SOME CARDIOPROTECTIVE EFFECTS OF AQUEOUS EXTRACT OF GINGER AGAINST MONOSODIUM GLUTAMATE INDUCED TOXICITY IN THE HEART OF ADULT WISTAR RATS
1*Fakunle, PB., 1Mene, A A and 2Kehinde, BD
1Department of Anatomy, Technology Ogbomoso, Oyo State, Nigeria
2Department of Biochemistry, Ladoke Akintola University of Technology Ogbomoso, Oyo State, Nigeria
* Corresponding author
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ABSTRACT:
Research findings have indicated cardiovascular disease (CVD) to be the major morbidityand mortality in adult man.The protective effect of ginger extract against Monosodium Glutamate-induced cardiotoxicity was evaluated in 48 wistar rats (weighing 150-250 g) classified into 6 groups (8 rats per each group). The rats in control group (Group A) received distilled water for 21 successive days. The rats in treated (Group B) were treated with 4g/kg/day of MSG orally for 21 successive days, rats in treated group (Group C) were treated with 1g/kg/day of ginger extract orally for 21 successive days, rats in treated group (Group D) received 2g/kg/day of ginger extract orally for 21 successive days, rats in treated group (Group E) received 4g/kg/day of MSG and 1g/kg/day of ginger extract orally for 21 successive days, rats in treated group (Group F) received 4g/kg/day of MSG and 2g/kg/day of ginger extract orally for 21 successive days. Results show that MSG administration resulted in changes in body weight, significant increase in the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) and distortions to cardiac tissue as compared to rats treated with ginger extract.
KEYWORDS
Liposome, GelElectrophoresis, surfactantand Evans blue.
INTRODUCTION
New drug development is an expensive and incurs a great amount of time. The average cost for INTRODUCTION
Cardiovascular Disease (CVD) has been shown from various reports to be the major morbidity and mortality in adult human being (John et al, 1999). Various drugs, food addictives, toxins and plant extracts have been reported to have contributed to the increase of Cardiovascular Diseases. Monosodium Glutamate (MSG) is one of the commonly and widely used food spices in our daily diets (Walker and Lupein, 2000). Its consumption has increased world wide as flavouring and food additive in our daily cooking (Chaudari and Roper, 1998) to improve palatability and food peference in a meal (Bellisle et al, 1996). However, consumption of this food addictive has been shown to cause metabolic disorders including hyperlipidaemia, hyperglycaemia and oxidative damage of tissues (Dinz et al, 2005; Nagata et al, 2006) which may possibly be responsible for the pathophysiology of many diseases like cancer, diabetes, endothelial dysfunction (Naderali et al, 2004; Dinz et al, 2005), brain lesion (Mallick, 2007) and Coronary Heart Disease (CHD) (Dinz et al, 2005; Nagata et al, 2006; Singh and Pushpa, 2005).
Monosodium Glutamate, an example of food addictive and flavour enhancer has been reported to possibly induce oxidative stress in the heart tissue of adult wistar rats when subcutaneously administered by altering the activities of SOD, XOD and CAT, which invariably predisposes to Coronary Heart Disease/Atherosclerosis (Singh and Pushpa, 2005).
Nayira et al (2009) reported that administration of MSG to adult albino rats resulted in oxidative stress and cardiac tissue damage with pronounced increase in the activities of diagnostic serum marker enzymes: CPK and AST as compared to that of the control rats. Histopathological examination of the heart tissue also showed that MSG induced myocardial infarction observed as areas of necrotic lesion in the cardiac tissue of the rats treated with MSG.
Chronic administration of chloroquine, widely used anti-malarial and anti- rheumatic drug may result in cardiac tissue damage. Histological results suggested toxicity of myocardial cells of wistar rats upon chronic oral administration of chloroquine and this was shown by the moderate hypertrophy of cardiomyocytes (Izunya et al, 2011).
Cisplatin, a platinum- based drug is one of the most effective anti-neoplastic agents used for the treatment of testicular, ovarian, bladder, cervical, lung and cancers (Abu-surrah and Kettunen, 2006). Moreover, this substance has been associated with several toxic side effects including cardiotoxicity and hepatoxicity (Abdulhakeem, 2006). Administration of Cisplatin to albino rats resulted in degeneration and necrosis of the cardiac muscle fibres with fibrous tissue reaction on histological examination and also significant increase in AST and ALT activities (P<0.05) (Ahmed and Sajida, 2012).
Histopathological changes have been observed in foetal heart of rats that was treated with enalapril maleate, an anti-hypertensive drug that reduces blood pressure (Khaki et al, 2005.)
Rodents that were exposed to isoproterenol treatment showed myocardial infarction (Upaganlawar et al, 2011). Similarly, aqueous extract of Ocimum gratissimum administered to adult wistar rats resulted in degenerative changes of cardiac tissue which might consequently impair some cardiac function (Ajibade et al, 2011).
Man has evolved a highly sophisticated and complex antioxidant protection system in order to protect the cells and organs of the body against reactive oxygen species, which involves a variety of endogenous and exogenous substances that have interactive and synergistic impact to neutralize free radicals (Jacob, 1995).
Monosodium glutamate also known as sodium glutamate or MSG is the sodium salt of glutamic acid, one of most abundant naturally occurring non-essential amino acids (Ninomiya, 1998). Harmful effects of different environmental chemicals, industrial pollutants and food addictives have been reported by previous investigators (Moore, 2003). Much controversy has been associated with safety of MSG consumption locally and globally (Biodun and Biodun, 1993).In Nigeria, most communities and individuals often use MSG as a bleaching agents for the removal of stains from cloth
Though MSG improves taste stimulation and enhances appetite, reports indicate that it is toxic to human and experimental animals (Belluardo et al, 1990). It has been reported that MSG has neurotoxic effects resulting in brain cell damage, retinal degeneration, endocrine degeneration and some pathological conditions such as stroke ,epilepsy, brain-trauma, neuropathic pain, schizophrenia and many other pathological conditions(Eweka and ,2007).It has also been reported that MSG has toxic effects on the kidney causing disruptions and distortions of the cyto-architecture of the kidneys which resulted in the cellular in the cellular necrosis and sparsely distribution of the Bowman’s spaces(Eweka, 2007).
Ginger or ginger root is the rhizome of the plant Zingiber officinale, which is consumed as a delicacy, medicine, or spice. Ginger cultivation began in south Asia and has since spread to East Africa and Caribbean (Spices, 2007).Ginger as an herb, spice and preservative have been reported to have many medicinal values .Preliminary researches indicates that nine compounds found in ginger may bind to human serotonin receptors which may influence gastrointestinal functions (Nievergelt et al, 2010)..Ginger also have effect on the cardiovascular system by lowering blood pressure(Ghayur et al, 2005).It has also been reported that ginger had protective effect against liver damage induced by Adriamycin and this is due to its antioxidant activities(Sakr et al, 2010).
MATERIALS AND METHODS
Location and duration of study
This study was conducted at the animal house of the Department of Human Anatomy, Ladoke Akintola University of Technology, Ogbomosho, Oyo state, Nigeria. The preliminary studies animal acclimatization, actual animal experiment and evaluation of results, lasted for a period of three months. However, the actual administration of MSG and ginger extract to the test animals lasted for three weeks.
Wistar rats weighing 150-250g were used for the experimental design. A total number of 48 rats (male and female) were involved. The experimental animals were housed in standard plastic cages, fed with rat chow, and water daily. The experimental animals were divided into six groups.
Preparation of Ginger extract and MSG
MSG was purchased in an open market in Ogbomosho. Ginger roots were also purchased in an open market in Ogbomosho, Nigeria. The plant was authenticated to be ginger root by a seasoned Botanist; Dr Ogunkunle from the Department of Biology, LAUTECH. The ginger roots were washed and cut into pieces and was air-dried for two weeks after which it was grinded. The fine powder was taken to the Department of Food Science and Engineering, LAUTECH for further processing into a coarse powdery form.The powder form of the ginger was macerated in distilled water following the methods of Morakinyo et, 2010 .
Experimental design and Grouping
After the acclimitization period, rats were weighed and randomly divided into six groups comprising eight animals in each group. Animals were administered with Ginger (1g/kg and 2g/kg for low and high dose respectively) and MSG (4g/kg ).The groups are as follows:
Group A: Rats were given stock diet and water, they served as control.
Group B: Experimental animals were given stock diet and 2.5mlof MSG orally for 3weeks.
Group C: Experimental animals were given stock diet and 2ml of ginger extract (low dose) orally for 3weeks.
Group D: Experimental animals were given stock diet and 3ml of ginger extract (high dose) orally for 3weeks.
Group E: Experimental animals were given stock diet, 2mlof ginger extract (low dose) and 1.9ml of MSG orally for 3weeks.
Group F: Experimental animals were given stock diet, 4ml of high dose of ginger extract and 2.2ml of MSG orally for 3weeks
Procedures for animal sacrifice and tissue harvest
The animals were sacrificed by cervical dislocation on the 22nd day. Blood was collected from the heart for biochemical analysis of enzymes and the tissue (heart) was harvested immediately, weighed and fixed in 10 % formol saline for histological analysis using Hand E
Statistical analysis
Data obtained were analysed using the analysis of variance and tested for significance by the unpaired one-tailed student’s t-test
Table 1Composition, entrapment efficiency and invitro release character of the formulation.
Formulation / Formulation Code / Lipid composition(Molar Ratio)
Le:Ch:Span 20 / S20 / 9:1:1
Le:Ch:Span 40 / S40 / 9:1:1
Le:Ch:Span 80 / S80 / 9:1:1
Le:Ch: Span 80: Tween 80 / ST 80 / 9:1:2:1
Le:Ch / NS / 9:1
RESULTS
Particle size analysis
Graph 1 Particle size analysis of the formulation
ES 40 formulations showed a wider distribution of particles in the size range of 0 – 5 µm (about 58 %) rather than other formulations but showed very less distribution in the size range of 6 -10 µm and more (Fig 1). It was very much less or negligible in the size range of 26 – 30 µm. But this was reverse in the case of non surfactant, as it showed very less size distribution in the size range of 0 – 5 µm but showed more prominence of its presence in size range greater than 21 µm. ES 20 showed greater size distribution within the size range of 6 – 10 µm but was very much minimal in 26 – 30 µm. In the case of ES 80 the maximum size was within the range of 11 – 15 µm but was very minimum in the size range of 21 – 25 µm. All showed an average presence in the size range of 6 – 10 micron. The EST 80 formulation had maximum of its vesicles in the size range of 11 – 15 µm but was minimal in the size range of 26 – 30 µm. S 80 rises its particle size from the range of 0 – 5 reaching peak at the range of 11 – 15 and abruptly falls to a very low size distribution in the range of above 15 micron.
Microtomized brain slicing
A macroscopically discernible blue area of dye extravasation was present on brain for the animal administered with EST 80 formulation. The blue staining was completely absent for the normal isolated brain (Fig 1). The basal part of the sliced cerebral cortex were more stained for the EST 80 ( TS ST 80) formulation when their 5 micron microtome sliced picture were seen , but staining were absent for the other formulation (Fig 2). This was clearly evident when observed under 40 X magnification (Fig 3). The slow invagination of Evans blue dye ofEST 80 slowly through the peripheral brain parenchyma in to the interior brain cortex region could be clearly observed form the microtomized brain picture of TS ST 80 (EST 80) (Fig 3).
Isolated whole Brain picture of normal and ST 80 formulation administered rat
Figure 1 Normal brain do not show any blue staining by the dye but ST 80 (EST 80) formulation administered brain shows extravasation of the dye in to the brain bypassing blood brain barrier)
Figure 2 T.S Microtomized section (5µm thickness) of isolated brain slices showing absence of evans blue dye in TS Normal (ENS) , TS S20 (ES20) , TS S40 (ES40) and TS S80 (ES80). But in the case of TS ST 80 (EST 80) extravasation of the Evans blue dye could be clearly observed.
T.S Microtomized section (5µm thickness) of isolated brain slices
Figure3 The Microtomized isolated brain section of ST 80 when viewed under optical microscope under 10 X magnification shows clear penetration of Evansblue dye.
Histopathological studies
Eosin - haematoxylin-counterstaining (blue) revealed distinct populations of stained cells with astrocytic morphology in the brain cortex for ST 80 formulation (Fig 4). These staining of cells were completely absent for other formulation. The Blue staining (Haematoxylin) for the nuclei and the pink (Eosin) for the cytoplasm were clearly visualized for all the formulation under 400 X magnification. But in the case of ST 80 formulation the brain parenchyma cells along with the eosin – haematoxylin staining showed blue large sized granular shaped structures which are a clear evident for the presence of Evans blue dye. These granular shaped dyes were completely absent or negligible in other formulations.
Histology slides
Figure 4Histology slides showing haematoxylin and eosin stained cells. In Non surfactant Liposomes (ENS) , S 80 (ES80) , S 40 (ES 40) and S 20 (ES 20) the stained cells shows absence of Evans blue dye grains .But in the case of ST 80 (EST 80) Evansblue dye presence is clearly evident.
SDS –PAGE GEL electrophoresis
The gel photograph revealed the presence of prominent Evans blue dye band in the brain homogenate samples of ST-80 and control (0.1 %Evans blue 0.05µl). The blue dye band was prominent in the 10 % SDS gel under electrophoretic potential of 100 V and the position of the sample (ST 80) was inline with the control band. The other formulations brain homogenate failed to show any such kind of band (Fig 5).
SDS PAGE Gel
Figure 5 SDS PAGE Gel shows the presence of Evans blue dye (shown with an arrow) for ST 80 (EST 80) which is inline with pure Evans blue dye (ED) run at the same time on the same gel cast (shown with an arrow).
DISCUSSION
The result of particle size distribution shows that S 40 surfactant was efficient enough to achieve more amounts of less particle sized liposomes within the size range of 0 – 5 micron which was superior to S80 surfactant. The liposomes in the absence of surfactant fails to achieve smaller size distribution on comparing to others and this proves that the emulsification using surfactant during the preparation is needed to achieve maximum reduction in the liposomal size and this reduced size has more potential in extravasating the blood capillaries towards targeting vital sites. Non surfactant modified liposomes mostly would form large unilamellar vesicles. Most of all the formulation showed an average population in the size range of 6 – 10 micron which proved to be the stable size in this manufacturing technique. The ST80 formulation showed a maximum population in the size range of 11 – 15 micron but falls down on either side proves that this formulation favors stability in this size range.
From the isolated brain pictures (Fig 1) it is clearly evident that the formulation EST80 was a very good carrier in carrying Evans blue dye to brain cortex thus staining the brain parenchyma. Further in the brain sliced picture (Fig 2) among the other formulation, the EST80 formulation was good enough to stain the brain which was further proved from the 40X magnification picture of the same which shows clear impregnation of the dye (Fig 3). The histopathological studies showed that there were no damage in the brain parenchyma due to these formulations but instead haematoxylin stained nuclei blue and eosin stained cytoplasm pink which were evident from those pictures. Among the other formulation, ES80 formulation histopathological picture showed the presence of blue colored dye grains but was absent in other formulations.SDS PAGE gel electrophoresis procedure provided a clear evidence (Fig 5) of presence of Evans blue dye by developing a blue band for ES80 formulation which is inline with the control – Evans blue dye itself ( showed by arrow marks ) .
The above results and discussion confers that the liposomes prepared by reverse phase evaporation technique and if modified with surfactant particularly span 80 and tween 80 at a molar concentration of 2:1 would make the entrapped liposomal drug extravasate through the brain capillaries and could achieve detectable presence in the brain parenchyma. More over those optimized liposomal formulation can have its particle size reduced to the need of targeting sites if above said surfactant combination used.
CONCLUSION:
Thus the liposomes if modified using surfactant particularly span 80 and tween 80 at molar concentration of 2:1 could be a very good candidate to ferry the desired impermeable drug to brain without any pathological damage to the site. This formulation if developed further could also be a very good carrier for most of the neuropathological drugs which otherwise has access through intrathecal or implants or any other painfulprocedure. In conclusion, research work can further be initiated towards validating and standardizing this formulation for clinical and preclinical studies.
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