesting honey combs (right) and packing honey (left) ca. 1450 BC, West Bank, Luxor

  5. The earliest record of man’s interest in honey comes from cave paintings like this one in Spain circa 4000 BC. 7

  6. Prehistoric relations • Prehistoric records show a honey bee/human connection going back 6,000 BP • Humans were essentially another predator of the honey bee Smoking the hive Mesolithic cave painting Cueva de la Arana, Bicorp, Spain Motopo Hills, Zimbabwe, ca. 10,000 yrs ago

  7. BEE VENOM

  8. Insect Sting = Injection of up to 100 g protein (or 0.10 mg or 0.0001 gram)

  9. What is Bee Venom? • Bee venom is a complex composition of enzymes, proteins and amino acids. • It is a colorless clear liquid, with a sweet taste and a little bitter. • It is soluble in water, insoluble in alcohol and ammonium sulphate. • If is comes in contact with air, it forms, opaque or grayish-white crystals.

  10. Bee venom pH 5-5.5 B:Dried A:liquid Yellowish brown Colorless LD50:2.8mg/kg (IV) (In mice) sharp-bitter tasting Cold resistance Heat resistance (when dry)

  11. Bee venom composition melittin (family) melittin F apamin mast-cell degranulation peptide 401 (MCD) secarpin tertiapin adolapin protease inhibitor procamine A, B minimine cardiopep PEPTIDES PEPTIDES

  12. phospholipase A2 hyaluronidase acid phosphomonoesterase glucosidase lysophospholipase ENZYMES ENZYMES histamine dopamine norepinephrine leukotriens ACTIVE ACTIVE AMINES AMINES

  13. carbohydrates like: Glucose Fructose NON NON- -PEPTIDE PEPTIDE COMPONENTS COMPONENTS 6 phospholipids LIPIDS LIPIDS r-aminobutyric acid B-aminoisobutyric acid AMINO AMINO- -ACIDS ACIDS

  14. Dried bee venom composition ENZYMES MOL. Wt. % (Dry Venom) COMPONENT MOL. Wt. % (Dry Venom) Hyaluronidase 38,000 1.5-2.0 Phospholipase A2 19,000 10-12 PEPTIDES Glucosidase 170,000 0.6 Melittin 2,840 40-50 Acid Phosphomono- esterase 55,000 1.0 Apamin 2,036 2-3 MCD-Peptide 401 2,588 2-3 Lysophospholipase 22,000 1.0 Adolapin 11,500 1.0 ACTIVE AMINES Protease inhibitor 9,000 < 0.8 Secarpin 0.5 Histamine Tertiapin 0.1 Dopamine 0.13-1.0 Melittin F 0.01 Norepinephrine 0.1-0.7 Procamine A, B 1.4 NON-PEPTIDE COMPONENTS Minimine 6,000 2-3 Carbohydrates: Glucose & Fructose < 2.0 Cardiopep < 0.7

  15. B.V. SUBSTANCES AND THEIR EFFECTS: B.V. SUBSTANCES AND THEIR EFFECTS: •radioprotective activity; •mastocytolitic; •histamine release; •blood pressure depressants •antigenic properties; it is the major BV allergen ; •antagonistic effect on staphylococic alfa-toxin and tetanus toxin; •antitumoural effect •acts on biological membranes Phospho lipase A (enzyme)

  16. B.V. SUBSTANCES AND THEIR EFFECTS: B.V. SUBSTANCES AND THEIR EFFECTS: •selectively attacks tissue hyaluronic acid polymers; •increase the capillary permeability (Neumann and Habermann); •immune response and tissue- spread properties; •antigenic; •anaphylactogene Hyaluronidase Apamin (a polypeptide with 18 amino acids) •antigenic and; •anti-inflammatory properties

  17. B.V. SUBSTANCES AND THEIR EFFECTS: B.V. SUBSTANCES AND THEIR EFFECTS: In many animal studies, in comparison studies with hydrocortisone, this peptide was 100 times more potent as an anti- inflammatory agent in suppressing the development of adjuvant- induced arthritis. (Simics p 13) & quot. Mast Cell Degranulating peptide •analgesic (Shkenderov, 1982); •anti-inflammatory (Shkenderov, 1982) Adolapin

  18. B.V. SUBSTANCES AND THEIR EFFECTS( B.V. SUBSTANCES AND THEIR EFFECTS(continued continued) ) •antibacterial; •antifungal; •anti-lyme disease (in vitro experiment) •antitumoural; •central nervous system inhibitory; •block nerve muscle and ganglial synapses; •contraction of the striated and smooth muscles; •histamine releasing; •mastocytololysic; •radio protecting (against X-irradiation; study on mice, Shipman and Cole, 1967); •vascular permeability increasing; •haemolysis; •lowers blood pressure; •anti-inflammatory; •mellitin (which represents 40-60 % from the B.V. substances) has no antigenic properties (Orlov); otherwise, according to Artemov, the bee enemies would have gotten a specific immunity; •stimulate the pituitary - adrenal axis to release both cathecolamines and cortisol (Brooks et al.); •increase plasma cortisol levels •acts on biological membranes Presently, it is one of the most potent anti-inflammatory agents known, and it can be useful in treating arthritis and rheumatism. Melittin (a polypeptide also consisting of 26 amino acids which represents 40-60% of the bee venom)

  19. B.V. SUBSTANCES AND THEIR EFFECTS( B.V. SUBSTANCES AND THEIR EFFECTS(continued continued) ) •increase both the force of contraction (beta-adrenergic) and the heart rate with little or no effect on coronary circulation (Brooks et al.); •anti-arrhythmic properties (Brooks et al.); •stimulate the pituitary - adrenal axis to release both cathecolamines and cortisol (Brooks et al.) Cardiopep

  20. Bee Venom •Uses –As Pure Bee Venom for use in desensitization –As treating a variety of neurological disorders.

  21. Biomedical Applications: Folk remedies and anecdotal reports suggest that honey bee venom has beneficial effects for patients with: Multiple Sclerosis Arthritis Infectious Diseases

  22. RESEARCH FOCUS: Does Bee Venom (BV) have useful antimicrobial activity?

  23. • Antibacterial activity of snake, scorpion and bee venoms: a comparison with purified venom phospholipase A2enzymes • Perumal et al., 2007 • indicate that have significant antibacterial effects against gram (+) and gram (−) bacteria, which result of the primary antibacterial components of the PLA2enzymes.

  24. Atomic Force Microscopy Study of the Effect of Antimicrobial Peptides on the Cell Envelope of Escherichia coli M. Meincken1 1, D. L. Holroyd2 2 and M. Rautenbach2 2, 2005 005 • bee venom melittin on Escherichia coli as the target cell were studied by atomic force microscopy (AFM). • peptides are lytic to E. coli, peptide causes distinct morphological changes in the outer membrane and in some cases the inner membrane, • probably as a consequence of different mechanisms of action..

  25. Peptides Volume 33, Issue 1, January 2012, Pages 18–26 Toxicity study of antimicrobial peptides from wild bee venom and their analogs toward mammalian normal and cancer cells Jiřina Slaninová, Veronika Mlsov. Hilda Kroupová, Lukáš AlánTereza Tůmová, Lenka Monincová, Lenka Borovičková, Vladimír Fučík, Václav Čeřovský isolated and characterized remarkable antimicrobial peptides (AMPs) from the venom reservoirs of wild bees. display high antimicrobial activity against Gram-positive and -negative bacteria, antifungal activity

  26. Modulation of the Activity of Secretory Phospholipase A2by Antimicrobial Peptides Hongxia Zhao and Paavo K. J. Kinnunen, 2003 • the concerted action of antimicrobial peptides and sPLA2could improve the efficiency of the innate response to infections.

  27. Antimicrobial Activity of Honey Bee Venom against Select Infectious Fish Pathogens North American Journal of Aquaculture Volume 75, Issue 3, 2013 • Sang Mi Hana, Kwang Gill Leea, Kwan Kyu Parkb& Sok Cheon Pakc BV inhibits the growth and survival of bacterial strains and that BV may be a useful complementary antimicrobial agent against fish pathogenic bacteria.

  28. • A strong antibacterial activity of bee venom against both Gram negative and Gram positive bacteria has been reported [Perumal et al., 2007]. • Nakatuji et al. [2009] also reported that bee venom could control the growth of Staphylococcus aureus.

  29. • Bee venom exhibited antibacterial activities against skin bacteria such as • Propionibacterium acnes, • Staphylococcus epidermidis and • Streptococcuspyogenes pyogenes [Han et al., 2010].

  30. Antibacterial activities of bee venom, propolis, and royal jelly produced by three honey bee, Apis mellifera L., hybrids reared in the same environmental conditions Annals of Agric Sci., Moshtohor, Vol. 45, No. 2: 895-902 pp., 2007. Khaled M. Attalla , Ayman A. Owayss and Karem M. Mohann There Gram (+) bacteria; Staphylococcus aureus, Bacillus subtilis and Listeria monocytogenes and two Gram (-); Escherichia coli and Salmonella enteritidis were used • Bee venom seemed to be the most active followed by propolis then royal jelly. • Gram (+) bacteria was more sensitive to these products than Gram (-) ones. • The use of these, natural, cheap and safe bee products as alternative food preservatives and in some pharmaceutical application is promising,

  31. Sung-Won Park et al., Altern Integ Med 2013, 2:10 Antimicrobial activities of honey bee venom against pathogens isolated from clinical bovine mastitis in Korea • In this study, the concentration of three major active components had a little frustration among three venom products, • It was clearly demonstrated that the honey bee venom inhibited the growth of seventeen gram positive bacteria strains and two gram negative strains isolated from bovine mastitis in Korea. However, the pore forming on cell membrane wasn’t observed by using bee venoms and sensitive gram positive bacteria.

  32. African Journal of Microbiology Research Vol. 5(18), pp. 2765-2772, 16 September, 2011 N. S. Surendra, G. N. Jayaram and M. S. Reddy • Antimicrobial activity of crude venom extracts in honeybees (Apis cerana, Apis dorsata, Apis florea) tested against selected pathogens The selected bacteria and fungal species were Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Xanthomonas subtilis, Proteus vulgaris, Salmonella typhimurium and Candida albicans • The results showed that, ABV has significant antimicrobial effects and could be a potential alternative antibiotic.

  33. Antimicrobial Effect of Melittin Isolated from Syrian Honeybee (Apismellifera) Venom and its Wound Healing Potential Int. J. Pharm. Sci. Rev. Res., 21(1), Jul – Aug 2013; n° 54, 318-324 Omran Alia, Massouh Laila , Al-DaoudeAntonious • In this study, bee venom (BV) was collected using electric shock method. • Melittin, was isolated and identified using RP-HPLC C18 column and MALDI-TOF-MS analysis. • The obtained melittin exhibited a potent antibacterial activity particularly against Gram-positive bacteria as its MIC was 12.5µg/ml for Listeria monocytogenes compared with 200µg/ml for Yersinia kristensenii (a Gram-negative bacterium) indicating that melittin has significant antibacterial effects.

  34. World Applied Sciences Journal 30 (3): 266- 270, 2014 Evaluation of the Antibacterial Activity of Bee Venom from Different Sources Ahmed Hegazi, Amr M. Abdou, Sherein. Abd El- Moez and Fyrouz Abd Allah • evaluate the antibacterial activity of bee venom from different sources against selected Gram-positive and Gram-negative bacterial strains of medical importance. • Three different samples of bee venom (BV) collected from the honeybee Apis mellifera as well as whole bee extract were assessed for their potential use as antibacterial agents against five pathogenic bacterial strains

  35. • pathogenic bacterial strains including • Staphylococcus aureus, • Streptococcus pyogenes, • Klebsiella pneumoniae, • Escherichia coli • Pseudomonas aeruginosa. • Both bee venom and whole bee extract exhibited antibacterial activity against all five bacterial strains with different levels according to the type. • The results of the current study indicate that BV inhibits the growth and survival of bacterial strains and that BV can be used as complementary antimicrobial agent against pathogenic bacteria

  36. Table (1): Influence of bee venom on growth inhibition of different bacteria Staphylococcus aureus Streptococcus pyogenes Klebsiella Pneumoniae Pseudomonas aeruginosa Escherichia Coli Treatment Bacterial Normal growth 1.701± 0.015* 0.167 ± 0.003 0.267 ± 0.001 0.351 ± 0.001 0.401 ± 0.009 0.369 ± 0.004 0.901 ± 0.15 0.106 ± 0.001 0.116 ± 0.001 0.501± 0.001 0.306 ± 0.001 0.349 ± 0.001 1.559 ± 0.005 0.155 ± 0.003 0.305 ± 0.007 0.552± 0.002 0.415 ± 0.005 0.095 ± 0.001 1.400 ± 0.001 0.125 ± 0.007 0.325 ± 0.004 0.317± 0.006 0.457± 0.005 0.057± 0.002 1.450 ± 0.005 0.114 ± 0.029 0.664 ± 0.009 0.910 ± 0.012 0.681 ± 0.004 1.049 ± 0.003 Apitox Vacsera Ethanolic sac extract Whole Bee extract Tetracycline (50ug)

  37. Table (2): The minimal inhibitory concentration (MIC) of bee venom of different bacteria Staphylococcus aureus Streptococcus pyogenes Klebsiella Pneumoniae Pseudomonas aeruginosa Escherichia Coli Treatment Bacterial Normal growth ---- ---- ---- ---- ---- Apitox 1600 * 2800 3600 2600 1000 1000 1000 2400 1000 1800 1200 1000 2400 1700 1800 2400 2600 3600 2400 2100 2800 2100 3800 2400 4400 Vacsera Ethanolic sac extract Whole Bee extract Tetracycline (50ug)

  38. Int.J.Curr.Microbiol.App.Sci (2015) 4(4): 141-1 Antibacterial Activity of Bee Venom Collected from Apis mellifera Carniolan Pure and Hybrid Races by Two Collection Methods Ahmed G. Hegazi, EL-Feel M. A. , Eman H. Abdel-Rahman and Abed Al-Fattah M. A The objective of this investigation was to evaluate the antibacterial activity of bee venom against selected Gram-positive and Gram-negative bacterial strains of medical importance.

  39. Ahmed G. Hegazi, EL-Feel M. A. , Eman H. Abdel- Rahman and Abed Al-Fattah M. A • Antibacterial activity of bee venom evaluated against five pathogenic bacterial strains, including • Staphylococcus aureus, • Streptococcus pyogenes, • Klebsiella pneumoniae, • Escherichia coli • Pseudomonas aeruginosa.

  40. Table 1 Bee venom collected weight gm. / colony from pure and hybrid Carniolan honey bee race. Treatment Apis mellifera Carniolan race Fiber 52 Hybrid race Fiber 82 Latex 89 Latex 74 1stweek Sample 2ndweek Sample 3rdweek Sample 4thweek Sample Over all mean46±10.03 75±17.47 32 41 84 100 72 111 132 145 29 42 111 105 102±11.91 106±14.66

  41. Table (2): Influence of bee venom collected by two different methods (fiber and latex) on growth inhibition of different bacteria Staphylococcus aureus Streptococcus pyogenes Klebsiella Pneumoniae Pseudomonas aeruginosa Escherichia coli Treatment Fiber 1.550 ± 0.005 Latex 1.550 ± 0.005 Fiber 1.400 ± 0.001 Latex 1.400 ± 0.001 Fiber 0.900 ± 0.150 Latex 0.900 ± 0.15 Fiber 1.700 ± 0.150 Latex 1.70 ± 0.150 Fiber 1.550 ± 0.005 Latex 1.55 ± 0.005 Normal growth 0.095 ± 0.001 0.057 ± 0.002 0.057 ± 0.002 0.349 ± 0.001 0.349 ± 0.001 0.069 ± 0.004 0.069 ± 0.004 0.049 ± 0.003 0.049 ± 0.003 Tetracycline (50ug) 0.095 ±0.001 0.104± 0.005 0.155 ± 0.005 0.500± 0.008 0.125 ± 0.002 0.178± 0.003 0.101 ± 0.001 0.197± 0.0025 0.167± 0.001 0.173± 0.004 0.114 ± 0.009 1stweek Sample 0.155 ± 0.005 0.305 ± 0.005 0.257± 0.002 0.325 ± 0.002 0.106 ± 0.001 0.116 ± 0.001 0.187± 0.001 0.267± 0.001 0.114 ± 0.009 0.134 ± 0.009 2ndweek Sample 0.352± 0.002 0.552± 0.002 0.3117± 0.002 0.3117± 0.002 0.201± 0.001 0.109 ± 0.001 0.201± 0.009 0.401± 0.009 0.510 ± 0.012 0.510 ± 0.012 3rdweek Sample 0.404± 0.005 0.115 ± 0.005 0.615± 0.008 0.257± 0.002 0.173± 0.003 0.106 ± 0.001 0.143± 0.002 0.267± 0.001 0.273± 0.004 0.104 ± 0.009 4thweek Sample

  42. Table (3): Influence of bee venom collected by two different methods (fiber and latex) on minimal inhibitory concentration Staphylococcus aureus Fiber Latex Fiber Streptococcus pyogenes Klebsiella Pneumoniae Fiber Pseudomonas aeruginosa Fiber Escherichia coli Treatment Latex Latex Latex Fibe Late x r Tetracycline (50ug) 1stweek Sample 2ndweek Sample 3rdweek Sample 4thweek Sample 1.000 1.00 1.60 1.60 1.80 1.80 1.200 1.200 4.40 4.40 1.600 2.80 1.80 2.40 2.20 2.20 1.600 1.900 2.10 2.20 2.800 2.80 2.40 2.60 2.00 2.40 2.600 1.700 2.80 2.40 3.600 3.20 3.60 3.60 2.40 2.50 1.800 1.800 3.80 3.80 2.600 2.80 2.30 2.40 2.70 2.60 2.600 1.900 2.40 2.60

  43. Ahmed G. Hegazi, EL-Feel M. A. , Eman H. Abdel- Rahman and Abed Al-Fattah M. A • The results revealed that the amount of bee venom collected from the pure Carniolan race was 46±10.03 mg / colony of venom vz 102 ±11. 91 mg / colony of venom in hybrid. • Both bee venom of pure and hybrid bees exhibited antibacterial activity against all five bacterial strains and differs according to the type.

  44. Ahmed G. Hegazi, EL-Feel M. A. , Eman H. Abdel- Rahman and Abed Al-Fattah M. A • Bee venom exhibited antibacterial activity against all five bacterial strains. • The minimum inhibitory concentration of BV was determined. • These results indicate that BV inhibits the growth and survival of bacterial strains and that BV may be a useful complementary antimicrobial agent against pathogenic bacteria even if bee venom collected by different methods

  45. Synergy with Antibiotics Normal Bacterial Growth BV only Optical Density Penicillin only With Penicillin and BV Time

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