AQUEOUS EXTRACTS OF VERNONIA AMYGDALINA AND OCIMUM GRATISSIMUM PROTECT AGAINST ELECTROLYTE DERANGEMENT IN SALT-LOADED RATS

AQUEOUS EXTRACTS OF VERNONIA AMYGDALINA AND OCIMUM GRATISSIMUM PROTECT AGAINST ELECTROLYTE DERANGEMENT IN SALT-LOADED RATS

Ugochukwu Vincent Igbokwe ¹, Ejike Daniel Eze ^{* 2}, Moses Dele Adams ³ and Chidimma Felicia Chukwuegbo ¹

Department of Physiology ¹, Faculty of Basic Medical Sciences, College of Health Sciences, Nnamdi Azikiwe University, Nnewi Campus, Anambra State, Nigeria.

Department of Physiology ², Faculty of Biomedical Sciences, Kampala International University, Western Campus, P. O. Box 71, Ishaka, Bushenyi, Uganda.

Department of Biochemistry ³, Faculty of Computing and Applied Sciences, Baze University, Abuja, Nigeria.

ABSTRACT: The study aimed at checking the effects of aqueous extracts of Vernonia amygdalina and Ocimum gratissimum on the electrolyte level of salt-loaded rats. 25 male rats weighing 160-220g were shared into 5 groups of 5 rats. Group 1 animals that were fed standard feed and water served as the control. Animals in group 2-5 which were salt-loaded orally with 2 ml of 4% sodium chloride solution for 2 weeks, were left untreated, treated with 1 ml of 300 mg/kg body weight of aqueous bitter leaf extract, 300 mg/kg body weight of aqueous extract of scent leaf and 300 mg/kg body weight of both extracts in ratio 1:1 respectively. Treatment of salt-loaded animals with the extract was done orally once daily for two weeks after which plasma electrolyte levels were determined. There was significant (P<0.05) increase in Na⁺ and Cl⁻ levels in all the salt-loaded groups when compared with the control group. Bitter leaf extracts significantly (P<0.05) decreased the sodium level only when compared to the group with no treatment while scent leaf extract did not affect (P>0.05) on any of the electrolytes. Treatment with both extracts reduced Na⁺ and Cl⁻ levels significantly (P<0.05). Individual and co-treatment with bitter leaf and scent had no change (P>0.05) in the levels of K⁺ and HCO₃⁻. The co-administration of aqueous extract of Vernonia amygdalina and Ocimum gratissimum has synergistic effect that might be of importance in reduction of blood electrolytes of sodium and chloride in salt-induced derangements. This might be useful in managing salt-induced hypertension.

Vernonia amygdalina, Ocimum gratissimum, Asteraceae, Labiatae, Electrolyte, Salt-induced hypertension

INTRODUCTION: Globally, according to the World Health Report, hypertension is one of the heart killer illnesses, and this is so especially in rural and developing communities where there are gradual changes in lifestyles to those of more developed and urban societies.

Diet poses as the major cause of hypertension in the present-day population, ranging from high fatty foods to high salt intake. Throughout evolution, the human race only consumed sodium and chloride naturally present in food and the daily intake was about 10 mmol/day ¹.

Not less than 5000 years ago, salt began to be added to food, and the present intake has increased between 100-400 mmol/day ². Dietary salt appears to be an important single factor in raising the blood pressure ³. Excess dietary salt has harmful effects, which include increasing the mass of the left ventricle, thickening and stiffening conduit arteries and resistance arteries, including the coronary and renal arteries. It also increases the number of stroke incidence, the severity of cardiac failure and the tendency of platelet aggregation. Salt-induced increase in blood pressure has often been observed to occur within a period of several days to weeks, a time course approximately paralleling the re-establishment of electrolyte balance.

Electrolytes are naturally occurring elements and compounds in the body. They are any substance that produces an electrically conducting solution when dissolved in water. Electrolytes include sodium (Na⁺), potassium (K⁺), calcium (Ca²⁺), bicarbonate (HCO³⁻), magnesium (Mg²⁺), chloride (C1⁻), and hydrogen phosphate (HPO₄²⁻). They control important physiologic functions ⁴. These substances are present in the blood, body fluids, and urine and are gotten from food, drinks, and supplements. Excess salt-intake raises the level of electrolytes in the bloodstream and wrecks the delicate fluid balance between the extracellular and intracellular fluids. Electrolytes have been demonstrated by some studies to have an inverse relationship with renal function and cardiovascular events ⁵. Electrolyte imbalance due to kidney failure alters the normal physiological functions by altering the level of body fluids and blood thereby increasing the blood pressure; this is directly connected to the kidneys which have a big role to play in the regulation and maintenance of the body electrolytes. The kidneys remove wastes, control the body's fluid balance, and keep the right levels of electrolytes thereby maintaining a constant acid-base balance. Long-time electrolyte imbalance thickens and narrow walls of arteries, damage the kidneys giving rise to kidney diseases, cause stroke, etc which are precursors in the development of hypertension.

Hypertension is defined by a systolic blood pressure that is ≥ 140mmHg and a diastolic blood pressure that is ≥ 90mmHg ⁶. High blood pressure is the greatest cause of strokes and heart failure and also a major contributor to coronary heart disease. Essential hypertension e.g. salt-induced hypertension also results from a complex interaction of genes and environmental factors such as diet, (high fatty foods intake), age, physical changes, etc. Another type of hypertension known as secondary hypertension results from identifiable causes like kidney diseases, endocrine abnormalities, obesity, apnoea, pregnancy, excessive alcohol intake, etc. A dispute which lasted about 100 years over the evidence that suggested that hypertension is in part due to the present high intake of salt is now resolved ⁷. Hypertension is diagnosed using a device called sphygmomanometer whose cuff is wound around the upper part of the arm above the elbow.

In view of the importance of a healthy heart to the body and the essence of the body homeostatic mechanisms, there is a need to explore available natural products ⁸. A medicinal plant is any plant which, in one or more of its organs, contains substances that can be used for the therapeutic purposes or which are precursors for the synthesis of useful drug and the main therapeutic activity depends upon the plant or fungal metabolites which it contains ⁹.

Vernonia amygdalina, a member of the Asteraceae family, is a shrub or small tree of 2–5m with a petiolate leaf of about 6 mm diameter and elliptic shape. The leaves are green with a characteristic odor and a bitter taste. No seeds are produced and the tree is propagated through cutting. It is known in Nigerian local languages as etidot (Efik), uzi (Ebira), onugbu (Igbo), ewuro (Yoruba) and chusar duki (Hausa). Elsewhere in Africa, it is called muop or ndole (Cameroon), tuntwano (Tanzania) and mululuza (Uganda) ¹⁰. The bitter taste is due to antinutritional factors such as alkaloids, saponins, tannins, and glycosides. The leaf of Vernonia amygdalina extract is reported to possess a number of chemotherapeutic potentials ^{11, 12}. Other activities include antidiabetic ^13-16, antioxidant ^{17- 19}, anticancer ²⁰, hepatoprotective ²¹, cardioprotective ⁸, antimalarial ²², antihyperglycaemic ²³, anti-hypercholesterolemia and antialbuminemia ²⁴, antimicrobial ²⁵, toxicity ²⁶, hepatoprotective ²⁷, antihyperlipidaemic ¹⁵, hematological effect  ²⁸ as well as amelioration of electrolyte and renal disorders ²⁹.

Ocimum gratissimum is an herbaceous shrub that belongs to the Labiatae family. In Nigeria, the Nupe tribe calls it Tan-motsungi-wawagi, Ebira: Ireru; in the southern part of Nigeria, the plant is called “effinrin-nla” by the Yoruba speaking tribe. It is called “Ahuji or Nchanwu” by the Igbos, while in the Northern part of Nigeria, the Hausas call it “Daidoya” ³⁰. The plant contains alkaloids, tannins, phytates, flavonoids, and oligosaccharides ³¹. In Nigeria, it is used in treatment of epilepsy, diarrhea, mental illness and fever ³². Other reported effects are antihyperglycemic ^{33, 34, 35, 36}, anti-hyperlipidemic ^{37, 38}, renal function ³⁹, anti-microbial ⁴⁰, antidiabetic ^{41, 42}, antioxidant ^{43, 44}, toxicity ³⁰, anti-diarrheal ⁴⁵ and ameliorate diabetic disorders ⁴⁶.

The combination of Vernonia amygdalina and Ocimum gratissimum has been reported to possess antidiabetic ⁴⁷, cardioprotective ⁴⁸, kidney restorative ^{49, 50} as well as angiotensin-converting enzyme inhibitory, hypolipidemic and antioxidant properties ⁵¹ without possible effect of same combination on electrolyte level in hypertensive rats. Therefore, this work seeks to study the effect of aqueous extracts of both plants on electrolyte level of salt-loaded male Wistar rats.

MATERIALS AND METHODS:

Materials:

Plant materials and Authentication: The fresh species of Vernonia amygdalina and Ocimum gratissimum which were obtained from the Ose Market, Onitsha, Anambra State, Nigeria, were identified and authenticated at the Forestry Research Institute of Nigeria, Ibadan. Voucher Specimen Number of F101863 for V. amygdalina and 110026 for O. gratissimum was assigned.

Experimental Animals: Male rats weighing (160-220g) were purchased from a commercial supplier. The rats were housed in well-ventilated cages in the animal house of the College. The animals were allowed free access to feed (growers fighter feed) and clean water according to the Barthold’s guidelines of the National Research Council (US) Committee for the Update on the Guide for the Care and Use of Laboratory Animals ⁵². The animals were acclimatized to the housing and feeding conditions for fourteen days.

Methods:

Ethical Approval: Ethical approval number was allocated under the Animal Research Ethics Review Committee of the Faculty of Basic Medical Sciences, College of Health Sciences, Nnamdi Azikiwe University, Nnewi Campus, Anambra State, Nigeria (NAUTH/CS/66/Vol. 2/149) in a letter dated September 15, 2017. This is in conformity with guidelines that are in compliance with National and International Laws and Guidelines for Care and Use of Laboratory Animals in Biomedical Research.

Preparation and Administration of Sodium Chloride Solution: 4 g of sodium chloride was dissolved in 100 ml of distilled water to obtain a concentration of 4% sodium chloride. 2 ml of the 4% sodium chloride solution was orally administered by means of calibrated syringe with attached cannula to the male Wistar rats once a day for two weeks to cause elevated electrolyte levels.

Preparation of Aqueous Extracts of Verononia amygdalina and Ocimum gratissimum: Large quantities of the fresh specimens of Verononia amygdalina and Ocimum gratissimum were washed free of soil and debris. The leaves were plucked from the stems and air-dried for three (3) weeks. The dried leaves were milled to powder using a mechanical grinder and kept in airtight containers. Vernonia amygdalina and Ocimum gratissimum leaf powders (100 g) each was soaked in 1 liter of distilled water. The mixture was allowed to stand for 48 hours with intermittent stirring. Following filtration, the filtrate was stored in a refrigerator after which it is subjected to a lyophilizer yielding the extract which was reconstituted to give 300 mg/kg body weight. The same procedure was adopted to obtain extract individual extract of Verononia amygdalina and Ocimum gratissimum.

Experimental Design: Twenty-five male Wistar rats were divided into five (5) groups of five rats.

Group A (control) was fed with only feed and water throughout the duration of the experiment.

Group B (negative control) induced with 4% sodium chloride solution for two weeks and was left untreated until the end of the experiment.

Group C received 4% sodium chloride solution for two weeks. Then after, they were treated with 300 mg/kg/day of aqueous extract of Occimum gratissimum for two weeks.

Group D received 4% sodium chloride solution for two weeks and then treated with 300 mg/kg/day of aqueous extract of V. amygdalina for two weeks.

Group E received 4% sodium chloride solution for two weeks and treated with 300mg/kg/day in ratio 1:1 of combined aqueous extract of O. gratissimum and Verononia amygdalina for two weeks.

Blood Collection: The rats were placed in a closed cylinder containing a large ball of cotton soaked with chloroform and allowed to stand for a minute for them to lose consciousness. Syringes were then used to collect 2 ml of blood via cardiac puncture into plain bottles and centrifuged at 300 rpm for 10 minutes. The plasma was then collected and kept at 20 ºC until analysis.

Measurement of Electrolyte Level:

Sodium and Potassium Analysis using Flame Emission Spectrometry:

Requirement: Serum sodium and serum potassium were measured against a standard with 140 mmol/L sodium and potassium 5 mmol/L. Stock potassium standard (1.OM) 58.45% NaCl was dissolved in distilled water and made up to 1 liter. Stock potassium standard (1.OM) 74.55% KCl was dissolved in distilled water and made up to 1litre.

Method: The solution was mixed well, and the air compressor was switched on, the air pressure was adjusted, then deionized water was introduced through the auto-miser and the gas turned on, the flame was adjusted to give fine sharp cones as described by ⁵³. Then appropriate filters were placed for simultaneous sodium and potassium estimation, after which the machine was set at zero with deionized water, then the standard (120/2) adjusted 120.0 for sodium, and 2.0 for potassium were introduced. Then the display of the standard was checked, which showed the exact concentration for sodium and potassium, after which dilute test serum was introduced the readings for sodium and potassium were noted.

Plasma Chloride using Coulometry Method: Reagents Used: diluting fluid, glacial acetic acid (100ml), concentrated nitric acid (6.4ml), deionized water (1 litre).

Solution: Unflavoured gelatin (6.0g), Thymol blue (0.1g), Thymol (0.1g).

Method: 0.1ml of plasma with 10ml of diluting fluid was put in a beaker, and 2-3 drops of the indicator solution were added as described by ⁵⁴. Then the electrolyte was immersed. The machine was switched on, and the digital display started. The concentration of chloride in mmol/L was indicated when the digital display stopped finally, the procedure was repeated with 0.1ml of standard to confirm the accuracy of the result.

Plasma Bicarbonate Test: Reagents used: Saline (1%), dissolved 1g of NaCl in 100mls of distilled water, HCl, 0.01N:

Method: The method of ⁵⁵ was adopted. Briefly, 0.1ml of plasma bicarbonate was added to 1.5 ml of 1% saline and 2 drops of phenol red indicator and they were mixed well.4.0 ml of 1% saline was put in another tube and then 1.0 ml of 0.1N HCl, was added.0.1ml of plasma bicarbonate was added to 2 drops of phenol red indicator and titrated against 0.01N NaOH till the color changed from yellow to red.

Statistical Analysis: Data analysis was done using the SPSS (Version 20.0) software. The results were expressed as mean value ± SEM of five determinations. Differences between mean and the main effects of treatment group were determined by the one-way analysis of variance (ANOVA) with Tukey’s posthoc test. The values of P<0.05 was considered statistically significant.

RESULTS:

Acute Toxicity for both Extract: The aqueous leaf extract of Verononia amygdalina and Ocimum gratissium have median lethal dose of 288.5mg/kg body weight in rats when administered orally.

TABLE 1: ELECTROLYTE LEVELS OF THE DIFFERENT TEST GROUPS (GROUP 2-5) AND THE CONTROL GROUP (GROUP 1) AFTER SALT LOADING

Result is significant at p=<0.05

From Table 1, there was a significant (P<0.05) increase in sodium ion level in groups 2, 4, and 5 when compared with group 1 after salt-loading except in group 3. Significant (P<0.05) increase was noticed in chloride ion level in all the salt-loaded groups (2, 3, 4, and 5) and increase in bicarbonate ion level in group 2 only and none for the other groups. There were no statistically significant (P>0.05) changes in potassium ion levels in all the salt-loaded groups.

TABLE 2: ELECTROLYTE LEVELS OF THE DIFFERENT TEST GROUPS (GROUP 2-5) AND CONTROL GROUP (GROUP 1) AFTER ADMINISTRATION OF EXTRACTS

Result is significant at p=<0.05

From this table, it is shown that there was a significant (P<0.05) decrease in sodium ion, chloride ion, and bicarbonate ion levels and none for potassium in the untreated group when compared with the positive control. Significant (P<0.05) decrease occurred only in chloride ion level in group administered with bitter leaf and no change (P>0.05) in sodium ion, bicarbonate ion, and potassium ion. Also there was a significant (P<0.05) decrease in sodium ion and chloride ion and none for potassium ion and bicarbonate ion in groups treated with scent leaf. The group treated with both extracts showed a decrease (P<0.05) in chloride ion level only.

TABLE 3: ELECTROLYTE LEVELS OF THE TREATED GROUPS (GROUP 3-5) AND UNTREATED GROUP (GROUP 2) AFTER ADMINISTRATION OF EXTRACTS

Result is significant at p=<0.05

From the table above, the result showed a significant (P<0.05) decrease in sodium ion level in group treated with bitter leaf extract. No Significant (P>0.05) changes occurred for the other electrolyte levels of the other groups.

TABLE 4: ELECTROLYTE LEVELS OF TEST GROUPS (GROUP 2-5) BEFORE AND AFTER ADMINISTRATION OF EXTRACT

Result is significant at p=<0.05

From the table, the result shows that a significant (P<0.05) decrease occurred in sodium ion and chloride ion levels in the group treated with both extracts only. Results also show no significant (P>0.05) change for bicarbonate ion level in all the test groups, but there was a significant (P<0.05) decrease in potassium ion in the untreated group.

DISCUSSION: Studies have indicated a positive association between dietary salt intake, level of BP and prevalence of hypertension in humans and animals ⁷. High salt intake raises the blood pressure by causing the kidney to retain sodium and water thereby increasing the blood volume. This study investigated the effect of aqueous extracts of Ocimum gratissium and Vernonia amygdalina on electrolyte level of salt-loaded male Wistar rats.

Electrolytes regulate the nerve, muscle function, body hydration, blood pressure and rebuilding of damaged tissues ⁵⁶. Nerves, heart, muscles all use electrolytes to maintain voltages across their cell membranes in order to carry electrical impulses to other cells. Electrolyte imbalance can, therefore, result in blood pressure alterations ⁵⁶. Sodium-ion is the major cation of extracellular fluids ⁵⁷. The rate of Na⁺ excretion by the kidney is related to the glomerular filtration rate (GFR). When the GFR falls, less Na⁺ is excreted and vice versa ⁵⁸. Potassium ions play an important role in the way in which nerve impulses are propagated along the nerve cells and transmitted to receptor cells ⁵⁹. The significant increase in sodium and chloride ion levels in group 2, 4 and 5 when compared to group 1 after salt-loading except in group 3 which showed no increase following administration of 2 ml of 4% of sodium chloride solution once per day for two weeks comes as no surprise and could be as a result of retention and sodium overload culminating in elevated sodium levels, as variable increase in salt-intake from 1-8% for 1-16 weeks increases the blood volume ⁶⁰. The observed significant increase in sodium and chloride ion level after salt-loading with NaCl may also imply that it is possible NaCl causes water retention which releases a digitalis-like substance that increases the contractile activity of the heart and blood vessels. It may also be that sodium itself penetrates the vascular smooth muscle cells causing it to contract ⁶¹.

The absence of increase in the sodium level in group 3 is suggested to be a result of the degree of salt-sensitivity which varies widely among individuals ⁶². According to ⁶³, high salt-intake increases the ECF volume which in turn causes an increase in blood volume. This increases the mean circulatory filling pressure and venous return of blood to the heart resulting in an increase in cardiac output and arterial blood pressure.

Chloride ion is essential for water balance, regulation of osmotic pressure and acid-base balance. Since Na⁺ and Cl⁻ act almost parallel, abnormalities of sodium metabolism are generally accompanied by abnormalities in chloride metabolism ⁵⁸. Serum bicarbonate is a measure of the base that remains after all acids, stronger than carbonic acid, have been neutralized. It represents the reserve of alkali available for the neutralization of such strong acids, and it has been termed the alkali reserve. The rise in blood HCO₃⁻ is compensated by increased renal excretion of HCO₃^{− 64}. Chloride, potassium, and bicarbonate showed no significant changes after salt-loading as a way of compensating for the increase in sodium and chloride levels (salt retention by the kidneys). Observational studies that demonstrated there is an inverse relationship between potassium intake and BP ⁶⁵ were not observed in this study. Recent meta-analyses indicated that adequate intake of minerals, e.g. potassium and probably calcium, rather than restriction of sodium, should be the focus of dietary recommendations in salt-induced hypertension.

Limitation of sodium chloride in food has historically been considered the critical change for reducing blood volume that might lead to hypertension. The reduction in the serum levels of sodium and chloride ions after the two weeks of salt withdrawal correlated with the study of the three meta-analyses by ^{60, 66, 67} which revealed that a reduction in sodium intake over periods ranging from few days to years lowers blood pressure.

Treatment with Vernonia amygdalina showed a significant decrease in serum level of sodium ion in group 3 without a resultant change in chloride, potassium or bicarbonate levels. Group 4 treated with Ocimum gratissimum had no significant decrease in all the electrolyte levels. There was a significant decrease in the sodium and chloride levels in the group treated with both extracts. This might be a result of their phytochemical components which chemically triggered the reduction in the serum levels of sodium and chloride ion, thus are regarded as been synergistic.

In summary, the result of this study showed that taking Vernonia amygdalina alone has a positive role to play in reduction of sodium ion levels in any case of electrolyte imbalances ^{68, 69}. Also that combining both leaf extracts of Vernonia amygdalina and Ocimum gratissimum can be beneficial in management and treatment of salt-induced hypertension because of their impact in reducing the plasma levels of sodium and chloride ions which are the major contributor to salt-induced elevation of blood pressure ^{4, 70}.

CONCLUSION: This study has thus demonstrated that the combination of the aqueous leaf extracts of Vernonia amygdalina and Ocimum gratissimum has synergistic effect that might be of importance in reduction of blood electrolytes of sodium and chloride in salt-induced derangements. This might be of use in the management of salt-Induced hypertension. However, further studies are required to be carried out to explain the mechanism of action of the leaf extracts.

ACKNOWLEDGEMENT: The authors of this work wish to acknowledge the technical assistance of the chief technologist and other laboratory assistants of the Department of Physiology, Faculty of Basic Medical Sciences, Nnamdi Azikiwe University, Nnewi Campus, Anambra State, Nigeria, towards ensuring that this research study was completed in good record time. Indeed, we are sincerely grateful.

CONFLICTS OF INTEREST: The authors have not declared any conflict of interest.

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    How to cite this article:

    Igbokwe UV, Eze ED, Adams MD and Chukwuegbo CF: Aqueous extracts of Vernonia amygdalina and Ocimum gratissimum protect against electrolyte derangement in salt-loaded rats. Int J Pharm Sci & Res 2020; 11(4): 1889-97. doi: 10.13040/IJPSR.0975-8232.11(4).1889-97.

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