INTRODUCTION:

Blood, one of the main homeostatic systems of the body, supports normal viability, integrity, and adaptive responses¹, with the functional state of its systems changing dynamically in accordance to the nature, strength, and duration of exposure to internal (e.g. disease) and external (e.g. stress, toxins, drugs, etc.) factors. Adverse alterations in hematological, electrolytes and other biochemical parameters have been associated with hypertension^2-6. Such alterations include increases in hematocrit⁷, plasma activities of alanine and aspartate transaminases⁸, and plasma concentrations of sodium^2,6,9 and chloride¹⁰; as well as reductions in plasma calcium^3,11,12 and potassium^2,10.

Abnormal hematological findings such as low hemoglobin¹³, low¹³ or high⁵ hematocrit, and elevated white blood cells count¹⁴; and increases in plasma sodium⁶ are associated with higher risks of mortality and morbidity. In fact, increased hematocrit and hemoglobin are independent risk factors for ischemic heart diseases and stroke⁵. In addition, red cell distribution width, a novel predictor of mortality, often reported as part of complete blood count in routine clinical practice, is a measure of variability in size of the erythrocytes in circulation¹⁵.

Several antihypertensive drugs have been reported to reverse (at varying degrees), the adverse changes accompanying hypertension^16,17, and substantially reduce the risk of hypertension-related morbidity and mortality¹⁸. Natural products (especially those derived from plants) have been a very rich source of new medicines, and continue to provide a great variety of structural templates for drug discovery and development¹⁹. Plant-derived substances have recently become of great interest due to their versatility²⁰, and relative cheapness. Among the great number of plants currently used by traditional health care practitioners in the management of hypertension is Chromolaena odorata (family Asteraceae), a plant found throughout the tropics²¹, and commonly called siam weed²². Consequent upon the use of this plant in traditional health care as an antihypertensive and diuretic, Ikewuchi et al.²³ investigated the hypotensive effect of aqueous extract of the leaves. However, due to the paucity of information in the literature regarding its mechanism of antihypertensive action and the effect of administration of the extract on the plasma chemistry and hematological indices of the hypertensive, the present study was undertaken to investigate the effect of aqueous extract of the leaves on the plasma chemistry and hematological indices of normal and sub-chronic salt-loaded Wistar rats.

MATERIALS AND METHODS:

Preparation of plant extract:

Samples of fresh Chromolaena odorata were collected from within the Abuja campus of the University of Port Harcourt, Nigeria. The identity was confirmed by Dr. Michael C. Dike of Taxonomy Unit, Department of Forestry and Environmental Management, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria; and Mr. John Ibe, the Herbarium Manager of the Forestry Department, National Root Crops Research Institute, Umuahia, Nigeria. The leaves were removed, cleaned of soil, oven dried at 55 °C and ground into powder. The powder was soaked in hot, boiled distilled water for 12 h, after which the resultant mixture was filtered and the filtrate was stored in the refrigerator for subsequent use. A known volume of this extract was evaporated to dryness, and the weight of the residue was used to compute its concentration, which was in turn used to determine the dose of administration of the extract. The resultant residue of the crude aqueous extract was used for the phytochemical study.

Determination of the phytochemical profile of the aqueous extract:

General procedures:

Gas chromatography was carried out at Multi Environmental Management Consultants Limited, Igbe Road, Ikorodu, Lagos, with a Hewllet-Packard HP6890, gas chromatograph apparatus, coupled to a flame ionization detector, and powered with HP Chemstation Rev A 09.01 [1206] software, to identify and quantify compounds. Standard solutions were prepared in methanol (for alkaloids, allicins and benzoic acid derivatives), acetone (for lignans), methylene chloride (for terpenes) and ethanol (for glycosides and saponins). The linearity of the dependence of response on concentration was verified by regression analysis. Identification was based on comparison of retention times and spectral data with standards. Quantification was performed by establishing calibration curves for each compound determined, using the standards.

Determination of benzoic acid derivatives’ profile:

The extract was obtained as reported by Ndoumou et al.²⁴, and subjected to gas chromatography on a capillary HP 1 column (30 m × 0.25 mm × 0.25 μm film thickness). The inlet and detection temperatures were 250 and 320 °C. Split injection (split ratio of 20:1) was adopted. The carrier gas was nitrogen, at a pressure of 2.11 kg cm^-2. The hydrogen and compressed air pressures were 1.97 and 2.25 kg cm^-2. The oven was programmed initially at 60 °C for 5 min, then ramped at 15 °C min^-1 for 15 min, maintained for 1 min, and again ramped at 10 °C min^-1 for 4 min.

Determination of the terpenes profile:

The extract was obtained as reported by Ortan et al.²⁵, and subjected to gas chromatography on a capillary HP 5MS column (30 m × 0.25 mm × 0.25 μm film thickness). The inlet and detection temperatures were 150 and 300 °C. Split injection (split ratio of 20:1) was adopted. The carrier gas was hydrogen, at a flow rate of 1.0 mL min^-1. The hydrogen and compressed air pressures were 1.55 and 1.97 kg cm^-2. The oven was programmed initially at 40 °C, ramped at 5 °C min^-1 to 200 °C, and ran at 200 °C for 2 min.

Determination of the allicins profile:

The extract was obtained as reported by Roy et al.²⁶, and subjected to gas chromatography on a capillary DB-5MS column (30 m × 0.32 mm × 0.25 μm film thickness). Injector and detector temperatures were 220 ºC and 250 ºC. Split injection (split ratio of 20:1) was adopted. The carrier gas was helium, at a flow rate of 1.0 mL min^-1. The hydrogen and compressed air pressures were 1.55 and 1.97 kg cm^-2. The column was held initially at 110 °C for 2 min and then increased by 5 °C per min up to 280 °C.

Determination of the lignans profile:

The extract was obtained as reported by Chapman et al.²⁷, and subjected to gas chromatography on a ZP-5 column (30 m × 0.32 mm × 0.25 μm film thickness). One microliter of sample was injected. The initial oven temperature was 40 °C, while the injector and transfer line temperatures were 250 and 280 °C. A solvent delay of 2.00 min was followed by ramping at 10 °C min^-1 to a final temperature of 230 °C and held for 1.00 min.

Determination of the saponins profile:

The extract was obtained as reported by Guo et al.²⁸, and subjected to gas chromatography on a capillary DB-225MS column (30 m × 0.25 mm × 0.25 μm film thickness). The inlet and detection temperatures were 250 and 320 °C. Split injection (split ratio of 20:1) was adopted. The carrier gas was nitrogen. The hydrogen and compressed air pressures were 1.97 and 2.81 kg cm^‑2. The oven was programmed initially at 60 °C for 5 min, ramped at 12 °C min^-1 for 18 min, before ramping again at 15 °C min^-1 for 5 min.

Determination of the glycosides profile:

The extract was obtained as reported by Oluwaniyi and Ibiyemi²⁹, and subjected to gas chromatography on a capillary DB-225MS column (30 m × 0.25 mm × 0.25 μm film thickness). The inlet and detection temperatures were 250 and 320 °C. Split injection (split ratio of 20:1) was adopted. The carrier gas was nitrogen. The hydrogen and compressed air pressures were 1.97 and 2.81 kg cm^-2. The oven was programmed initially at 60 °C for 5 min, ramped at 12 °C/min for 18 min, before ramping again at 15 °C min^-1 for 5 min.

Determination of alkaloid profile:

The extract was obtained as reported by Tram et al.³⁰, and subjected to gas chromatography on a capillary DB-5MS column (30 m × 0.25 mm × 0.25 μm film thickness). The inlet and detection temperatures were 250 and 320 °C. Split injection (split ratio of 20:1) was adopted. The carrier gas was nitrogen. The hydrogen and compressed air pressures were 1.97 and 2.67 kg cm^-2. The oven was programed initially at 60 °C for 5 min, ramped at 10 °C min^-1 for 20 min, and ramped again at 15 °C min^-1 for 4 min.

Bioassay:

Experimental design for the salt-loading experiment:

Wistar albino rats (180-210 g at the start of the study) were collected from the animal house of the Department of Physiology, University of Nigeria, Enugu Campus. Studies were conducted in compliance with applicable laws and regulations for handling experimental animals. The rats were weighed and sorted into seven groups (Table 1) of five animals each, so that their average weights were approximately equal. The animals were housed in plastic cages. After a 1-week acclimatization period on guinea growers mash (Port Harcourt Flour Mills, Port Harcourt, Nigeria), they were weighed, before commencing the experiment. Hypertension was induced by giving 8% salt-loaded feed for six weeks, to the appropriate rats. The 8% salt-loaded regimen was adopted from Ikewuchi et al.³¹ and Obiefuna et al.³². At the end of six weeks, the administration of the extract was commenced, after weighing the animals. The Moditen^TM (amyloride hydrochloride-hydrochlorothiazide; product of Greenfield Pharmaceutical Co. Ltd, Jiang Su Province, China) and the extract were orally administered daily, for ten days. The dosage of administration of the extract was adopted from Ikewuchi et al.²³. The animals were allowed food and water ad libitum. At the end of the treatment period, the rats were fasted overnight and anaesthetized by exposure to chloroform. While under anesthesia, they were painlessly sacrificed and blood was collected from each rat into heparin and EDTA sample bottles. The heparin anti-coagulated blood samples were centrifuged at 3000 r/min for 10 min, after which their plasma was collected and stored for subsequent analysis, while the EDTA anti-coagulated blood samples were used for the hematological analysis.

Table 1-Experimental design for the salt-loading experiment

S/N	ID	Treatment
1	Normal	Normal feed and water
2	Test control	8% salt-loaded feed and water
3	Reference	8% salt-loaded feed and moduretic (0.1 mg kg^-1 body weight)
4	Treatment I	8% salt-loaded feed and extract (100 mg kg^-1 body weight)
5	Treatment II	8% salt-loaded feed and extract (200 mg kg‑1 body weight)
6	Treatment control I	Normal feed and extract (100 mg kg^-1 body weight)
7	Treatment control II	Normal feed and extract (200 mg kg^-1 body weight)

Determination of the plasma biochemical indices:

The plasma activities of alanine and aspartate transaminases, and concentrations of albumin, calcium, creatinine, total protein and urea were determined using Randox test kits (Randox Laboratories, Crumlin, England, UK). The activities of alanine and aspartate transaminases were respectively measured by monitoring at 546 nm, the concentrations of pyruvate and oxaloacetate hydrazones formed with 2,4-dinitrophenylhydrazine. The wavelengths for the determination of urea and creatinine were 546 nm and 482 nm. Plasma total protein was determined by the Biuret method, at 560 nm, whilst plasma albumin was determined using the bromocresol green dye binding method, at 630 nm. Plasma calcium concentration was determined by monitoring at 575 nm, the concentration of cresol phthalein complex formed. Plasma albumin ‘corrected’ calcium levels were computed with the following formula³³:

Corrected calcium (mg/dL)

= 4{measured calcium (g/L)+0.02[40-albumin (g/L)]}

Plasma sodium and potassium contents were determined by colorimetric methods using Atlas Medical test kits (ATLAS Medical, William James House, Cowley Road, Cambridge, UK); while bicarbonate and chloride were determined by titrimetric methods³⁴.

Determination of the hematological indices:

Hematological indices were determined using Medonic M16 Hematological Analyzer (Nelson Biomedical Limited., UK).

Statistical analysis of data:

All values are reported as the mean ± standard deviation (s.d.). The values of the variables were analysed for statistically significant differences using the Student’s t-test, with the help of SPSS Statistics 17.0 package (SPSS Inc., Chicago Ill). P<0.05 was assumed to be significant. Graphs were drawn using Microsoft Office Excel, 2010 software.

RESULTS:

Phytochemical profile:

Eight known benzoic acid derivatives were detected, consisting of 86.83% ferulic acid, 13.14% vanillic acid and 0.02% gallic acid (Figure 1). Twenty four known terpenes were detected, consisting of 74.26% limonene, 2.50% sabinene, 2.49% borneol acetate, 2.05% camphor, 1.94% camphene, 1.59% 1,8-cineole, 1.41% neral, 1.37% terpinen-4-ol, 1.28% borneol, 1.29% β-pinene, 1.02% geranyl acetate, 0.89% citronellol, 0.82% lupeol, 0.80% taraxeron, 0.80% β-amyrin, 0.80% neryl acetate, 0.79% α-pinene, 0.77% α-amyrin, 0.69% α-thujene, 0.67% α-terpineol, 0.61% allo ocimene, 0.51% myrcene, 0.48% nerol (geraniol) and 0.17% cis ocimene (Figure 2). Three known allicins were detected, consisting of 84.16% diallyl thiosulphinate, 12.54% methylallyl thiosulphinate and 3.30% allyl methyl thiosulphinate (Figure 3). Eight known lignans were detected consisting of 71.80% retusin, 17.23% galgravin, 5.69% dehydroabietic acid, 1.93% apigenin-4΄,7-dimethyl ether, 1.35% 2-allyl-5-ethoxy-4-methoxyphenol, 1.09% sakuranin, 0.52% epieudesmin and 0.40% (9E, 12E, 15E)-9, 12, 15-octadecatrien-1-ol (Figure 4). Four known saponins were detected, consisting of 85.80% avenacin-B1, 13.95% avenacin A-1, 0.16% avenacin B-2 and 0.09% avenacin A-2 (Figure 5). Forty two alkaloids were detected consisting of 51.02% indicine-N-oxide, 4.37% buphanidrine, 3.92% akuammidine, 3.44% undulatine, 3.17% oxoassoamine, 3.02% crinane-3α-ol, 2.11% choline, 2.01% ambelline, 1.91% cinchonidine, 1.77% trigonelline, 1.70% 6-hydroxybuphanidrine, 1.57% crinamidine, 1.56% theobromine, 1.55% augustifoline, 1.25% 6-hydroxyundulatine, 1.24% echitamine, 1.22% augustamine, 1.20% ellipcine, 1.16% cinchonine, 1.09% 6-hydroxypowelline, 0.90% dihydro-oxo-demethoxyhaemanthamine, 0.89% camptothecin, 0.82% voacangine, 0.79% theophylline, 0.66% acronycine, 0.64% sparteine, 0.59% caffeine, 0.52% nitidine, 0.56% mitraphylin, 0.49% 13-α-hydrorhombifoline, 0.47% 9-octadecenamide, 0.43% monocrotaline, 0.36% 1β,2β-epoxyambelline, 0.35% powelline, 0.33% tetrandrine, 0.26% emetine, 0.24% thalicarpin, 0.19% colchicine, 0.13% epoxy-3,7-dimethyoxycrinane-11-one, 0.08% paclitaxel, 0.02% echitammidine and 0.02% lupanine (Figure 6). Three known glycosides were detected, consisting of 99.95% arbutin, 0.03% amygdalin and 0.02% salicin (Figure 7).

The plasma content of albumin corrected calcium in the test control group was significantly lower (P<0.05) than those of treatments 1 and 2, and treatment controls 1 and 2, but not significantly different from those of normal and reference. The plasma level of chloride in the test control group was significantly lower (P<0.05) than those of treatment 2, and treatment controls 1 and 2, but not significantly different from those of normal, reference and treatment 1. The plasma level of bicarbonate in the test control group was significantly higher (P<0.05) than those of the normal, reference, treatment 2 and treatment control 2, but not significantly different from those of treatment 1 and treatment control 1.Figure 10 shows the effect of the extract on the red cell indices of normal and chronic salt-loaded rats. The hematocrit and hemoglobin concentration of the test control group were significantly lower (P<0.05) than those of normal and treatment control 2, but not significantly lower than those of reference, treatment 1 and treatment 2 and treatment control 1. The red cell count of the test control group was significantly lower (P<0.05) than those of the other groups, except the reference, which was not significantly higher. The mean cell volume of the test control group was significantly higher (P<0.05) than that of treatment control 2, but not significantly higher than those of the other groups. The mean cell hemoglobin concentration of the test control group was significantly higher (P<0.05) than those of treatment 2 and treatment control 1, but not significantly higher than those of normal, reference, treatment 1 and treatment control 2. The mean cell hemoglobin of the test control group was significantly higher (P<0.05) than those of normal, treatment 2, treatment control 1, and treatment control 2, but not significantly higher than those of reference and treatment 1.

The effect of the extract on the platelets, total and differential white cell counts of normal and chronic salt-loaded rats is shown in Figure 11. The platelets count of the test control group was significantly lower (P<0.05) than normal, treatment 1, treatment 2 and treatment control 2, significantly higher than treatment control 1, but not significantly higher than that of reference. The total white cells count of the test control group was significantly lower (P<0.05) than those of reference, treatment 2 and treatment control 1, significantly higher (P<0.05) than that treatment control 2, but not significantly lower than those of normal and treatment 1. The neutrophils count of the test control group was significantly higher (P<0.05) than those of normal, reference and treatment 2, but not significantly higher than those of treatment 1, treatment control 1 and treatment control 2. The lymphocytes count of the test control group was significantly lower (P<0.05) than those of normal, reference, treatment 1 and treatment control 2, but not significantly lower than those of treatment 2 and treatment control 1. The monocytes count of the test control group was significantly higher (P<0.05) than those of the other groups except treatment 2, which was not significantly lower.

DISCUSSION:

The extract did not have any adverse effect on the liver and kidney of the test animals. This is shown by its reduction of the plasma markers of liver and kidney functions/integrity (such as plasma activities of alanine and aspartate transaminases, concentrations of urea and creatinine). The extract also countered the lowering of plasma calcium levels induced by salt-loading. It may have done this by altering parathyroid hormone secretion which is involved in the regulation of calcium metabolism³³. Since changes in calcium fluxes underlies several intracellular signaling pathways^17,33, it may have great impact on arterial muscle tone, due to the reliance of cardiac muscle on extracellular calcium for contraction^3,12,35. Therefore, the extract may have effected its anti-hypertensive action through changes in calcium flux-moderated alterations in muscle tone.

In this study, the extract produced low plasma sodium and increased plasma potassium levels. This effect is akin to that of the potassium-sparing diuretics, which are known to inhibit either aldosterone directly, or the Na⁺/K⁺ exchange mechanisms in the distal tubules and collecting ducts^33,36, and by so doing, induce loss of sodium in the urine and the retention of potassium in the blood, with resultant lowered plasma sodium and raised plasma potassium levels. This suggests that the extract may be a potassium-sparing diuretic and may contain a β-antagonist. The increased plasma potassium may also have resulted from decreased filtration and decreased secretion of potassium in distal tubule due to renal failure³⁷; however, the plasma urea and creatinine profiles, seems to negate the latter supposition.

The extract had a positive effect on the hemopoietic system of the test rats. It increased red cells count, without increasing the hematocrit, hemoglobin concentration and mean cell volume. Hematocrit determines blood viscosity, regulating peripheral vascular resistance and therefore, in principle, blood pressure^5,38. Therefore, that the observed increase in red cell count was not accompanied by corresponding increases in hematocrit and hemoglobin concentration could only be explained by the fact that the contribution of the increase in red cell number was nullified by the reduction in their sizes in other to reduce the effect on blood pressure, hence the non-significant effects on hematocrit and hemoglobin concentration. According to Tanindi and colleagues¹⁵, higher red cell width is strongly correlated with higher systolic and diastolic blood pressures, and vice versa. Earlier, Ikewuchi and Ikewuchi³⁹ and Ikewuchi et al.²³ had reported that the leaves and its extract are rich in iron and quercetin. These compounds may account for the ability of the extract to raise red cells count in the treated animals, since both of them are anti-anemic agents^40,41.

The raised white blood cells count observed in the treated rats in this study, has two implications. First, protection against the onset of acute coronary syndrome^42,43, and secondly, increased risk of coronary artery disease^44,45,46,47. The increased white cells count may have been produced by the immune-modulatory activity of tannic acid⁴⁸, detected in the extract. The treatment reversed the salt-loading induced thrombocytopenia. Apostol et al.⁴⁹ reported a similar anti-thrombocytopenic effect by Euphorbia hirta, and attributed the effect to presence of polyphenolic compounds. The increased platelets count produced by the extract implies, its ability to increase clotting and protect against bleeding⁵⁰.

These results suggest that the extract may be a diuretic that causes raised red cells count and leukocytosis, is anti-thrombocytopenic, and has a tonic effect on the liver and kidney, at least at the doses at which it was administered in this study.

ACKNOWLEDGEMENTS:

The authors thank Bello Atanda Akeem, the Managing Consultant of Multi Environmental Management Consultants Limited, Igbe Road, Ikorodu, Lagos, for carrying out the phytochemical analysis in his laboratory.

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*Corresponding Author E-mail: ecoli240733@yahoo.com