Hepatoprotective Effect of Origanum Vulgare on CCl4 Induced Mice Experimental Model

1. Hepatoprotective Effect of Origanum Vulgare on CCl4 Induced Mice Experimental Model Al V-lea Congres al Medicilor Fitoterapeuti din Romania, “Cercetare si rezultate clinice in fitoterapie”   15 - 17 Mai 2015, Sibiu, Romania Bogdan SEVASTRE1, Ioana BEDECEAN1, Alexandra DREANCĂ1, Ioan MARCUS1, Laurian VLASE2, Alexandra C. SEVASTRE-BERGHIAN2, Radu SILAGHI-DUMITRESCU3, Daniela HANGANU2, Daniela BENEDEC2 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 11A, Cluj-Napoca, Romania, *corresponding author:bogdan.sevastre@usamvcluj.ro 1

2. 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 2 http://en.wikipedia.org/wiki/Oregano

3. 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 (ursolicacid, oleanolicacid ) Pharmacology • antiseptic, digestive, antispastic, diuretic • Infections (aerways, digestive, urinary tract) • local – antiitchy, trophyc, protector • Spice 3 http://en.wikipedia.org/wiki/Oregano

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

5. Sevastre B. et al. - Hepatoprotective effect of Origanum vulgare Materials and methods Plant materials • Aerial parts (Origani herba) Ariesului Valey, Cluj, Romania, in July 2014 • ethanolic extract 10%, heat 80°C, 30 min, centrifugation • Flavonoids: spectophotometry AlCl3 → 2.19 % expressed in rutozid • Total polyphenols: spectophotometry (Arnow sol.) → 4.99% in cafeic acid • Conclusion → high polyphenol content

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

7. Sevastre B. et al. - Hepatoprotective effect of Origanum vulgare Materials and methods Experimental design Toxicity tests Swiss mice Control (5 mice) 4 weeks (20 mice) 6 weeks (20 mice) Origanum vulgare alcoholic extract CCl4 (1 ml/ kg) (10 mice) O. vulgare (1 mg/kg) + CCl4 (1 ml/ kg) (10 mice) O. vulgare (3.5 mg/kg) + CCl4 (1 ml/ kg) (10 mice) 45 Swiss mice O. vulgare (7 mg/kg) + CCl4 (1 ml/ kg) (10 mice)

8. Sevastre B. et al. - Hepatoprotective effect of Origanum vulgare Materials and methods 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)

9. Sevastre B. et al. - Hepatoprotective effect of Origanum vulgare Materials and methods 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). • GraphPad Prism version 5.0 for Windows, GraphPad Software, San Diego California USA.

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

11. 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 3rd ed., Blakwell Publiching, USA 2007

12. Sevastre B. et al. - Hepatoprotective effect of Origanum vulgare Results and discussions Induced hepatic enzymes Fig. 2. Effects of Origanum vulgareextract on GGTplasma activity (* = p>0.05: ** = p>0.01; *** = p>0.001) (mean ± SEM) (5 animals / group)

13. Sevastre B. et al. - Hepatoprotective effect of Origanum vulgare Results and discussions Hepatocellular insufficiency A B Fig. 3. Effects of Origanum vulgareextract 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 3rd ed., Blakwell Publiching, USA 2007

14. 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.) (109cells/L) S.E.M = Standard Error of the Mean (5 animals / group) Normal values (103cells/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 3rd ed., Blakwell Publiching, USA 2007

15. Sevastre B. et al. - Hepatoprotective effect of Origanum vulgare Results and discussions Hematology Table 2 The effect of O. vulgarealcoholic extract on the RBCs parameters (mean ± S.E.M.) S.E.M = Standard Error of the Mean (5 animals / group) (** = p>0.01, *** = p>0.001 as compared to CCl4) Normal values: RBC 7-12.5 1012/L, HGB 102-166 g/L, HCT 39-40 %, Hrapkiewicz and Medina, Clinical Laboratory Animal Medicine 3rd ed., Blakwell Publiching, USA 2007

16. Sevastre B. et al. - Hepatoprotective effect of Origanum vulgare Results and discussions Hematology Table 3 The effect of O. vulgarealcoholic extract on the platelets (mean ± S.E.M.) S.E.M = Standard Error of the Mean (5 animals / group) (** = p>0.01, *** = p>0.001 as compared to CCl4) Normal values: PLT 800-1100 109/L, Hrapkiewicz and Medina, Clinical Laboratory Animal Medicine 3rd ed., Blakwell Publiching, USA 2007

17. Origanum vulgare had toxic effects only in very high doses (<2000 mg/ kg) reduced the CCl4 toxicity on mice experimental model (↓ALT, ↓AST, ↓GGT) Improvement of A/G ratio A dose dependent effect could not be established Sevastre B. et al. - Hepatoprotective effect of Origanum vulgare Conclusions

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