Al Vlea Congres al Medicilor Fitoterapeuti din Romania

Al V-lea Congres al Medicilor Fitoterapeuti din Romania, “Cercetare si rezultate clinice in fitoterapie” 15 - 17 Mai 2015, Sibiu, Romania Hepatoprotective Effect of Origanum Vulgare on CCl 4 Induced Mice Experimental Model Bogdan SEVASTRE 1, Ioana BEDECEAN 1, Alexandra DREANCĂ1, Ioan MARCUS 1, Laurian VLASE 2, Alexandra C. SEVASTRE-BERGHIAN 2, Radu SILAGHI-DUMITRESCU 3, Daniela HANGANU 2, Daniela BENEDEC 2 1 University of Agricultural Science and Veterinary Medicine, Faculty of Veterinary Medicine, Manastur Street. 3 -5, Cluj-Napoca, Romania, 2 University of Medicine and Pharmacy “Iuliu Hateganu”, Emil Isaac Street 13, Cluj-Napoca, Romania 3 "Babeș-Bolyai" University, Janos Street 11 A, Cluj-Napoca, Romania, *corresponding author: bogdan. sevastre@usamvcluj. ro 1

Sevastre B. et al. - Hepatoprotective effect of Origanum vulgare Introduction Origanum vulgare L. , (Laminacea) – oregano, (maghiran salbatic) native to warm temperate western and southwestern Eurasia and the Meditarranean region. perennial herb, growing from 20– 80 cm tall http: //en. wikipedia. org/wiki/Oregano 2 2

Sevastre B. et al. - Hepatoprotective effect of Origanum vulgare Introduction ORIGANI HERBA: ¨ Volatile oils 1% - Origani aetheroleum: carvacrol, timol, p-cimen, pineni etc. ¨ Tanins 8% ¨ Flavonoids (derivatives of apigenin, luteolin) ¨ Anthocyani (peonidol, malvidol) ¨ cafeic acids derivatives ¨ Triterpenes (ursolic acid, oleanolic acid ) Pharmacology antiseptic, digestive, antispastic, diuretic Infections (aerways, digestive, urinary tract) local – antiitchy, trophyc, protector Spice http: //en. wikipedia. org/wiki/Oregano 3 3

Sevastre B. et al. - Hepatoprotective effect of Origanum vulgare Objective Investigate the hepatoprotective effect of Origani Herba 4

Sevastre B. et al. - Materials and methods Hepatoprotective effect of Origanum vulgare Plant materials Aerial parts (Origani herba) Ariesului Valey, Cluj, Romania, in July 2014 ethanolic extract 10%, heat 80°C, 30 min, centrifugation Flavonoids: spectophotometry Al. Cl 3 → 2. 19 % expressed in rutozid Total polyphenols: spectophotometry (Arnow sol. ) → 4. 99% in cafeic acid Conclusion → high polyphenol content 5

Sevastre B. et al. - Materials and methods Hepatoprotective effect of Origanum vulgare Toxic hepatitis carbon tetrachloride (Sigma), 1 ml / kg, “per os” 3 times / week, 4/6 weeks 6

Sevastre B. et al. - Hepatoprotective effect of Origanum vulgare Materials and methods Experimental design Toxicity tests Swiss mice Control (5 mice) 4 weeks 6 weeks (20 mice) Origanum vulgare alcoholic extract CCl 4 (1 ml/ kg) (10 mice) O. vulgare (1 mg/kg) + CCl 4 (1 ml/ kg) (10 mice) 45 Swiss mice O. vulgare (3. 5 mg/kg) + CCl 4 (1 ml/ kg) (10 mice) O. vulgare (7 mg/kg) + CCl 4 (1 ml/ kg) (10 mice) 7

Sevastre B. et al. - Materials and methods Hepatoprotective effect of Origanum vulgare Laboratory analysis Blood hematology - EDTA anticoagulated blood (Abacus Junior Vet, automatic analyzer Diatron Messtechnik, Budapest, Hungary) Plasma biochemistry – serum – ALAT, ASAT, GGT, total proteins, albumins, ureea, creatinin - ( semiautomatic analyzer, STAT – FAX 1904 Plus, Global Medical Instrumentation, Inc. 6511 Bunker Lake Blvd. Ramsey Minnesota, 55303 USA, by using special determination kits) 8

Sevastre B. et al. - Materials and methods Hepatoprotective effect of Origanum vulgare Statistical analysis one -way analysis of variance ANOVA, followed by post hoc Dunnett’s range test two-way ANOVA followed by Bonferroni post test Statistical significance was at p<0. 05 (95% confidence interval). Graph. Pad Prism version 5. 0 for Windows, Graph. Pad Software, San Diego California USA. 9

Sevastre B. et al. - Hepatoprotective effect of Origanum vulgare Results and discussions Toxicity tests 5 mice / group 450 mg / kg b. w. group i. p. 900 mg / kg b. w. group (5 animals /group) 1250 mg / kg b. w. group 1500 mg / kg b. w. group 1750 mg / kg b. w. group 2000 mg / kg b. w. group 2500 mg / kg b. w. group 3000 mg / kg b. w. group 4000 mg / kg b. w. group 2 -2 h, 1 -12 h, 1 -24 h lethargy, spasms, abdominal tenderness 2 -1 h, 2 -2 h, 5 -30 min, 10

Sevastre B. et al. - Hepatoprotective effect of Origanum vulgare Results and discussions Hepatocellular leakage enzymes A B Fig. 1. Effects of Origanum vulgare extract on ALT (A) and AST (B) plasma activity (* = p>0. 05: ** = p>0. 01; *** = p>0. 001) (mean ± SEM) (5 animals / group) Normal values (UI/L): ALT 26 -77, AST 54 -269 Hrapkiewicz and Medina, Clinical Laboratory Animal Medicine 3 rd ed. , Blakwell Publiching, USA 2007 11

Sevastre B. et al. - Hepatoprotective effect of Origanum vulgare Results and discussions Induced hepatic enzymes Fig. 2. Effects of Origanum vulgare extract on GGT plasma activity (* = p>0. 05: ** = p>0. 01; *** = p>0. 001) (mean ± SEM) (5 animals / group) 12

Sevastre B. et al. - Hepatoprotective effect of Origanum vulgare Results and discussions Hepatocellular insufficiency A B Fig. 3. Effects of Origanum vulgare extract on plasma proteins: total proteins (A), and albumins (B) (* = p>0. 05: ** = p>0. 01; *** = p>0. 001) (mean ± SEM) (5 animals / group) Normal values (g/L): Proteins 35 -72, Albumins 25 -48 Hrapkiewicz and Medina, Clinical Laboratory Animal Medicine 3 rd ed. , Blakwell Publiching, USA 2007 13

Sevastre B. et al. - Hepatoprotective effect of Origanum vulgare Results and discussions Hematology Table 1 The effect of O. vulgare alcoholic extract on the WBC count and differential count (mean ± S. E. M. ) (10 9 cells/L) WBCs LYM MID GRA Control 6 weeks 6. 91± 3. 18 4. 38± 1. 84 0. 23± 0. 19 2. 29± 1. 59 CCl 4 4 weeks 11. 9± 8. 29 7. 15± 5. 84 0. 63± 1. 18 4. 11± 3. 8 6 weeks 6. 53± 2. 86 4. 31± 1. 26 0. 215± 0. 17 2. 01± 1. 5 4 weeks 5. 01± 2. 88 3. 08± 1. 79 0. 17± 0. 13 1. 76± 1. 49 6 weeks 5. 42± 2. 97 3. 01± 0. 96 0. 21± 0. 09 2. 2± 2. 18 4 weeks 7. 4± 3 5. 37± 1. 81 0. 2± 0. 18 1. 82± 1. 28 6 weeks 5. 79± 2. 3 4. 24± 1. 87 0. 08± 0. 05 1. 47± 0. 49 4 weeks 9. 19± 2. 83 4. 76± 0. 91 0. 49± 0. 19 3. 94± 3. 37 6 weeks 6. 33± 3. 86 4. 79± 2. 7 0. 14± 0. 16 1. 38± 1. 47 CCl 4+ O. vulgare 1 mg/kg CCl 4+ O. vulgare 3. 5 mg/kg CCl 4+ O. vulgare 7 mg/kg S. E. M = Standard Error of the Mean (5 animals / group) Normal values (103 cells/µl): WBC 2. 61 -10. 05, N 0. 4 -2. 0, L 1. 27 -8. 44 M 0 -0. 29, E 0 -0. 17, B 0 -0. 02 Hrapkiewicz and Medina, Clinical Laboratory Animal Medicine 3 rd ed. , Blakwell Publiching, USA 2007 14

Sevastre B. et al. - Hepatoprotective effect of Origanum vulgare Results and discussions Hematology Table 2 The effect of O. vulgare alcoholic extract on the RBCs parameters (mean ± S. E. M. ) RBC (1012/L) HGB (g/L) HCT (%) VEM (f. L) HEM (pg) CHEM (g/L) Control 6 weeks 8. 08± 0. 76 138± 13. 0 41. 31± 3. 18 51. 16± 1. 16 17. 11± 0. 39 334± 5. 93 CCl 4 4 weeks 7. 4± 1. 17 121± 33. 37 40. 75± 6. 33 55± 1. 87 16. 06± 2. 63 291. 8± 46. 05 CCl 4+ O. vulgare 1 mg/kg 6 weeks 7. 34± 0. 68 127. 83± 7. 6 39. 7± 1. 95 54. 16± 2. 99 17. 5± 0. 97 322. 33± 7. 42 4 weeks 8. 46± 6. 08 96. 33± 39. 1 44. 17± 28. 39 54. 16± 3. 97 14. 23± 5. 46 260. 5± 96. 75 6 weeks 7. 2± 0. 66 128. 16± 11. 25 40. 18± 3. 2 55. 83± 1. 32 17. 83± 0. 67 319. 5± 5. 99 4 weeks 7. 59± 3. 41 119. 5± 50. 18 40. 25± 17. 2 53. 5± 1. 37 15. 75± 1. 08 294. 83± 22. 96 6 weeks 8. 11± 0. 41 141. 8± 6. 34 42. 48± 1. 28 52. 6± 1. 94 17. 46± 0. 23 333± 8. 03 4 weeks 6. 39± 0. 66 94. 25± 12. 01 33. 45± 3. 54 52. 25± 0. 5 15. 55± 0. 98 296. 75± 17. 23 6 weeks 6. 29± 1. 8 99. 66± 37. 92 32. 88± 8. 87 52. 33± 1. 63 15. 51± 2. 73 296. 16± 52. 88 CCl 4+ O. vulgare 3. 5 mg/kg CCl 4+ O. vulgare 7 mg/kg Control S. E. M = Standard Error of the Mean (5 animals / group) (** = p>0. 01, *** = p>0. 001 as compared to CCl 4) Normal values: RBC 7 -12. 5 1012/L, HGB 102 -166 g/L, HCT 39 -40 %, Hrapkiewicz and Medina, Clinical Laboratory Animal Medicine 3 rd ed. , Blakwell Publiching, USA 2007 15

Sevastre B. et al. - Hepatoprotective effect of Origanum vulgare Results and discussions Hematology Table 3 The effect of O. vulgare alcoholic extract on the platelets (mean ± S. E. M. ) PLT (109 cells /L) PCV (%) MPV (f. L) Control 6 weeks 755. 16± 602. 05 0. 43± 0. 24 6. 38± 1. 11 CCl 4 4 weeks 607. 4± 250. 91 0. 432± 0. 15 7. 2± 0. 46 CCl 4+ O. vulgare 1 mg/kg 6 weeks 485. 16± 171. 08 0. 31± 0. 1 6. 68± 0. 73 4 weeks 605. 6± 577. 7 0. 416± 0. 4 7. 33± 0. 98 6 weeks 706. 33± 140. 19 0. 46± 0. 08 6. 63± 0. 21 4 weeks 323. 16± 226. 17 0. 22± 0. 14 7. 08± 0. 57 6 weeks 487± 100. 43 0. 326± 0. 06 6. 66± 0. 11 4 weeks 340. 75± 144. 22 0. 24± 0. 09 7. 25± 0. 37 6 weeks 357. 33± 224. 06 0. 24± 0. 14 7± 0. 38 CCl 4+ O. vulgare 3. 5 mg/kg CCl 4+ O. vulgare 7 mg/kg Control S. E. M = Standard Error of the Mean (5 animals / group) (** = p>0. 01, *** = p>0. 001 as compared to CCl 4) Normal values: PLT 800 -1100 109/L, Hrapkiewicz and Medina, Clinical Laboratory Animal Medicine 3 rd ed. , Blakwell Publiching, USA 2007 16

Sevastre B. et al. - Hepatoprotective effect of Origanum vulgare Conclusions Origanum vulgare had toxic effects only in very high doses (<2000 mg/ kg) reduced the CCl 4 toxicity on mice experimental model (↓ALT, ↓AST, ↓GGT) Improvement of A/G ratio A dose dependent effect could not be established 17

Sevastre B. et al. - Hepatoprotective effect of Origanum vulgare Thank you ! 18 18