Pharmaceutical Biology 2001, Vol. 39, No. 6, pp. 435–439 1388-0209/01/3906-435$16.00 © Swets & Zeitlinger Evaluation of Hemorrheologic Parameters and Biliary Secretion in Ligaria cuneifolia (Argentine mistletoe) Extract-Treated Rats Guillermo Mengarelli1, Alicia Dominighini1, Mariana Ferrero1, María de Luján Alvarez2, Marcelo Wagner3, Alberto Gurni3, Cristina Carnovale2 and Alejandra Luquita1 1 Department of Physiology, Faculty of Medical Sciences, University of Rosario, Argentina; 2Department of Physiological Sciences, Faculty of Biochemical and Pharmaceutical Sciences, University of Rosario, IFISE-CONICET, Argentina and 3 Department of Pharmacobotanical, Faculty of Pharmacy and Biochemistry, University of Buenos Aires, Argentina Abstract Introduction Ligaria cuneifolia (R. et P.) Tiegh. (Loranthaceae) (Argentine mistletoe) is usually used in local folk medicine “to produce an increase of blood fluidity and a decrease of plasma cholesterol level”. The present work was carried out to evaluate the effect of L. cuneifolia treatment on blood fluidity and biliary secretion. Adult male Wistar rats were divided into five groups (n = 5): control animals were injected intraperitoneally with water and treated rats received 1.5, 2.5, 3.5 and 5.5 mg/100 g body weight of aqueous extract of L. cuneifolia each 24 h during 3 days. The relative viscosity of blood [(hr)45/Hct] was measured and the rigidity index (RI) in the diluted red cell suspensions was determined. L. cuneifolia treatment produces a significant increase in (hr)45/Hct and in RI at doses of 2.5, 3.5 and 5.5 mg/100 g body weight. Also, a decrease of plasma cholesterol level was observed, which showed a negative association with RI increase (rs: -0.65; p < 0.05). Increases in bile flow and biliary output of bile acids and cholesterol were observed in treated rats (2.5, 3.5 and 5.5 mg/100 g body weight). Ligaria cuneifolia-treatment produces a decrease in the plasma cholesterol level increasing red blood rigidity index and producing an increase in blood viscosity. The observed increase of bile acid and cholesterol biliary output causes a decrease of plasma cholesterol level. Preparations from leaves and stems of several plants belonging to the formerly known “Loranthaceae family” have been widely used in traditional medicine. Plants of this family have long been recognised as therapeutic herbs. The Loranthaceae is currently divided into three families: Loranthacea (sensu strictu), Viscaceae and Eremolepidacea. However, many authors still refer to the traditional division including them all as Loranthaceae (sensu latu) (Barlow, 1964; Kujit, 1988). Ligaria cuneifolia (R. et P.) Tiegh. (Loranthaceae) grows in the Northwest and central regions of Argentina and is widely employed in Argentine folk medicine. Infusions of L. cuneifolia leaves and stems have been used as a substitute for Viscum album L. (Viscaceae) based on its alleged ability to decrease high blood pressure. In addition, both herbs are hemiparasite species usually employed in folk medicine “to produce an increase of blood fluidity and a decrease of plasma cholesterol level”. Few studies have been carried out on L. cuneifolia (Wagner et al., 1998; Varela & Gurni, 1995). L. cunefolia infusions were originally reported to decrease high blood pressure (Domínguez, 1928; Ratera et al., 1980; Taira et al., 1994). The purpose of the study was to investigate the effect of L. cuneifolia treatment on Wistar rat blood fluidity through the hemorrheological behaviour. On the other hand, it is already known that the bile acid biosynthesis and secretion in combination with the excretion of free cholesterol into bile is the major route for the elimination of cholesterol from the Keywords: Ligaria cuneifolia, whole blood viscosity, plasma cholesterol, erythrocyte deformability, biliary secretion. Accepted: March 3, 2001 Address correspondence to: Cristina Carnovale, Ph.D., Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Instituto de Fisiología Experimental (IFISE)-CONICET, Suipacha 570-2000 Rosario, Argentina. Fax: 54-341-4399473. E-mail:ifise1@citynet.net.ar, carnovale@ciudad.com.ar 436 G. Mengarelli et al. mammalian body (Björkhem, 1985; Carey & Duane, 1967). It was of the utmost importance to study the effect of L. cuneifolia on liver physiology. Materials and methods Plant material Specimens were collected from different host plants and identification was done by one of us (A.A.G.). Voucher specimens were kept at the Museo de Farmacobotánica “Juan A. Domínguez”, Faculty of Pharmacy and Biochemical of University of Buenos Aires (BAF 9018). Classification of the species was performed by means of the key according to Abbiatti (1946). calculated using of the equation of Matrai et al. (Chailley et al., 1981): hr = (hb hp ) 45 Hct = (hr ) 45 Hct Filterability measurements were performed with minor modification of the technique described by Reid et al. (1976). The whole blood was centrifuged at 1900 g for 5 min at 25 °C, and plasma and buffy coat were removed. Red blood cells (RBC) were washed 2 times with phosphate-buffered saline (PBS), pH 7.4, 285 mOsm/L, and containing 0.25% bovine albumin. After washing, 1 ml of a 10% RBC suspension was placed in a graduated cylinder. Using a negative filtration pressure of 10 cm H2O, the suspension was passed through a 13 mm polycarbonate filter (Nucleopore Corr. USA) with a pore size of 5 mm. Results were expressed as a rigidity index (RI): RI = (Ts - Tb ) (Tb ) ¥ 100 Hct Extracts The plant material was properly air-dried and powdered before use. Powdered dried leaf material (5 g) was extracted with boiling water for 30 min. Extracts were evaporated under reduced pressure and residues dissolved in water. Animals and treatment Male Wistar rats weighing 400 to 450 g were housed two per cage and maintained under a 12 h light/dark period. Rats were fed ad libitum with a normal standard diet and water. All the experimental protocols were performed according to the “Guide for the Care and Use of Laboratory Animals” (National Institutes of Health, Publication No. 86-23, revised 1985). The animals were divided into five groups of five rats each. Tail blood was obtained for both determination of serum cholesterol and hematocrit (basal values). Then, the aqueous extracts of L. cuneifolia were administered intraperitoneally once daily for three days. The treated rats received a volume of 0.2 ml of the following doses (mg/100 g body weight): 1.5, 2.5, 3.5 and 5.5. The control group was injected with the vehicle alone (water). Experimental procedures On the day of the experiment, the rats were weighed and anaesthetized with pentobarbital (5 mg/100 g body weight, i.p.). The bile duct was cannulated and the bile was collected, on ice into preweighed tubes, every 15 min for 60 min. Rectal temperature was maintained at 38.0 ± 0.5 °C throughout the experiments to prevent hypothermic alterations of bile flow, as stated elsewhere (Roberts et al., 1967). At the end of bile collection, blood was withdrawn by heart puncture, and the liver was promptly removed and weighed. Whole blood viscosity (hb) and plasma viscosity (hp) were measured with a Wells-Brookfield LVT cone-plate viscometer at shear rate of 230 s-1 and at 37 °C. The relative viscosity of blood at a standard hematocrit (Hct) of 45% was Ts corresponds to the passage of cell suspension; Tb is the passage of PBS medium. Hct is the hematocrit of 10% RBC suspension. RI is an estimation of erythrocyte deformability. The heparinized blood samples were used to determine plasma cholesterol concentration (pre- and post- injection of L. cunefolia) by the enzymatic esterase-oxidase method (Henry et al., 1974). Serum samples were used to determine activities of alkaline phosphatase (ALP), glutamate-pyruvate aminotransferase (GPT; ALAT), glutamate-oxalacetate aminotransferase (GOT; ASAT), cholinesterase (CHE) and lactate dehydrogenase (LDH). Standard kinetic kits (Wiener Lab., Rosario, Argentina) were used to determine enzyme activities in serum. The bile flow (BF) was estimated by gravimetry, assuming a density for bile 1.0 g/ml; BF was expressed as ml/min g of liver. Bile acid (BA) concentration in bile was assayed with 3a-hydrosteroid dehydrogenase according to Talalay’s method modified by Berthelot (Talalay, 1960; Berthelot et al., 1970). Protein concentration was measured in bile samples with the Lowry’s method (Lowry et al., 1951), using bovine albumin as standard. Bile cholesterol concentration was determined by the enzymatic esterase-oxidase method (Fawcett & Menkes, 1994). The biliary outputs of bile constituents were calculated as the product of BF times the bile concentration. Histology Liver fragments were placed in formaldehyde 10%; 24 h later they were embedded in paraffin, cut and stained with hematoxylin – eosin and trichromica Masson – Alcian blue. Statistical analysis The results obtained were expressed as mean ± S.E. Significance of the differences was tested by two-way ANOVA, and, in the case of significance, a Newman-Keul’s test also was applied. Hemorrheologic properties of Ligaria cuneifolia 437 Results Blood samples Enzyme activities Enzyme activities of ALP, GPT (ALAT), GOT (ASAT), CHE and LDH did not vary with the different doses of aqueous extract of L. cuneifolia. Hemorrheological parameters The hp did not vary with the different doses of aqueous extracts of L. cuneifolia. Figure 1 illustrates the effect of different doses of intraperitoneal administration of aqueous extracts of L. cuneifolia on hemorrheological parameters. Panel A shows values of relative viscosity of blood [(hr)45/Hct]. The results clearly demonstrate that (hr)45/Hct increases throughout all doses of L. cuneifolia, reaching a maximum value at 2.5 mg/100 g body weight. As indicated in Panel B, the three doses (2.5, 3.5, 5.5 mg/100 g body weight) of L. cuneifolia significantly increased the RI. Table 1 shows the values of changes in plasma cholesterol level between pre- and post-injection of different doses of L. cuneifolia. Determination of bile flow and biliary outputs of bile components Data on bile flow and biliary output are presented in Figure 2. Bile flow and bile acid output (Panel A, B) increased significantly in treated rats from the dose of 2.5 mg/100 g body weight. The output of cholesterol (Panel C) exhibits a significant increase in animals treated with 2.5 and 3.5 mg/ 100 g body weight. At 1.5 and 5.5 mg/100 g body weight the increase observed was not statistically significant. Histology Histological examination of livers demonstrated that the hepatic acinus remained unchanged in spite of the treatment in all the cases. The size of the nucleus was increased in only few hepatocytes in the rats treated with 2.5, 3.5 and 5.5 mg/100 g body weight, but no significant difference was detected. Discussion The results of our study indicate that L. cuneifolia treatment did not produce liver damage at the doses used. This is indicated by the lack of variations in all the serum enzyme activities with the different doses of L. cuneifolia. The absence of hepatotoxic action is also demonstrated by histological examination. The result of (hr)45/Hct shows the blood viscosity without the influence of hematocrit and the hp. The (hr)45/Hct increases Figure 1. Effect of different doses of L. cuneifolia on hemorrheological parameters. Panel A: The relative viscosity of blood at a standard hematocrit (Htc) of 45% was calculated for shear rate 230 s-1 using the equation of Matrai et al. (1987). Panel B: rigidity index determined by filtration through micropore membrane of the diluted red cell suspension (hematocrit 10%). C: control group. Data are shown as the mean of five rats ± SE. * Significant difference p < 0.05 vs. the control rats. ** Significant difference p < 0.01 vs. the control rats. 438 G. Mengarelli et al. in L. cuneifolia-treated rats with doses of 2.5, 3.5 and 5.5 mg/100 g body weight, by a loss of the erythrocyte deformability estimated by RI determination. The treatment with 1.5 mg/100 g body weight of L. cuneifolia shows a significant diminution of plasma cholesterol without producing an alteration in (hr)45/Hct (see Table 1 and Fig. 1B). Our results show that the diminution of plasma cholesterol levels are associated with elevated erythrocyte rigidity indicated by a negative association (rs: -0.65, p < 0.05). This observation is consistent with other studies in patients with myocardial infarction or unstable angina pectoris, where it has been described that a diminution of total cholesterol is associated with an increase in RI (Fawcett & Menkes, 1994). Moreover, studies in vitro have demonstrated that the plasma cholesterol loss is accompanied by a decrease in cholesterol of erythrocytes and is associated with an increase in the proportion of nondiscocytic erythrocytes, which are known to be less deformable and thus contributes to an increase in RI (Chailley et al., 1981). On the other hand, the reduction of plasma cholesterol levels is accompanied by a significant increase in the biliary output of bile acids and cholesterol for the doses of 2.5, 3.5 and 5.5 mg/100 g of body weight (Fig. 2B, C). In this connection, it is known that the two major output pathways responsible for elimination of cholesterol from the body and maintenance of cholesterol homeostasis involve degradation of cholesterol to bile acids (their biliary excretion) and the canalicular secretion of cholesterol (Russel, 1992). Since the bile acids provide the primary stimulus for bile flow (Turley & Dietschy, 1982), we can assume that the increase observed in bile flow (see Fig. 2A) is a result of augmented biliary bile acid excretion. From these results, we can conclude that L. cuneifolia has hypocholesterolaemic activity as compared to the nontreated group, due to one or more of its constituents (Fernández et al., 1998). We have observed that L. cuneifolia does not produce an increase of blood fluidity as the folk medicine suggest; quite the contrary, the plant appears to increase blood viscosity or to decrease blood fluidity. This contradiction probably is due to the lack of studying hemorrheological behaviour in the blood of the persons treated with L. cuneifolia. On the other hand, since a decrease in plasma cholesterol level is usually associate with an increase in plasma fluidity, it is therefore reasoned that blood fluidity increases too. However, in our work, the plasma viscosity was not modified and the decrease in blood fluidity (increase in blood viscosity) is due to elevated erythrocyte rigidity. Further pharmacological investigations are needed to ensure the safe use of this natural folk preparation. Figure 2. Effect of different doses of L. cuneifolia on bile flow and biliary parameters. Panel A: Bile flow expressed in ml/min/g of liver. Panel B: Bile acid output expressed in nmol/min/g of liver. Panel C: Biliary cholesterol output expressed in nmol/min/g of liver. C: control group. Data are shown as the mean of five rats ± SE. * Significant difference p < 0.05 vs. the control rats. ** Significant difference p < 0.01 vs. the control rats. Acknowledgements This work was supported by research grants from Universidad Nacional de Rosario (Argentina) and Instituto de Fisiología Experimental (CONICET, Argentina). The authors wish Hemorrheologic properties of Ligaria cuneifolia 439 Table 1. Diminution of plasma cholesterol level after injection of different doses of L. cuneifolia. Doses of L. cuneifolia (mg/100 g body weight) 0 (C) 0.0 ± 0.9 1.5 2.5 3.5 5.5 -19.7 ± 1.9* -17.3 ± 1.0* -15.0 ± 2.0* -18.6 ± 1.1* Note: The diminution of plasma cholesterol (Cho) level was calculated using the equation: DCho (mg/ml) = Chobasal - Chopost-Lc. Chobasal: Cho plasma cholesterol concentration determined in blood pre-injection of extract. Chopost-Lc: Cho plasma cholesterol concentration determined in blood post-injection of extract of L. cuneifolia. C: control group was injected with the vehicle alone (water). Values are expressed as mean ± SE of at least five animals per group. * Significant difference vs. control group (p < 0.05). to acknowledge Dr. Tomás Telles for performing histological analyses. We also wish to thank Cecilia Larroca and Juan Monti for their technical assistance. References Abbiatti D (1946): Las Lorantáceas Argentinas. 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