24 growing male albino Wistar rats were obtained from the animal house of the Department of Medical Physiology, University of Calabar, Nigeria. The rats were initially weighing between 70g and 90g but their final weight was between 150g and 200g when they were used for the experiment. The rats were weighed before commencement of the feeding experiment and thereafter were weighed daily. They were nursed under controlled environmental condition.

2kg of fresh leaves of mistletoe were purchased from a local plantation in Akpabuyo Local Government Area of Cross River State, Nigeria during the rainy season and were identified as Viscum album by a botanist (Mr. Frank Adepoju) in the Department of Biological Sciences, University of Calabar, Nigeria.

Fresh leaves of Viscum album from the host plant (citrus) were collected. The leaves were first washed free of sand and debris. Wash water was blotted off and the leaves ground to paste. A quantity of the ground sample (50g) was weighed and soxhlet extracted with 150ml distilled water at 100°C for 9hr. Where larger ground samples were used, extraction was done under reflux with an appropriate volume of distilled water. The extract was slowly evaporated to dryness in vacuo at 40°C using a rotary evaporator. A total yield of 31% was obtained. Weighed samples of the extract were then used to prepare the stock solution after the method of Eno et al., ₂₀

High salt diet containing 8% sodium chloride was prepared using a standard diet containing 0.3% sodium chloride after the method of Obiefuna et al, ₁₉ and Akinyanjuola, ₂₁.

Seventy male white mice (18-20g) were used for the study. They were randomly selected and assigned to 7 cages of 10 animals per cage. They were allowed a week for adaptation. Each group then received one of the following doses: 0, 125, 250, 500, 1000, 2000 and 4000 mg/kg body weight of the extract, i.p. The maximum volume injected was 0.2mls. The groups were then returned to the home cages and allowed free access to food and drinking water. The mortality in each cage was assessed 24 hours after the administration of the extract. Percentage mortalities were converted to probits and plotted against the log₁₀ of the dose of the extract from which the LD₅₀ value was extrapolated following the method of Miller and Tainter ₂₂.

The twenty-four male albino Wistar rats were divided into 4 groups of 6 rats each. They were fed as follows: Group 1 (control) was fed on normal rat pellet + drinking water. Group 2 was fed on normal rat pellet + drinking water + 150mg/kg of mistletoe extract orally once daily. Group 3 was placed on high salt diet (8% sodium chloride) + 1% sodium chloride drinking water. The group 4 received same as the third group + mistletoe extract (150mg/kg body weight) orally once daily. The feeding regimens lasted for six weeks. At the end of the feeding period, the animals were sacrificed and blood sample collected for daily analysis. The animals were weighed daily.

The animals were made unconscious using chloroform anesthesia and blood collected via cardiac puncture (blood was drawn from the heart) a modification of the method by Ohwada ₂₃. The samples were collected by the help of 5mls syringe attached to needle (21 SWG) into plain capped bottles. The samples were immediately used for the estimation of the different variables.

The rats were sacrificed under chloroform anaesthesia. They were quickly dissected and their kidney, heart and blood vessels were removed and weighed using the mettler P163 weighing balance.

Permanent preparations using routine biopsy method was employed. Tissue sections were treated with traditional haematoxylin and eosin stains. The tissue blocks from the heart, kidney and blood vessels were fixed in 10% neutral formalin after which they were dehydrated using alcohol and then cleaned in xylene. They were then embedded in paraffin wax and thin sections cut at 5 microns. The sections were then stained with haematoxylin for 15 minutes, differentiated with 1% acid alcohol, counter stained in eosin for 2 mins and mounted with DPX. The sections were then viewed under the microscope (x400) and photomicrograph taken.

Blood samples from each rat was collected separately into clean capped plain tubes and allowed to stand for 30 minutes for clotting to occur. These were then centrifuged at 2500 revolution per minute for 15 minutes. The serum was extracted into clean test tube for the analysis of sodium, potassium, chloride, bicarbonate and calcium ions.

Sodium and potassium ions were measured using Flame photometry method at wave length of – 590nm for sodium and 770nm for potassium.

Bicarbonate ions were estimated by titration method. Calcium Ions were measured by photometric test using O-cresophethalein – complexene in an alkaline medium, ₂₄

Measurement of chloride ion was done by titration, using mercuric nitrate method of Schales and Schales ₂₅.

Data were presented as mean ± SEM. Data were analysed using a one way analysis of variance (Anova) then followed with post hoc test (Least Square Deviation). P value of less than 0.05 was declared as significant statistically.

The LD₅₀ value for the crude extract from V. album leaves was extrapolated to be 420.70mg/kg mice i.p (r = 0.9515; P<0.01, n = 10) from which a test (safe) dose of 150mg/kg body weight was derived for the chronic feeding experiment.

Figure 1 show mean body weight in control and test groups. In the control group, the initial (day 1) mean body weight was 84.00±6.26g. It rose to 174.17±14.76g on the last day (107.35%). This was equivalent to a growth rate of 2.15g per day. Animals in group 2 (normal + extract) had an initial body weight of 93.00 ± 2.25g and 123.17±5.17g on day 42 producing 30.17g (32.44%) change in body weight (growth rate of 0.72g/day). This shows a relative reduction in weight and growth rate in group 2 when compared with control rats.

Figure 1

Figure 1: Comparison of weekly change in body weight in the experimental groups. Values are mean ± SEM. n = 6.

The third group of animals (salt fed) had a loss in weight of 1.33g (1.13%) on the last day of the experiment (from an initial mean body weight of 118.00 ± 6.63g to 116.67± 9.03g final weight). Their growth rate was -0.03g/day. Rats in group 4 (salt fed + extract) had growth rate of 0.89g/day or total weight gain of 37.17g (39.77%). Their mean weight on the last day was 130.17±10.79g from an initial value of 93.00±2.25g.

In the control group, the mean weight of the left kidney was 0.58±0.11g. It was 0.53±0.03g, in group 2 rats, showing no significant difference from the control value. The salt fed group had a reduction in the mean weight of the left kidney of 0.45±0.03g, their mean weight was reduced when compared to the control; this was also not significantly different. But in the salt fed + extract group, there was a significant (P<0.05) increase in the mean weight of the kidney (0.53±0.03g) when compared to salt fed group, (Fig. 2).

Figure 2

Figure 2: Comparison of weights of the heart and left kidney in control and test groups. Values are mean ± SEM. n = 6. ** = P

The mean heart weights in the control group were 0.61±0.02g (with mean body weight of 247.7±8.16g). The mean heart weight, 0.55±0.03g (having a mean body weight of 189.967±7.24g) in the salt fed group was significantly (P<0.01) lower compared with control value. The elevation in mean heart weight of group 2 (0.66±0.03g, with 181.37±7.66g as mean body weight) and group 4 (0.64±0.02g (with mean body weight of 190.2±5.45g) were not significant compared with control value, (Fig. 2).

Table 2 shows comparison of serum electrolyte concentration in the experimental groups. The mean value of sodium ion in control group was 142.17±0.65Meq/L; group 2 had slight reduction in sodium ion concentration (141.67±0.21Meq/L) compared to control. Salt fed (untreated) group had a significant (P<0.01) increase in sodium ion concentration (144.33±0.42Meq/L) compared with normal control group. Group 4 (salt fed + extract) had a significant reduction in sodium level (139.5±0.67Meq/L) compared with control (P<0.05) and salt fed rats (P<0.001).

The mean value of potassium ion in control group was 4.18 ± 0.07Meq/L. The mean potassium level in group 2 (4.60 ± 0.04Meq/L) was significantly (P<0.001) higher than control value. The salt fed group had a potassium ion level of 3.96±0.42mEq/L which was significantly lower than that of the control group (P<0.05). Potassium ion concentration was significantly (P<0.001) reduced in the salt + extract group (5.03±0.05mEq/L) compared with both control and salt fed groups.

The mean chloride ion concentration of the normal control group was 103.00 ± 0.44mEq/L. Normal + extract (group 2) had a mean chloride ion concentration of 101.33±0.42 mEq/L, which was significantly lower than that of the control group (P<0.05). The mean chloride ion concentration of the salt fed group (105.00±0.44mEq/L) was significantly higher (P<0.01) than that of the control group. The salt fed + extract group had a mean chloride ion concentration of 102.66±0.84mEq/L was not significantly lower than that of the control group but was significantly lower than that of the salt fed group (P<0.01).

The mean bicarbonate ion concentration of the normal + extract group was 24.00±0.36 mEq/L) which was not significantly different from the normal control group. The salt fed group had also a slightly higher bicarbonate ion concentration (24.66±0.21mEq/L) which was not significantly different from the control group. However, the salt fed + extract group had a bicarbonate ion concentration of 26.66±0.42 mEq/L which was significantly different from both that of the control group and salt fed group (P<0.001 and P<0.01, n = 6 respectively). See Table 1.

The mean value of calcium ion concentration in the control group was 3.2±0.10mEq/L. The normal + extract group had a calcium ion concentration of 3.23±0.04mEq/L which was slightly higher than that of the normal control group. The salt fed group had a calcium ion concentration of 0.02±0.10mEq/L which was insignificantly lower than that of the control group. The calcium ion concentration in the salt fed + Extract group (3.26±0.10mEq/L) was only slightly higher than control value but was significantly higher when compared with salt fed group (P<0.05).

Plate 1a shows the photomicrograph of the longitudinal section (x 400) of the cardiac muscle in control group. It is characterized by cross striation and inter-digitations of the myofibrils with well stained myoplasm and nuclei and showing an outline of the normal heart. In the extract fed group the myocardium had blood stains and myofilbrils can not be properly distinguished as individual cells compared with control although, the nuclei are well stained, (Plate 1b). In high salt fed (untreated) group, the cardiac muscles had thicker muscular wall, showing alignment of the myofibrils with numerous nuclei indicating hyperplasia of the nuclei, the myofibrils were fused together, (Plate 1c). Also seen are the cross striations observed in the control plate. Plate 1d shows that in the high salt + extract fed group, the cardiac muscle had cross striation similar to control but with slightly thicker muscular wall than control.

Figure 3

Plate 1: Photomicrograph of the section of the heart in (a) control, (b) normal + extract fed, (c) salt fed and (d) salt + extract fed groups stained with H & E (x 400) showing the cardiac muscles.

Plate 2a shows the photomicrograph of the section of a descending aorta in normal control revealing the three basic layers of the blood vessel, the tunica intima (TI) with distinct epithelial lining resting on the basement membrane, tunica media (TM) of prominent stained nuclei and smooth muscles interlocked with elastic membrane. The tunica adventitia (TA) is also seen as a thin connective tissue layer which is also present with collagen and elastic fibers. In the group 2 animals, the three basic layers are intact with epithelial lining of the tunica intima, the tunica media has fewer stained nuclei with intact elastic tissues, and the tunica adventitia is less prominent than in control plate (Plate 2b). The high salt fed group also shows the three basic layers of the blood vessel but with slight erosion of the epithelial lining, the TM is thicker compared to the control plate but with fewer stained nuclei comparatively. TA is also less predominant, (Plate 2c). Plate 2d shows a photomicrograph of the section of the descending aorta in high salt fed plus extract group stained with H and E (x 400) showing the three basic layers with similar architecture as control.

Figure 4

Plate 2: Photomicrograph of the section of the descending aorta in (a) control, (b) normal + extract fed, (c) salt fed and (d) salt + extract fed groups stained with H & E (x 400) showing the cardiac muscles.

As shown in Plate 3a, the section of the kidney in control group shows segments of the renal cortex and intact glomerulus, which are normally placed; the nuclei of the glomeruli are well distinguished. In the normal + extract fed group (Plate 3b) the glomerulus and renal cortex are obliterated, creating a wider renal bowman space for filtration. Although, the nuclei of the cells and juxtaglomerular capsules were not distinctly shown, rather shows well stained cells of the glomerular apparatus which were also very well outlined. Tubular vessels are also well outlined. The section of the kidney in salt fed group is shown in Plate 3c, characterized by obliteration of some segments of the glomerulus, with sparse interstitium, creating greater renal space for excretion. This section also shows enormous tubular necrosis with massive degeneration of the cells of the glomerular apparatus (atrophic changes) showing yellow pigments, this plate has low staining intensity, and the renal vessels are not well outlined. Plate 3d shows the section of the kidney of group 4, showing segments of the renal cortex having well outlined glomerular tuft, creating a small renal space compared to plate 3c. The Juxtaglomerular apparatus is not clearly outlined and the tubules cannot be clearly identified and nuclei not well stained. Blood vessels are not properly distinguished and slight necrosis is also present.

Figure 5

Plate 3: Photomicrograph of the section of the kidney in (a) control, (b) normal + extract fed, (c) salt fed and (d) salt + extract fed groups stained with H & E (x 400) showing the cardiac muscles.

Figure 6

Table 1: Comparison of mean values of serum electrolytes in control and test groups.

The authors of this article wish to acknowledge Mr. Inah Igiri a Laboratory Technologist in Histo-Pathology Department, University of Calabar-Nigeria for preparing our tissue slides, Dr. Theophilus Ugbem of Histo-Pathology Department, Mr. E. Ije of Anatomy Department, University of Calabar-Nigeria for their independent interpretation of the photomicrographs and Miss Iya Ntui of Chemical Pathology Department, University of Calabar-Nigeria for her technical support.