MATERIALS AND METHODS:

Collection of Animals and Preparation of Plant Extract: Albino rats were collected from the animal house of the Department of Physiology, University of Nigeria, Enugu Campus, Enugu, Nigeria. Samples of fresh Sansevieria liberica plants were procured from: a horticulturist by Air Force Gate, Aba Road; Alikor Estate Choba; a horticultural garden by Uniport main gate, Abuja campus; behind Ofrima complex, Abuja campus, Uniport, all in Port Harcourt, Nigeria. After due identification at the University of Port Harcourt Herbarium, Port Harcourt, Nigeria, they were rid of dirt and the leaves were removed, oven dried at 55⁰C and ground into powder. The resultant powder was soaked in boiled distilled water for 12h, after which the resultant mixture was filtered and the filtrate, hereinafter referred to as the aqueous extract was stored for subsequent use. A known volume of this extract was evaporated to dryness, and the weight of the residue used to determine the concentration of the filtrate, which was in turn used to determine the dose of administration of the extract to the test animals.

Experimental Design: Studies were conducted in compliance with applicable laws and regulations. The rats were randomly sorted into five groups of five animals each, so that the average weight difference was ±1.7g. The animals were housed in plastic cages. After a one-week acclimatization period on guinea growers mash (Port Harcourt Flour Mills, Port Harcourt, Nigeria), the treatment commenced and lasted for seven weeks. The control group received a diet consisting 100% of the commercial feed, while the four test groups received a diet consisting 8% salt and 92% commercial feed. The 8% dietary salt-loading was adapted from Obiefuna et al.⁸. At the end of the sixth week, the rats were weighed, before commencing the administration of the extract, while the reference treatment group had its salt-loading discontinued. The first test group (Test 1) received daily by intra-gastric gavages 200mg/kg body weight of the Sansevieria liberica extract; the second group (Test 2) received 250mg/kg body weight of the Sansevieria liberica extract; while the other three groups, test control, reference treatment (reference) and control groups received appropriate volumes of water by the same route. The dosage of administration of the extract was adapted from Adeyemi et al.⁷. The animals were allowed food and water ad libitum. At the end of the one week treatment period, the rats were anaesthetized by exposure to chloroform. While under anesthesia, they were painlessly sacrificed and blood was collected from each rat into EDTA and heparin sample bottles. The EDTA anti-coagulated blood samples were used for the hematological studies: while the heparin anti-coagulated blood samples were centrifuged at 1000g for 10min, after which their plasma was collected and stored for subsequent analysis.

Enzyme Assays: The plasma activities of alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP), were determined using Randox Test kits (Randox Laboratories, Crumlin, England). ALT was measured by monitoring at 546nm, the concentration of pyruvate hydrazone formed with 2,4-dinitrophenylhydrazine. AST was measured by monitoring at 546nm, the concentration of oxaloacetate hydrazone formed with 2,4-dinitrophenylhydrazine. The activity of alkaline phosphatase (ALP) was determined by monitoring the degradation of p-nitrophenylphosphate to p-nitrophenol, at 405nm.

Determination of Plasma Electrolytes: Plasma sodium and potassium concentration was determined by flame photometry. When elements or their compounds are heated at high temperatures, they gain energy and become excited, and so, when they fall back to their ground or original state, produces an emission spectrum which is characteristic of the element. The intensity of the emission is within certain limits, proportional to the concentration of the element in the solution.

Plasma calcium concentration was determination by the cresol phthalein complexone method⁹. Colour is developed with cresol phthalein complex at pH 12. The magnesium interference is eliminated by complexing it with 8-hydroxyquinoline, after measuring the optic density a 575nm. An excess of ethyleneglycol (diamine) tetra acetic acid (EDTA) is added for washing the calcium and the optical density is measured again. The difference is proportional to calcium level.

Plasma chloride concentration was determination by the titrimetric method¹⁰. Mercuric nitrate is titrated against chloride to form mercuric chloride in the presence of an indicator diphenyl carbazone. Light violet colour is observed when the entire chloride ion in the sample has been used up and excess mercuric nitrate produces a violet colour. The end point of reaction is proportional to chloride concentration.

Plasma bicarbonate concentration was determination by the titrimetric method¹⁰. HCl reacts with bicarbonate and liberates carbon (IV) oxide leaving excess unreacted HCl in solution. NaOH is titrated against the HCl in the presence of phenol red indicator and point of neutralization is where orange colour is produced. The amount of bicarbonate is inversely proportional to unreacted HCl.

Determination of Hematological Indices

These were carried out as reported by Cheesbrough¹⁰. Plasma Hemoglobin concentration (Hb) was measured with DTH Haemoglobinometer^TM. Packed cell volume (PCV) was measured with micro-haematocrit, with 75×16mm capillary tubes filled with blood and centrifuged at 3000rpm for 5 minutes. The red blood cell (RBC) and total white blood cell (WBC) counts were estimated by visual methods. Differential WBC count was carried out using Leishman staining technique. The mean cell hemoglobin (MCH), mean cell haemoglobin concentration (MCHC) and mean cell volume (MCV) were also calculated, as follows:

· Mean cell hemoglobin (pg/cell) =

· Mean cell haemoglobin concentration (g/100mL) =

· Mean cell volume (fL) =

Determination of Plasma Chemistry: Plasma total and conjugated bilirubin, urea and creatinine concentrations were determined using Randox test kits (Randox Laboratories, Crumlin, England). Direct (conjugated) bilirubin reacts with diazotized sulphanilic acid in alkaline medium to form a blue coloured complex, whose intensity can be monitored at 546nm. Total bilirubin is determined in the presence of caffeine, which releases albumin bound bilirubin, by the reaction with diazotized sulphanilic acid, with intensity of the resultant solution monitored at 578nm. Urease hydrolyzes urea to ammonia, which can be measured photometrically at 546nm, by Berthelot’s reaction. In the presence of a strong alkali, creatinine reacts with picric acid to form picramic acid which imparts a yellow-red color on the solution, whose intensity can be monitored at 492nm. The amount of the complex formed is directly proportional to the creatinine concentration. Plasma total protein was determined by the Biuret method, while plasma albumin was determined using bromcresol green (BCG) dye binding method¹¹. Bromcresol green, a yellow dye, binds selectively to albumin at pH 4.2, to form an intense blue protein-dye complex with a maximum absorbance at 630nm. Alkaline copper solutions react with peptide bonds in protein to produce a violet color whose intensity at 560nm, is directly proportional to the amount of protein present.

Statistical Analysis of Data: All values are quoted as the mean ± SD. The values of the various parameters for the various groups were analyzed for statistical significant differences using the student’s t-test, with the help of SPSS Statistics 17.0 package. P<0.05 was considered to be significant.

RESULTS:

The effect of aqueous extract of Sansevieria liberica on plasma marker enzymes of salt-loaded rats is given in Table 1. The plasma aspartate transaminase activity of Test 1 was significantly (P<0.05) lower than test control, but not significantly lower than control, reference and Test 2. There were no significant differences in the plasma alanine transaminase activities of all the groups. The alkaline phosphatase activity of Test 2 was significantly (P<0.05) higher than test control, reference and Test 1, but not different from control.

Table 1 shows the effects of aqueous extract of Sansevieria liberica on plasma chemistry of salt-loaded rats. The plasma creatinine concentration of Test 1 was significantly (P<0.05) lower than control, but not significantly different from test control, reference and Test 2. There were no significant differences in the plasma urea, total protein, total bilirubin and unconjugated bilirubin levels, and unconjugated/conjugated bilirubin ratios of all the groups. The plasma conjugated bilirubin level of test control was significantly (P<0.05) higher than control and reference, but not significantly higher than the test groups. The plasma albumin level of Test 1 was significantly (P<0.05) lower than Test 2, but not significantly lower than control, test control and reference.

Table 2 shows the effect of aqueous extract of Sansevieria liberica on plasma electrolyte levels of salt-loaded rats. There were no significant differences in the plasma bicarbonate concentrations of all the groups. The plasma calcium levels of the test groups were significantly (P<0.05) higher than test control and reference, but not significantly higher than control. The plasma chloride concentrations of the test groups were significantly lower than test control and reference, but not significantly higher than control. The plasma potassium levels of the test groups were significantly (P<0.05) higher than test control and reference, but not significantly lower than control. The plasma sodium concentrations of the test groups were significantly (P<0.05) lower than test control and reference, but not different from control (except Test 2 that was significantly higher than control).

Table 1: Effect of aqueous extract of Sansevieria liberica on plasma marker enzymes of salt-loaded rats

Enzymes

Activity (U/L)

Control

Test control

Reference

Test 1

Test 2

Aspartate transaminase

Alanine transaminase

Alkaline phosphatase

56.42±35.77^a,b

14.61±6.66^a

10.76±7.31^a,b

90.16±25.16^a

21.23±11.06^a

8.05±3.54^a

48.63±28.31^a,b

23.51±8.72^a

6.44±2.68^a

41.25±15.91^b

21.17±13.80^a

6.56±2.61^a

56.07±34.72^a,b

23.16±9.21^a

56.07±34.72^b

Values are mean ± SD, n=5, per group. Values in the same row with the different superscripts are significantly different at P<0.05.

Table 2: Effect of aqueous extract of Sansevieria liberica on plasma chemistry of salt-loaded rats

Parameter

Magnitude

Control

Test control

Reference

Test 1

Test 2

Creatinine (mg/dL)

Urea (mg/dL)

Total bilirubin (mg/dL)

Conjugated bilirubin (mg/dL)

Unconjugated bilirubin (mg/dL)

Unconjugated/conjugated bilirubin ratio

Total protein (mg/dL)

Albumin (mg/dL)

0.64±0.06^a

36.47±9.10^a

0.83±0.84^a

0.40±0.31^a

0.43±0.58^a

0.88±0.71^a

5.73±0.38^a

3.54±0.23^a,b

0.58±0.07^a,b

44.89±8.98^a

1.20±0.62^a

0.70±0.22^b

0.50±0.55^a

0.72±0.75^a

5.90±0.16^a

3.58±0.38^a,b

0.39±0.13^b

36.76±10.54^a

0.52±0.34^a

0.29±0.29^a

0.24±0.16^a

0.81±0.47^a

5.84±0.49^a

3.72±0.13^a,b

0.43±0.15^b

35.91±9.22^a

0.51±0.43^a

0.34±0.32^a,b

0.17±0.12^a

0.52±1.80^a

6.05±0.22^a

3.45±0.34^a

0.50±0.12^a,b

41.34±16.00^a

0.78±0.46^a

0.52±0.32^a,b

0.26±0.19^a

0.56±0.38^a

5.98±0.13^a

3.68±0.25^b

Values are mean ± SD, n=5, per group. Values in the same row with the different superscripts are significantly different at P<0.05.

Table 3: Effect of aqueous extract of Sansevieria liberica on plasma electrolyte levels of salt-loaded rats

Parameter

Concentration

Control

Test control

Reference

Test 1

Test 2

Bicarbonate (mmol/L)

Calcium (mmol/L)

Chloride (mmol/L)

Potassium (mmol/L)

Sodium (mmol/L)

25.10±1.43^a

2.42±0.07^a

99.70±3.70^a

4.49±0.77^a

139.40±1.14^a

24.40±1.14^a

2.16±0.05^c

107.40±1.14^b

3.48±0.25^b

149.00±2.12^c

23.60±1.14^a

2.07±0.05^d

111.40±1.14^c

3.22±0.08^c

155.20±4.15^b

24.50±1.66^a

2.47±0.13^a

102.25±1.48^a

4.25±0.21^a

134.00±16.84^a,b,c,d

24.75±0.43^a

2.57±0.16^b

102.75±1.30^a

4.175±0.238^a

143.75±0.83^d

Values are mean ± SD, n=5, per group. Values in the same row with the different superscripts are significantly different at P<0.05.

Table 4: Effect of aqueous extract of Sansevieria liberica on the hematological profile of salt-loaded rats

Parameter

Magnitude

Control

Test control

Reference

Test 1

Test 2

Packed cell volume (%)

Haemoglobin (g/dL)

Haemoglobin (%)

Mean cell haemoglobin concentration (g/dL)

Red cell Count (x10¹² cells/L)

Mean cell haemoglobin (pg/cell)

Mean cell volume (fL)

White blood cell count

a) Total (x10⁹ cells/L)

b) Differential (%)

i. Basophils

ii. Eosinophils

iii. Lymphocytes

iv. Monocytes

v. Neutrophils

39.50±3.74^a

13.28±1.37^a

91.00±9.38^a

33.51±0.59^a

4.78±0.51^a

28.62±2.95^a

85.41±8.80^a

5.54±0.84^a

0.10±0.22^a

1.10±0.89^a

44.40±5.18^a

3.50±3.35^a

50.90±4.90^a

35.40±3.36^a

12.00±1.04^a

82.00±7.18^a

33.88±0.70^a

3.96±0.50^b

30.43±1.50^a

89.70±4.37^a

4.96±0.71^a,c,d

0.00±0.00^a

0.80±1.30^a

47.20±5.36^a

3.00±2.35^a

49.00±5.66^a

35.00±6.56^a

11.78±2.13^a

80.60±14.38^a

33.92±0.65^a

4.06±0.90^a,b

29.21±1.47^a

86.14±4.39^a

4.24±0.23^d

0.00±0.00^a

0.80±0.84^a

47.80±8.04^a

2.20±1.48^a

48.80±8.90^a

41.20±3.70^a

13.50±1.47^a

92.60±9.71^a

32.73±0.73^a

4.76±0.55^a,b

28.39±0.98^a

86.84±4.09^a

6.74±1.12^b,c

0.00±0.00^a

1.60±1.14^a

47.80±6.57^a

1.00±1.23^a

50.20±5.40^a

38.00±5.43^a

12.62±1.82^a

86.40±12.38^a

33.42±0.57^a

4.54±0.78^a,b

29.13±1.98^a

87.15±6.85^a

7.18±0.98^b

0.20±0.45^a

0.60±0.89^a

46.80±4.44^a

2.00±2.83^a

50.60±5.03^a

Values are mean ± SD, n=5, per group. Values in the same row with the different superscripts are significantly different at P<0.05.

The effect of aqueous extract of Sansevieria liberica on the hematological profile of salt-loaded rats is given in Table 4. There were no significant differences in the packed cell volume, haemoglobin concentration, mean cell haemoglobin concentration, mean cell haemoglobin and mean cell volume, and basophil, eosinophil, lymphocyte, monocyte and neutrophil counts of all the groups. The red cell counts of test control were significantly (P<0.05) lower than control, but not significantly lower than reference and the test groups. The total white cell count of Test 2 was significantly (P<0.05) higher than control, test control and reference, but not significantly higher than Test 1: Test 1 was significantly (P<0.05) higher than control and reference, but not significantly higher than test control.

DISCUSSION:

The extract had no negative effects on the liver and kidney functions of the test animals. The concentration of Ca²⁺ in body fluids and its handling by cellular proteins are disturbed in patients and experimental animals with arterial hypertension^12,13. In the present study, the extract reversed the salt-loading induced lowering of plasma calcium levels. The extract may have achieved the present effect by affecting parathyroid hormone secretion. The parathyroid hormone increases the renal tubular reabsorption of calcium, promotes intestinal calcium absorption by stimulating the renal production of 1,25-dihydroxyvitamin D, and, if necessary, resorbs bone^14,15. Calcium fluxes are involved in neuromuscular activities and biosignaling^15,16. The increased plasma calcium may impart greatly on the tones of the arterial muscles, since according to Murray¹⁷, cardiac muscles rely on extracellular Ca²⁺ for contraction. There is a direct relationship between myogenic tone in isolated arteries and BP in intact animals.¹⁸ Thus, the mechanism of the antihypertensive action of the extract may be regulation of muscle tone, through increases in plasma calcium concentration, which in turn is produced by reducing its entry into the cells or increasing its removal from the cells into the extracellular space.

One of the mechanisms of action of antihypertensive drugs, especially the diuretics, is reduction in plasma sodium and chloride concentrations^15,19. These diuretics lower plasma levels of these electrolytes by reducing their reabsorption at different sites in the nephrons. Prominent amongst these, are the potassium-sparing diuretics, that inhibit either aldosterone directly, or the Na⁺/K⁺ exchange mechanisms in the distal tubules and collecting ducts^15,19. The effect is the loss of sodium in the urine and the retention of potassium in the blood, leading to lowered plasma sodium and increased plasma potassium levels. In this study, the leaf extract produced a low plasma sodium level and increased plasma potassium levels. This suggests that it may be a potassium-sparing diuretic and may contain a β-antagonist. It also prevented the salt-loading induced alkalosis.

The extract had a positive effect on the haemopoietic system of the test rats. It increased red cell mass, but had no significant effect on the haematocrit and haemoglobin concentration. Drug poisoning is one of the main causes of raised white blood cell count. According to some experimental and pathological studies, white blood cells play important roles in the destabilization of coronary artery plaques at the onset of acute coronary syndrome^20-22. However, an elevated white blood cell count in peripheral blood is a known risk factor of coronary artery disease²³. Thus, the observed higher white blood cell count in the test rats, in this study, has two implications. First, protection against the onset of acute coronary syndrome, and secondly, increased risk of coronary artery disease. The increased white cell count may have been produced by the immunostimulatory activity of saponins^24,25, that had earlier been reported to be abundantly present in the leaves of Sansevieria liberica²⁶.

In conclusion, this result suggests that the extract may be a diuretic that causes leukocytosis, without altering liver and kidney functions, at least at the doses at which it was administered in this study. It also supports the use of Sansevieria liberica in the management of hypertension, and in addition, suggests that its antihypertensive action may be mediated via alteration of plasma sodium and potassium levels or increases in muscle tone brought about by changes in plasma calcium levels.

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26. Ikewuchi CC, Ikewuchi CJ, Ayalogu OE and Onyeike NE Proximate and Phytochemical Profile of Sansevieria liberica Gérôme and Labroy. J Appl Sci Environ Manage. 2010; 14(2): 103 – 106.

Received on 30.08.2010

Accepted on 20.09.2010

Research J. Science and Tech. 2(5): Sept –Oct. 2010: 110-114

KEY WORDS: Hematological profile, plasma chemistry, plasma electrolyte, Sansevieria liberica.