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Ethnodietetics of Moringa oleifera leaves amongst the ethnic groups in Bida, Niger State, Nigeria and its hypoglycaemic effects in rats.

Introduction

Plants have played a significant role in maintaining human health and improving the quality of human life for thousands of years and have served humans well as valuable components of medicines, seasonings, beverages, cosmetics and dyes (Agaie, B., 2004). Herbal medicine is based on the premise that plants contain natural substances that can promote health and alleviate illness (Newman, 2000). One such plant, Moringa oleifera Lam., (Family: Moringaceae) is a multipurpose tree, used as vegetable, spice, a source of cooking and cosmetic oil and as a medicinal plant (Fahey, 2005; Fuglie, 1999). It is known as Drumstick in English, Zogallagandi in Hausa, Zogali in Nupe, Okwe oyibo in Igbo, Ewe igbale in Yoruba (Mann, 2003; Keay et al., 1964). All parts of the Moringa tree are edible and have long been consumed by humans. A plethora of its curative power and scientific validation of these claims are available in literature (Fahey, 2005; Fuglie, 1999). World Health Organization (WHO) estimated that there will be 221 million diabetic patients worldwide in 2010 (Zimmet et al., 2001). The global increasing prevalence of diabetes is a cause for concern both in terms of associated morbidity and increasing health costs (Cohen and Shaw, 2007). Diabetes is a syndrome of disorder of glucose metabolism usually caused by inherited and or acquired deficiency in production of insulin by the pancreas, or by the ineffectiveness of the insulin produced, which result in abnormally high blood sugar (hyperglycemia) (John, 2001). Hyperglycemia is a symptom and a cause of diabetes in which there is elevated level of blood glucose in the blood stream (Akah and Okafor, 1991). Diabetes mellitus that is characterized by persistent hyperglycemia from any cause is the most prominent disease that is related to failure of blood sugar regulation (John, 2001). Diabetes had since been considered as a disease of minor significance, but it is currently considered as one of the main threats to human health in 21st century (Al-Attar, 2010). Great changes in human environment, behaviour and lifestyle resulted in the raising rates of diabetes (Zimmet et al., 2001). More than 1% of the entire world populations are victims of diabetes and their numbers are gradually increasing (Webber, 2004). Lifestyle changes, particularly the modification of dietary habits and physical activity, are the cornerstone of the prevention and treatment of diabetes (Al-Attar, 2010). There is a considerable body of evidence that diabetes can affect many parts of the body and can lead to serious complications such as heart diseases and stroke, high blood pressure, blindness, kidney disease, liver disease, nervous system disease, amputations, endocrinepathies (endocrine system disorders), dental disease, sexual dysfunction and immunodeficiencies (Al-Attar, 2010). Alloxan is a toxic glucose analogue that selectively destroys insulin producing cells in the pancreas when administered to animals. This causes insulin-dependent diabetes mellitus called "Alloxan Diabetes" in these animals. This diabetes' characteristics are similar to type 1diabetes in human. Alloxan is selectively toxic to insulin-producing pancreatic beta cells because it preferentially accumulates in beta cells through uptake via the GLUT 2 glucose transporter (Lenzens, 2008; Lenzens and Panten, 1988).

In many cases, in-vitro (cultured cells) and in-vivo (animal) trials do provide a degree of mechanistic support for some of the claims that have sprung from the traditional medicine lore. Many herbal extracts or derivatives have been documented in traditional medicine as having clinical effectiveness in treating sugar imbalances in diabetes. Experimental studies of artificially-induced diabetic animals have demonstrated several sever abnormalities induction such as physiological, biochemical and histological alterations. Supplementation of the plant extracts in diabetic rats showed remarkable lowering effects of physiological abnormalities changes (Al-Attar, 2010). The hypoglycemic properties and anti-diabetic potential of medicinal plants have been well documented (Ahmed et al., 2007; Akah and Okafor, 1991; Gbolade, 2009; Modak et al., 2007; Pari and Latha, 2005; Singh et al., 2008). Several reports demonstrated significant reduction in blood sugar level in alloxan-induced diabetic (Kaleem et al., 2005; Nammi et al., 2003). Metabolites such as tannins, flavonoids and alkaloid demonstrated hypoglycemic activity in oral glucose tolerance test in rats (Islam et al., 2009). For instance, Scoparic acid D, a diterpenoid has been isolated from Scoparia dulcis used for treatment of diabetes (Lans, 2006) has been shown to demonstrate anti-diabetic effects in streptozotocin induced diabetic rats (Latha et al., 2009). Therefore, there is a strong need for safe and effective oral hypoglycaemic agents that provide the clinician with a wider range of options for preventing, treating and managing diabetes. However, we have not found any studies on the hypoglycaemic effects of locally grown Moringa oleifera in experimental rats. Thus, in the present study, we evaluated the effects of Moringa oleifera extracts on some biochemical variables of alloxan-induced albino rats.

Materials and Methods

Survey of ethnodietetics of Moringa oleifera leaves amongst the ethnic groups in Bida, Niger State, Nigeria:

A survey of the ethnodietetic uses of Moringa oleifera leaves amongst the ethnic groups in Bida, Niger State was conducted using structured questionnaires. Forty (40) questionnaires administered to both male and female with the age range between 10-79 years who lived in vicinity/habitats where Moringa are planted.

Plant materials:

The various parts of the locally grown Moringa oleifera (root bark, stem bark and leaves) were obtained from the Department (Botanical) garden, Department of Science Laboratory Technology, The Federal Polytechnic, Bida, Niger State, Nigeria.

Preparation of plant materials:

The plants collected were spread on a clean bench and allowed to air dry under room temperature. The plants' leaves, root barks and stem barks were grinded to powder.

Aqueous extraction:

100 g of the various parts powdered Moringa oleifera were separately weighed into different conical flask. 200ml of distilled water was added to each and stirred. Each mixture was covered and allowed to stand for 24 h. They were then filtered separately and the filtrates kept in water bottle (Mann et al., 2011).

Animals:

In the present study, forty (40) 2 weeks old healthy male Swiss albino rats of the Wistar strain, weighing 239-245 g were used. The experimental animals were obtained from National Institute for Pharmaceutical Research and Development (NIPRD), Federal Capital Territory, Abuja, Nigeria. All animals were kept in standard plastic cages and maintained under ambient temperature and humidity (64%) with 12 h light followed by a 12 h dark cycle. The animals were acclimatized for two week prior to actual experiments. Wistar rats were fed food and had free access to water.

Preparation of saline solution:

0.88g of NaCl was weighed and dissolved in 100ml of distilled water in a clean flask.

Preparation of alloxan:

0.8ml of methanol was added to 3.2ml of chloroform in the ratio 1:4. 1g of alloxan was dissolved in 4ml of chloroform--methanol solution and made up to 5ml with saline solution.

Administration of alloxan:

1ml of alloxan prepared as drug in chloroform--methanol--saline solution was administered intraperitoneally based on body weight to groups B to J animals. The blood glucose levels, body weights and temperature of the animals were determined before alloxan induction and 2 h after induction.

Diabetes Induction:

After fasting of 18 hours, the rats were intraperitoneally injected with alloxan (University of Jos, Plateau State, Nigeria) at a single dose of 45 mg/kg (body weight) in 0.5 ml saline solution. After injection, the rats had free access to food and water. Diabetes was allowed to develop and stabilize in these alloxan-treated rats over a period of four days. Diabetes mellitus was defined in these rats using determination of fasting blood glucose levels. The rats showing fasting blood glucose more than 230 mg/dl were considered diabetic and selected for the experimentation.

Experimental Treatment:

The animals were distributed into ten (10) different groups of four (4) animals in each group. The groups were labelled A, B, C, D, E, F, G, H, I, and J. Group A contain the control animals that were not induced with alloxan. The animals were marked with gentian violet at different parts of their body for identification. 5 ml of each extract was given to respective diabetic groups (B to J), six hours after induction with alloxan.

The control group was given only saline solution throughout the experiment.

Phytochemical Analyses:

The phytochemical analyses of the plants were performed using the method (Harbone, 1998; Evans, 1989) and Sofowora, 1986).

Body Weight Changes:

Rats' body weights were determined daily using a digital balance. This was recorded in gram. These weights were determined at the same time during the morning. The experimental animals were observed for signs of abnormalities throughout the period of study.

Body temperature determination:

The body temperature of the animals was determined using a clinical thermometer. This was done by placing the thermometer in between the hind limb and the abdomen. The temperature was taken after 60seconds and recorded in degree Celsius ([degrees]C).

Scarification of animals:

The animals were sacrificed using cardiac dislocation technique. Cotton wool was soaked in chloroform and placed in desiccators for 5 minutes to saturate the dessicator. The animal was then placed in the dessicator, covered and left for 10 minutes to weaken the animal. The animal was then removed and pinned on the board and was a dissected using dissecting material.

Biochemical Measurements: Blood glucose level determination:

The blood glucose level of the animal was determined daily using one touch glucometer machine. The tip of the animal's tail was cut using a sterile razor blade. A drop of the blood from the tail was spotted on the test spot region of the glucose strip after inserting it into the glucometer. The value obtained was recorded immediately and expressed in mg/dl. The glucose level determination was done early in the morning after withdrawal from food overnight and before extracts administration in the morning.

Full blood count determination: Haemoglobin determination:

5.0ml of cyanmethaemoglobin was dispensed into a test tube. 0.02ml of EDTA (10g of EDTA in 100ml of distilled water) was mixed with blood and added to the test tube containing the reagent. This was incubated at room temperature (20-25[degrees]C) for the three minutes. The absorbance of the solution was read at wavelength of 540mm against distilled water as blank using spectrophotometer.

Packed cell volume determination:

A heparinated capillary tube was filled with properly mixed EDTA blood. One end of the capillary tube was sealed with plasticine. This was spinned in a haematocrit centrifrige at 4000r.p.m for 5 minutes. The cells that were packed were read using haematocrit reader. Thus, the percentage of the packed cells was obtained as PCV.

Total white blood cells count determination:

0.02 ml of blood was obtained with an automatic pipette and added to 0.38 ml of diluting fluid or truk's fluid (2 ml of acetic acid in 100 ml distilled water) in a 2.0ml glass test tube. This was mixed and left for 10 minutes. The Neubauer counting chamber was filled and placed under a microscope objective to count the total white blood cells.

Determination of differential count:

A thin blood film was made on a microscope glass slide. After air drying, the film was stained using leischman's stain and air dried. This was read using x 100 objective of the microscope. The different cels were read and counted according to their morphological appearance in the strained film. A total of 5-10 fields were read. Thus, the percentage of each cell counted is calculated from the grand total of cells counted (ICSH, 1967).

Liver enzymes analyses: Glutamate Pyruvate Transaminase (GPT):

The GPT was determined using the method described by Young (1990). The liver homogenate sample was collected, centrifuged and the serum obtained. 1ml of the serum was placed in a clean test tube and 0.5 ml of reagent (R1) buffer in 1ml of distilled water was added. This was mixed and incubated for 20 minutes at 25[degrees]C. 5ml of 1.0 M NaOH was added and mixed. The absorbance was now taken against reagent blank at 546nm after minutes. The value obtained was now compared with GPT activity table.

Glutamate Oxaloacetate Transaminase (GOT):

The liver homogenate sample was collected and centrifuged to get the serum. 1 ml of serum was placed in a test tube and 0.5 ml of reagent (R1) Butter in 1 ml of distilled water. This was mixed and incubated for 30 minutes at 37[degrees]C. 0.5ml of reagent (R2) was added, mixed and incubated for 20 minutes at 25[degrees]C. 5 ml of 1.0m NOAH was added and mixed. The absorbance was now taken against reagent blank at 546 mm after 5 minutes (Young, 1990).

Alkaline Phosphatase (ALP):

The liver homogenate sample was collected, centrifuged and serum obtained. 1 ml of the working reagent was added into a clean test tube followed by 0.2 ml of the sample. This was mixed and incubated for 1 minute at 37[degrees]C. The change in absorbance at 546 nm wavelength was taken per minute for 3 minutes.

Statistical Analysis:

Data were statistically analyzed using Package for Social Sciences (SPSS) for Windows version 12.0 software. All experimental data were expressed as mean [+ or -] standard deviation (SD). Statistical analysis was performed by one-way analysis of variance (ANOVA) and two-way analysis of variance (ANOVA) of computer based Excel Software followed by the least significance difference (LSD) method. The P<0.05 level of probability was used as the criteria of significance.

Results and Discussion

The data obtained in the survey are shown in Table 1below.

Discussion

Ethnodietetics of Moringa oleifera leaves amongst the ethnic groups in Bida, Niger State:

The findings of the survey revealed that six ethnic groups are in close association with the distribution and usage of Moringa products. It is mainly consumed as salad by Hausa/Fulani ethnic group (15%), while other ethnic groups (Nupe 65%; Yoruba 10%; Kakanda 5%; Jaba 2.5%; Bwala 2.5%) (Table 1) used it as food as well as medicine in the treatment of diseases including diabetes. Only few are aware of its other potentials in the areas of water purification and as source of ink.

Assessment of the hypoglycaemic effects of various parts of Moringa oleifera:

The treatment of diabetic patients with naturally derived agents has the advantage that it does not cause the significant side effects as do chemical agents such as sulfonylurea. One of the side effects with sulfonylurea is that it causes a decreased amount of insulin production by putting too great a strain on the insulin producing beta cells. Treatment with herbal drugs has an effect of protecting beta cells and smoothing out fluctuations in glucose levels. Some agents, such as herbal extracts, can reduce the insulin resistance and, hence, improve the apparent insulin activity. Improved insulin activity leads to decreased circulating insulin, which leads to lower blood glucose and glycosylated haemoglobin levels as well as total cholesterol levels. Additionally, the use of these natural agents in conjunction with conventional drug treatments such as a chemical agent or insulin permits the use of lower doses of the drug and/or decreased frequency of administration which decreases the side effects most commonly observed.

The hypoglycaemic properties of Moringa oleifera extracts on the haematological and biochemical variables of alloxan--induced rats as well as the phytochemical screening of the extracts were carried out in this study. From the Table 2, all the extracts of M. oleifera contain alkaloids, glycoside, phenol and tannin. However saponin is absent in all the extracts. The results obtained show that the haemoglobin, the packed cells volume and the eosinophils were higher in the control than the experimental groups (Table 3). As shown in Table 4, the control group has mean GPT of 102 iu/L which is higher than other groups. The group treated with M. oleifera leave extract has mean GPT of 104 iu/L. The ALP of the M. oleifera treated group is lower than the control group (248.0 iu/L). The group treated M. oleifera exhibits significant GOT and GPT. From the Table 4, the high level of liver enzymes above the control in some of the groups could be because of the administered drug and this agrees with earlie report of Ajiboso (2005). The phytochemical screening of the extracts (Table 2) showed that they all contain glycosides and tannins which are often associated with toxicity (Soyinka, 2004). This can be the reason for the effects of the extracts on the haematological indices of the rats. The toxicity of glycosides remains a serious problem (Soyinka, 2004). The screening result is also in agreement with the findings of John (2008). The increase in blood glucose after alloxan induction could be due to damage of the pancreas by some chemicals as earlier described by Mohammed et al., (2007). The reduction in glucose level after extracts administration could be due to the triggering of the release of insulin as earlier shown by Lonescu et al., (1988). The rise in temperature of induced rats could be attributed to excessive urination which increases the osmotic pressure of the body. In addition, very high loss in weight recorded in the group treated with extract of M. oleifera root bark could be as a result of increase symptoms as explained by Webber (2004). This work concludes that administration of these extracts with hypoglycaemic properties can lower the blood sugar levels and could be used for the management of diabetes. However, toxicological studies are required before its utilization in dietary supplements.

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Abdulkadir Nda Umar, Abdullahi Mann, Olabode S.O. Ajiboso

Department of Science Laboratory Technology, The Federal Polytechnic, Bida, P. M.B. 55, Bida, Niger State, Nigeria

Corresponding Author: Abdullahi Mann, Director, Academic Planning Unit, Department of Science Laboratory Technology, The Federal Polytechnic, Bida, P. M.B. 55, Bida, Niger State, Nigeria.

E-mail: abdumann@yahoo.com; Phn: +2348034295656
Table 1: Ethnodietary survey of Moringa oleifera
in Bida metropolis

Ethnic         No              Percentage
group          Respondent      (%)

Nupe           26              65
Hausa          6               15
Yoruba         4               10
Kakanda        2               5
Jaba           1               2.5
Bwala          1               2.5

* All the ethnic groups encountered used
it as food and medicine

Table 2: Results of Phytochemical screening
of Moringa oleifera extracts

Samples   Alkaloids   Glycosides   Steroids   Phenols   Tannins

G         +           +            +          +         +
H         +           +            -          +         +
I         +           +            +          +         +

Samples   Saponins   Flavonoids

G         -          +
H         -          -
I         -          +

Key + = Detected, - = Not detected; G= Moringa oleifera leaves;
H= Moringa oleifera stem bark; I = Moringa oleifera root bark

Table 3: Showing the levels of blood parameters of normal
and alloxam-induced rats

G/BP    Hb (g/dl)      PCV(%)         WBC         NEU(%)

A      16.7 + 2.1    49.0 + 14.7   6.9 + 1.3     2.0 + 6.0
B      16.0 + 4.2    49.0 + 12.4   9.0 + 0.3    54.0 + 6.4
C      15.2 + 3.3    46.0 + 14.8   5.6 + 0.2    46.0 + 8.4
D      14.7 + 3.1    44.0 + 10.0   6.0 + 0.0    46.0 + 7.1

G/BP      L(%)          M(%)          E(%)
                                   (cells/ml)

A      52.0 + 16.8    3.0 + 0.2    3.0 + 1.0
B      43.0 + 14.0    2.0 + 0.1    2.0 + 0.2
C      47.0 + 20.0    2.0 + 0.3    2.0 + 0.4
D      48.0 + 12.4    2.0 + 0.2    4.0 + 1.2

* Mean values of determinations Results expressed in Mean
+ SEM A = Control; B = Moringa oleifera root bark extract;
C = Moringa oleifera stem bark extract; D = Moringa oleifera
leave extract. Hb = Haemoglobin estimation; PCV = Packed
Cell Volume; WBC= White blood Cell; NEU = Neutrophil;
I = Lymphocytes; M = Monocytes; E = Eosinophil and
BP = Blood Parameters

Table 4: Showing the level of liver enzymes ([u/dl)
of normal and alloxan-induced rats

G/LE          GOT           GPT           ALP

A      141.0+13.1    102.0+10.4    248.0+34.2
B      137.0+17.1     95.0+10.5    275.0+15.5
C      130.0+12.8     99.0+11.3    330.0+54.2
D      148.0+20.2    104.0+12.4    248.0+26.2

* Mean values of two determinations Results expressed in
Mean [+ or -] SEM; A = Control: B = Moringa oleifera root bark
extract; C = Moringa oleifera stem bark extract; D = Moringa
oleifera leave extract. GOT = Glutamate Oxaloacetate
Transaminase; GPT = Glutamate Pyruvate Transaminase; ALP =
Alkaline Phosphatase; G = Group; LE = Liver Enzyme
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Title Annotation:Original Articles
Author:Umar, Abdulkadir Nda; Mann, Abdullahi; Ajiboso, Olabode S.O.
Publication:Advances in Natural and Applied Sciences
Article Type:Report
Geographic Code:6NIGR
Date:Mar 1, 2011
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