Study of four weeks repeated-dose toxic test of Sweet Bee Venom in rats Original Articles

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(1)005~024 Sweet Bee.ps 2011.3.31 12:1 PM 페이지5 MAC-11. Sweet Bee Venom의 rat를 이용한 4주 반복 근육시술 독성시험. 5. DOI : 10.3831/KPI.2011.14.1.005. Original Articles. Sweet Bee Venom의 rat를 이용한 4주 반복 근육시술 독성시험. Study of four weeks repeated-dose toxic test of Sweet Bee Venom in rats. Received : 11. 02. 18 Revised : 11. 02. 24 Accepted : 11. 02. 25. Hae-Yon, Kwon1. ˝Ki-Rok, Kwon2. 1) Graduate School of Korean Medicine, Sangji University 2) Research Center of Korean Pharmacopuncture Institute. Key Words: Sweet Bee Venom, melittin, four weeksrepeated dose toxicity. ABSTRACT Objectives: This study was performed to analyse four weeks repeated -dose toxicity of Sweet Bee Venom (SBV-pure melittin, the major component of honey bee venom) in rats. Methods: All experiments were conducted under the regulations of Good Laboratory Practice (GLP) at Biotoxtech Company, a non-clinical study authorized institution. Male and female rats of 5 weeks old were chosen for the pilot study of four weeks repeated-dose toxicity and was injected at the level of 0.56 / body weight (eighty times higher than the clinical application dosage as the high dosage), followed by 0.28 and 0.14 / as midium and low dosage, respectively. Equal amount of normal saline was injected as the control group every day for four weeks. Results: 1. No mortality was witnessed in all of the experiment groups. 2. All experiment groups appealed pain sense in the treating time compared to the control group, and side effects such as hyperemia and movement disorder were observed around the area of injection in all experiment groups, and the higher dosage in treatment, the higher occurrence in side effects. 3. Concerning weight measurement, neither male nor female groups showed significant changes compared to the control group. 4. Concerning to the CBC and biochemistry, all experiment groups didn't show any significant changes compared to the control group. 5. Concerning weight measurement of organs, experiment groups didn't show any significant changes compared to the control group. 6. To verify abnormalities of organs and tissues, those such as cerebellum, cerebrum, liver, lung, kidney, and spinal cords were removed and we conducted histologocal observation with H-E staining. Concerning the histologocal observation of liver tissues, some fatty changes were observed around portal vein in 0.56 / experiment group. But another organs were not detected in any abnormalities. 7. The proper high dosage of SBV for the thirteen weeks repeated test in rats may be 0.28 one time.. /. in. Conclusion: Above findings suggest that SBV is relatively safe treatment medium. Further studies on the subject should be conducted to yield more concrete evidences.. Corresponding author. Ki Rok Kwon. Research Center of Pharmacopuncture Medicine, Korean Pharmacopuncture Institute, South Korea. Tel: +82-2-2658-9051. E-mail: [email protected].

(2) 005~024 Sweet Bee.ps 2011.3.31 12:1 PM 페이지6 MAC-11. 6. Journal of Pharmacopuncture 제 14권 제 1호( 2011년 3월). I. 서론. II. 재료 및 방법 1. Sweet BV. 4 se. mou-. rat. Sweet BV 230S, CP323S, Sartorius, Germany Normal Saline mixer Sweet BV. LA vortex. 2 2.1) 5. rat. Apis melifera 137.6 1. 111.9. 24 121.7. 125.3. 20.3 23.6 anaphylactic shock Sweet Bee Venom 10-0744755 Sweet BV Sweet BV 40-50 2840 melittin gel filtration melittin Sweet BV rat 4 Sweet BV rat Sweet BV. 4. 40.5 58.5 Teklad Certified Irradiated Global 18 Protein Rodent Diet 2918C 2005-79 Good Laboratory Practice GLP. 2.2 5. Sweet BV. 5 ( Group. ). ( / ). ( / ). G1. 0. 0.2. 5 (1101~1105). 5 (2101 ~ 2105). G2. 0.14. 0.2. 5 (1201 ~ 1205). 5 (2201 ~ 2205). G3. 0.28. 0.2. 5 (1301 ~ 1305). 5 (2301 ~ 2305). G4. 0.56. 0.2. 5 (1401 ~ 1405). 5 (2401 ~ 2405).

(3) 005~024 Sweet Bee.ps 2011.3.31 12:1 PM 페이지7 MAC-11. Sweet Bee Venom의 rat를 이용한 4주 반복 근육시술 독성시험. 2.3 1 Sweet BV 60. Epalyzer2, HITACHI, Japan HELENA, Japan AST, ALT, ALP, creatinine, total bilirubin, total protein, albumin, globulin, A/G ratio, total cholesterol, glucose, triglycerides, phosphorus, calcium. 0.1 0.4 0.007 80 0.56. 0.14. 7. 0.28. Sweet BV normal saline. 3.5 Lot No.: HAQ. 4. 8AV, thiopental sodium,. 1. 3 3.6 3.1 1. 1 1. 14. 2. 1 3.7. 3.2 1 ibular gland jejunum didymis 3.3. mandmandibular lymph node colon epi-. CBC 4. 16. 3 EDTA CBC bottle ADVIA 120, SIMENS, Germany RBC, HGB, hematocrit, RBC indices(MCV, MCH, MCHC), WBC, platelet count, reticulocytes 3.2 sodium citrate 9 : 1 3,000 rpm 10 ACL 7,000, Instrumentation Laboratory, USA prothrombin time, activated partial thromboplastin time. 10 neutral buffered formalin. Hematoxylin & Eosin H&E Calci-Clear-RapidTM tics, USA. National diagnos-. 4 3.4. Biochemistry 3 3,000 rpm. 10 7080,. SAS Institute Inc., U.S.A.. SAS version 9.1.3, Ba-.

(4) 005~024 Sweet Bee.ps 2011.3.31 12:1 PM 페이지8 MAC-11. Journal of Pharmacopuncture 제 14권 제 1호( 2011년 3월). 8. rtlett test. p 0.05 One-way analysis of variance ANOp 0.05 Dunnett's p 0.05, p 0. 01. VA t-test. 4. (Biochemistry) 0.28. 0.56. AST. III. 결과. Sweet BV Table 5.,. 1. Appendix 6. 5. abnormal gait hyperemia 0.56 convulsion. Sweet BV Table 6-7., Appendix 7, 8. 16 1 Table 1., Appendix 2 6 2. Sweet BV Fig. 1-2, Table 2-3., Appendix 3-4. 3. en, raised. 0.14 1. sple0.56. 0.28 Sweet BV Table 8.. (CBC). 0.14 HCT. Sweet BV. H&E 0.56 degeneration, hepatocyte, fatty, periportal 0.14. 0.28. lipid droplet. HGB. 90 0.56. PLT. Fig. 4 801. outer medulla cast, hyaline, focal,. 1908 10 cells 3. mild Sweet BV Table 4.. 1 0.14. 0.28. Table 9.,.

(5) 005~024 Sweet Bee.ps 2011.3.31 12:1 PM 페이지9 MAC-11. Sweet Bee Venom의 rat를 이용한 4주 반복 근육시술 독성시험. 9. Appendix 9-10 Fig. 2-6. 4. 3. IV. 고찰. susceptibility. i.p i.m. s.c topical. Apis melifera) 4. 24 1. 1 3. 1-3 4. poison, toxin, toxicant. 3. testing protocol guideline. GLP. 4 4 Sweet BV 40.

(6) 005~024 Sweet Bee.ps 2011.3.31 12:1 PM 페이지10 MAC-11. Journal of Pharmacopuncture 제 14권 제 1호( 2011년 3월). 10. 4 0.1 80. 0.4. Sweet BV Sweet BV human 60. 0.007 0.56. 0.14. 0.28 rat. 4 1. nidase. phospholipase A2 PLA2 histamine. hyaluro-. Sweet BV. melittin Sweet BV. Melittin. 26 40-50 Melittin. allergen. peptide 2840. PLA2. CBC 0.14 0.14. hematocrit hemoglobin. 0.28. melittin 0.56. PLT. melittin melittin nuclear factorB NF- B lipopolysaccharide LPS tumor necrosis factor- TNFsodium nitroprusside SNP. Sweet BV 0.28. 0.56. AST NF- B NF- B NF- B1 p50 NF- B2 p52 Rel RelA p65 RelB, c-Rel NF- B. Sweet BV. p50 Sweet BV Melittin. NF- B p50. 0.56.

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(10) 005~024 Sweet Bee.ps 2011.3.31 12:1 PM 페이지14 MAC-11. 14. Journal of Pharmacopuncture 제 14권 제 1호( 2011년 3월). Male. Female. Fig. 3 The tissue of cerebrum after 4 weeks repeated dose toxic test of Sweet Bee Venom(melittin-0.14, 0.28 and 0.56 / ) in SD rats. SD rats were treated one time a day with 0.56 / dose of Sweet Bee Venom during 4 weeks, there were not detected in any abnormalities compared with control group in Hematoxylin & Eosin (H&E) staining( 100). Fig. 1 Body weight changes of male and female in dose escalating four weeks repeated intramuscular toxicity study of Sweet Bee Venom in rats.. Fig. 2 The tissue of cerebellum after 4 weeks repeated dose toxic test of Sweet Bee Venom(melittin-0.14, 0.28 and 0.56 / ) in SD rats. SD rats were treated one time a day with 0.56 / dose of Sweet Bee Venom during 4 weeks, there were not detected in any abnormalities compared with control group in Hematoxylin & Eosin (H&E) staining( 100).. Fig. 4 The tissue of liver after 4 weeks repeated dose toxic test of Sweet Bee Venom (melittin-0.28(A&B) 0.56 / (C&D)) in SD rats. SD rats were treated one time a day with 0.14, 0.28 and 0.56 / dose of Sweet Bee Venom during 4 weeks, there were not detected in any abnormalities in 0.14, 0.28 / experiment group( 100), but 0.56 / group showed hepatocyte fatty change around portal vein compared with control group in Hematoxylin & Eosin (H&E) staining( 200)..

(11) 005~024 Sweet Bee.ps 2011.3.31 12:1 PM 페이지15 MAC-11. Sweet Bee Venom의 rat를 이용한 4주 반복 근육시술 독성시험. Fig. 5 The tissue of lung after 4 weeks repeated dose toxic test of Sweet Bee Venom(melittin-0.14, 0.28 and 0.56 / ) in SD rats. SD rats were treated one time a day with 0.56 / dose of Sweet Bee Venom during 4 weeks, there were not detected in any abnormalities compared with control group in Hematoxylin & Eosin (H&E) staining( 100).. 15. Fig. 6 The tissue of spinal cord after 4 weeks repeated dose toxic test of Sweet Bee Venom(melittin-0.14, 0.28 and 0.56 / ) in SD rats. SD rats were treated one time a day with 0.56 / dose of Sweet Bee Venom during 4 weeks, there were not detected in any abnormalities compared with control group in Hematoxylin & Eosin (H&E) staining( 100)..

(12) 005~024 Sweet Bee.ps 2011.3.31 12:1 PM 페이지16 MAC-11. 16. Journal of Pharmacopuncture 제 14권 제 1호( 2011년 3월).

(13) 005~024 Sweet Bee.ps 2011.3.31 12:1 PM 페이지17 MAC-11. Sweet Bee Venom의 rat를 이용한 4주 반복 근육시술 독성시험. 17.








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