Annals of RSCB Vol. XVI, Issue 1

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1 RESEARCHES REGARDING THE INFLUENCE OF APITHERAPY DIET ON ERYTHROCYTES IN WISTAR RATS WITH EXPERIMENTALLY CCl 4 INDUCED HEPATOTOXICITY C.V. Andriţoiu 1, V. Andriţoiu 2, Anca-Irina Prisăcaru 3, T. Petreuş 2, Carmen Cotrutz 2, I.M. Popa 1 1- DEPARTMENT OF PHYSICAL CHEMISTRY,,,GHEORGHE ASACHI TECHNICAL UNIVERSITY, IAŞI; 2- DEPARTMENT OF CELLULAR AND MOLECULAR BIOLOGY, UNIVERSITY OF MEDICINE AND PHARMACY,,GR. T. POPA, IAŞI; 3- DEPARTMENT OF NATURAL AND SYNTHETIC POLYMERS,,,GHEORGHE ASACHI TECHNICAL UNIVERSITY, IAŞI Summary The purpose of this experiment was to evaluate the influence of apitherapy diet in Wistar rats with experimentally carbon tetrachloride (CCl 4 ) induced hepatopathy by determining the evolution of hematological parameters (number of erythrocytes RBC; hemoglobin HGB; hematocrit HCT; mean corpuscular volume MCV, VEM; mean corpuscular hemoglobin - MCH, HEM; mean corpuscular hemoglobin concentration MCHC,CHEM; red cell distribution width RDW), under the conditions of chronic liver disease. In order to reduce the factors that accelerate the progression of liver damage apitherapy products (Apiregya, ApiImunomod, ApiImunostim, ApiImunostim Forte) have been administrated. The apitherapy products were purchased from,,stupina. The animals were handled under general anesthesia with thiopental. The determination of investigated parameters was achieved using an automatic analyzer and commercial kits. The experiment was unfold on a total of 60 white rats, Wistar breeding, divided into 6 groups: standard food group (group I), apitherapy diet group (group II), apitherapy diet - royal jelly group (group III), CCl 4 group (group IV), CCl 4 apitherapy diet group (group V), CCl 4 apitherapy diet royal jelly group (group VI). The hepatic injury was chemically induced by the intraperitoneal administration of CCl 4 (dissolved in paraffin oil, 10% solution). Two ml per 100 g were administered, once at 2 days, for 2 weeks. The administration of apitherapy diet, respectively of apitherapy diet and royal jelly in laboratory animals with CCl 4 induced hepatopathy resulted in the improvement of hematological parameters values to normal levels. Keywords: apitherapy, erythrocytes, hepatotoxicity. dr_calin_andritoiu@yahoo.com Introduction The liver functions as a hematopoietic organ (Steiff et al, 2003). The human stem cells for definitive hematopoiesis result from the endothelial cells of the hepatic vessels from the embryonic and fetal period (Peaul et al, 2003). In the conditions of hepatic injuries, hematological modifications clearly take place. Material and methods All the experimental proceedings achieved on laboratory animals (Wistar rats) in this study were in agreement with the international ethics regulations. Hepatic lesion was induced by intraperitoneal injection of CCl 4 (dissolved in paraffin oil, 10% solution). Two ml per 100 g were administered, once at 2 days, for 2 weeks. The experiment included six groups of Wistar rats. The first group served as control, the second one was fed with apitherapy diet, the third group was given apitherapy diet and royal jelly. The next three groups of animals were intoxicated with CCl 4 and fed with normal food (group IV), apitherapy diet (group V) and apitherapy diet with royal jelly (group VI). 216

The laboratory animals were given food supplements produced by S.C. STUPINA S.R.L, Bălăneşti, Gorj, Romania, supplements represented by Apiregya, ApiImunomod, ApiImunostim, ApiImunostim Forte.

These preparates included in their composition: honey, royal jelly, propolis, and pollen. The preparates were registered to OSIM with number AO 1242.

2 The laboratory animals were given food supplements produced by S.C. STUPINA S.R.L, Bălăneşti, Gorj, Romania, supplements represented by Apiregya, ApiImunomod, ApiImunostim, ApiImunostim Forte. The apitherapy products were administered for three weeks. The daily administered doses were 2g Apiregya, 1g ApiImunomod, 1g ApiImunostim, 1g ApiImunomod Forte. These preparates included in their composition: honey, royal jelly, propolis, and pollen. The preparates were registered to OSIM with number AO After the laboratory animals were anesthesiated with thiopental (dose of 1 ml/100 g from a 0.01% thiopental solution), blood samples were collected by the punction of the cord with a Vacuette system and submitted to biochemical analysis. The investigated parameters were: number of erythrocytes RBC; hemoglobin HGB; hematocrit HCT; mean corpuscular volume MCV, VEM; mean corpuscular hemoglobin - MCH, HEM; mean corpuscular hemoglobin concentration MCHC,CHEM; red cell distribution width RDW. The determination of the values for the investigated parameters were achieved with an automated analyzer (Aeroset, Abbott) and commercial kits (Abbott, USA). The statistical interpretation of the results was performed with One-Way ANOVA test and Tukey s post-hoc test. The results were given as mean ± standard deviation. The value of p<0.05 was considered significant. Results and discussions Number of erythrocytes RBC hepatopathy (group IV), there can be observed a significant decrease of RBC when compared to the control group standard food (group I) (8.51±0.04 versus 7.22±1.21, p<0.0033) (fig. 1). standard food produces an increase of RBC; ii) administration of apitherapy diet, 217 respectively of apitherapy diet + royal jelly (RJ) to the control healthy groups leads to the improvement of the RBC values, although the differences are not statistically significant; iii) administration of apitherapy diet and apitherapy diet + RJ to animals with CCl 4 induced hepatopathy brings RBC values in a range that can be compared to RBC values of the control groups (fig. 1). Hemoglobin - HGB hepatopathy (group IV) there can be observed a statistically significant decrease of the HGB values when compared to the control group fed with standard food (group I) (14.71±0.64 versus 13.27± 1.47, p<0.0361) (fig. 2). Fig. 1. Mean values of the erythrocytes (mii/μl) standard deviation (* a p< vs. control group standard food) Fig. 2. Mean values of hemoglobin and standard deviation (* a p< vs. control group standard food). CCl 4 determines the decrease of HGB; ii) HGB values didn t register any significant differences for the animals with CCl 4 induced hepatopathy that were given the apitherapy diet and apitherapy diet + RJ when compared to the control groups (fig. 2).

Hematocrit % - HCT hepatopathy (group IV) there can be observed a statistically significant decrease of the HCT values when compared to all the experimental groups: i) compared to the control group

71±1.28 versus 39.9±4.3, p<0.0001) (fig. 3). Fig. 3. Mean values of the hematocrit and standard deviation (* a p=0.0201 vs. lotul martor hrană standard; * b p=0.0413 vs. lotul cu apidietă; * c p=0.

3 Hematocrit % - HCT hepatopathy (group IV) there can be observed a statistically significant decrease of the HCT values when compared to all the experimental groups: i) compared to the control group standard food (group I) (44.65±2.05 versus 39.9±4.3, p<0.0201); ii) compared to the control group apitherapy diet (group II) (44.25±3.24 versus 39.9±4.3, p<0.0413); iii) compared to the control group apitherapy diet + RJ (group III) (45.71±1.28 versus 39.9±4.3, p<0.0001) (fig. 3). Fig. 3. Mean values of the hematocrit and standard deviation (* a p= vs. lotul martor hrană standard; * b p= vs. lotul cu apidietă; * c p= vs. lotul cu apidietă+lm; * d p<0.05 vs. lot CCl 4 ). an increase of the HCT values for the groups that were previously given CCl 4, compared to the group that received only CCl 4. The values obtained following the apitherapy treatment are comparable to the values obtained in the case of the control groups (healthy animals that received the same apitherapy treatment). Mean corpuscular volume MCV, VEM Administration of apitherapy diet (control group apitherapy diet) and of apitherapy diet + RJ (control group apitherapy diet + RJ) determines a significant increase of MCV values when compared to the control group standard food: i) 53.11±0.83 versus ± 2.01, p = ); ii) 53.11±0.83 versus 59.38±1.26, p=0.0053) (fig. 4). Administration of apitherapy diet, respectively of apitherapy diet + RJ leads to an improvement of the MCV values for the groups of animals that were previously given CCl 4. The values are comparable to those obtained for the control groups (healthy animals that were given the same apitherapy treatment) (fig. 4). Administration of apitherapy diet to animals with CCl 4 induced hepatopathy (group V) causes a statistically significant increase of the HCT values when compared to the CCl 4 group (group IV) (39.9±4.3 versus 44.01±2.44, p<0.0306). Administration of apitherapy diet and RJ to animals with CCl 4 induced hepatopathy (group VI) causes a statistically significant increase of the HCT values when compared to the CCl 4 group (group IV) (39.9±4.3 versus 44.16±2.54, p<0.0226). between groups V and VI could be observed regarding the HCT values. CCl 4 causes the decrease of HCT values; ii) administration of apitherapy diet and apitherapy diet + RJ, respectively, leads to 218 Fig. 4. Mean values of the mean corpuscular volume and standard deviation (* a p<0.05 vs. lotul martor hrană standard). Mean corpuscular hemoglobin - MCH, HEM Administration of apitherapy diet to animals with CCl 4 induced hepatopathy (group V) causes a statistically significant increase of the MCH values when compared to control group standard food (group I) (16.88±1.29 versus 18.79±0.77, p=0.0233) (fig. 5). Administration of apitherapy diet and RJ to animals with CCl 4 induced

hepatopathy (group VI) causes a statistically significant increase of the MCH values compared to the control group standard food (group I) (16.88±1.29 versus 18.89±1.05, p=0.0147) (fig. 5). Fig. 6.

05 vs. control group standard food). regarding the MCH values could be observed between groups V and VI.

values being comparable to those obtained for the control groups (fig. 5).

4 hepatopathy (group VI) causes a statistically significant increase of the MCH values compared to the control group standard food (group I) (16.88±1.29 versus 18.89±1.05, p=0.0147) (fig. 5). Fig. 6. Mean values of mean corpuscular hemoglobin concentration and standard deviation (* d p= vs. CCl 4 group). Fig. 5. Mean values of mean corpuscular hemoglobin and standard deviation (* a p<0.05 vs. control group standard food). regarding the MCH values could be observed between groups V and VI. standard food leads to a decrease of the MCH values; ii) administration of apitherapy diet and apitherapy diet + RJ improves the MCH values for the groups that had previously received CCl 4, the values being comparable to those obtained for the control groups (fig. 5). Mean corpuscular hemoglobin concentration MCHC, CHEM Administration of apitherapy diet and RJ to animals with CCl 4 induced hepatopathy (group VI) causes a statistically significant increase of the MCH values compared to the CCl 4 group (group IV) (31.94±0.77 versus 33.31±0.11, p<0.0108) (fig. 6). between groups V and VI could be observed regarding the MCHC values (fig. 6). Fig. 7. Mean values of the red cell distribution width and standard deviation (* a p<0.05 vs. control group standard food; * b p< vs. control group apitherapy diet; * c p< vs. control group apitherapy diet + RJ; * d p< vs. CCl 4 group). Red cell distribution width RDW hepatopathy (group IV) there can be observed a statistically significant decrease of the RDW values when compared to all the experimental groups: i) compared to the control group apitherapy diet (group II) (16.38±1.04 versus 19.51±1.62, p<0.0001); ii) compared to the control group apitherapy diet + RJ (group III) (16.42±0.57 versus 19.51±1.62, p<0.0001) (fig. 7). Administration of apitherapy diet to laboratory animals with CCl 4 induced hepatopathy (group V) determines the decrease of RDW when compared to: i) the control group standard food (group I) (18.74±0.21 versus 15.55±1.16, p<0.0001); ii) the CCl 4 group (group IV) (19.51±1.62 versus 15.55±1.16, p<0.0001) (fig. 7). 219

5 Administration of apitherapy diet + RJ to laboratory animals with CCl 4 induced hepatopathy (group VI) determines the decrease of RDW when compared to: i) the control group standard food (group I) (18.74±0.21 versus 15.44±0.9, p<0.0001); ii) the CCl 4 group (group IV) (19.51±1.62 versus 15.44±0.9, p<0.0001). between groups V and VI could be observed regarding the RDW values. apitherapy diet, respectively of apitherapy diet and RJ maintains the RDW values obtained for the animals that had previously been given CCl 4 in a range comparable to those obtained for the control groups; ii) administration of CCl 4 leads to an increase of RDW levels ; iii) administration of standard food also determines an increase of the RDW values, very close to those obtained for the CCl 4 group. The hematocrit depends on the erythrocyte mass, mean corpuscular volume and plasmatic volume. Generally, when the erythrocytes have a normal size, the modifications of HCT follow the RDW changes (Wallach, 1996; Perkins, 2004). MCV may suggest the physiopathologic mechanism of erythrocyte disorder. MCV depends on the plasmatic osmolarity and number of the erythrocyte fractions. Clinical significance in most cases of anemia, MCH correlates with MCV. Because of the similar behaviour of the MCV and MCH, MCHC remains constant in many hematopoietic disorders (Glader, 2004). The administered apitherapy preparates demonstrate the protective effect at the hepatic level. There are also studies regarding other natural products with protective effect in CCl 4 hepatopathy: Saccharomyces cerevisiae (Lai et al., 2009); lycopene from tomatoes (Kim et al., 2004); dehydrocavidine, an active compound from Corydalis saxicola Bunting (Yanhuanglian) (Wang et al., 2008); the diterpenes kahweol şi cafestol from coffee (Kyung Jin Lee et al., 2007); electrolysed wáter (Tsai et al., 2009); the flavone luteolin (3,4,5,7-tetrahydroxyflavone) 220 (Domitrović et al., 2009); hyaluronic acid and chondroitin-4-sulfate (Campo et al, 2004); olive oil (Fang et al., 2008); potato peel extract (Singht et al., 2008); resveratrolul (Fan et al., 2009). Conclusions The administration of apitherapy products Apiregya, ApiImunomod, ApiImunostim, ApiImunostim Forte is recommended for their hepatoprotective role against chronic liver disorders and for their positive effect upon the hematological parameters. Acknowledgement: This paper was supported by the project PERFORM-ERA "Postdoctoral Performance for Integration in the European Research Area" (ID-57649), financed by the European Social Fund and the Romanian Government. References Steiff, M.B., Spirak, J.L.: The liver and hematopoesis. In: Hepatology, A textbook of liver diseasese, Zakim D., Boyer Th. (eds)., 537, 542, Peault, B., Tavian, M.: Hematopoetic stem cell emergence in the human embryo and fetus. Ann NY Acad Sci, 996, 132, 140, Wallach, J.: Hematologic Diseases. In: Interpretation of Diagnostic Tests, Philadelphia ed., 293, 316, Perkins, S.: Examination of the Blood and Bone Marrow. In: Wintrobe's Clinical Hematology, Philadelphia, ed., 3, 21, Glader, B.: Anemia: General Considerations. In: Wintrobe's Clinical Hematology, Philadelphia, 948, 975, Lai, J.T., Hsieh, W.T., Fang, H.L., Lin, W.C.: The protective effects of a fermented substance from Saccharomyces cerevisiae on carbon tetrachloride-induced liver damage in rats. Clinical Nutrition, 28, 338, 345, Kim, Y., DiSilvestro, R., Clinton, S.: Effects of Lycopene-beadlet or tomato-powder feeding on carbon tetrachloride-induced hepatotoxicty in rats. Phytomedicine, 11, 152, 156, Wang, T., Sun, N.L., Zhang, W.D., Li, H.L., Lu., G.C., Yuan, B.J., Jiang, H., She, J.H., Zhang, C.: Protective effects of dehydrocavidine on carbon tetrachloride-induced acute hepatotoxicity in rats. Journal of Ethnopharmacology, 117, 300, 308, 2008.

6 Lee, K.J., Choi, J.H., Jeong, H.G.: Hepatoprotective and antioxidant effects of the coffee diterpenes kahweol and cafestol on carbon tetrachlorideinduced liver damage in mice. Food and Chemical Toxicology, 45, 2118, 2125, Tsai, C.F., Hsu, Y.W., Chen, W.K., Chang, W.H., Yen, C.C., Ho, Y.C., Lu, F.J.: Hepatoprotective effect of electrolyzed reduced water against carbon tetrachloride-induced liver damage in mice. Food and Chemical Toxicology, 47, 2031, 2036, Domitrović, R., Jakovac, H., Tomac, J., Šain, I.: Liver fibrosis in mice induced by carbon tetrachloride and its reversion by luteolin. Toxicology and Applied Pharmacology, 241, 311, 321, Campo, G.M., Avenoso, A., Campo, S., Ferlazzo, A.M., Micali, C., Zanghì, L., Calatroni, A.: Hyaluronic acid and chondroitin-4-sulphate treatment reduces damage in carbon tetrachlorideinduced acute rat liver injury. Life Sciences, 74, 1289, 1305, Fang, H.L., Lai, J.T., Lin, W.C.: Inhibitory effect of olive oil on fibrosis induced by carbon tetrachloride in rat liver. Clinical Nutrition, 27, 900, 907, Singh, N., Kamath, V., Narasimhamurthy, K., Rajini, P.S.: Protective effect of potato peel extract against carbon tetrachloride-induced liver injury in rats. Environmental Toxicology and Pharmacology, 26, 241, 246, Fan, G., Tang, J.J., Bhadauria, M., Nirala, S.K., Dai, F., Zhou, B., Li, Y., Liu, Z.L.: Resveratrol ameliorates carbon tetrachloride-induced acute liver injury in mice. Environmental Toxicology and Pharmacology, 28, 350, 356,

Researches Regarding the Influence of Apitherapy Diet on Leukocyte Formula in Wistar Rats with Experimentally CCl 4 Induced Liver Disease

Bulletin UASVM, Veterinary Medicine 68(1)/211 pissn 1843-527; eissn 1843-5378 Researches Regarding the Influence of Apitherapy Diet on Leukocyte Formula in Wistar Rats with Experimentally CCl 4 Induced