Determination of the Ferritin and Glucose Levels in Serum of Mice Treated With Ethanolic Leaf Extract of Phyllantusamarus
CHAPTER ONE
Objective of the Study
The purpose of this study is to determine the ferritin and glucose levels in serum of mice treated with ethanolic leaf extract of Phyllantusamarus which may indicate an advantage or disadvantage
CHAPTER TWO
REVIEW OF RELATED LITERATURE
Ferritin
Ferritin is a class of iron storage protein distributed in virtually all living kingdoms (Barnes and Sato 1980). Proteins of this class commonly form spherical protein nanocages, each of which is usually composed of similar or identical subunits. This cage-like 24-mer has a large inner cavity and shows highly symmetrical architecture, i.e., the 24 subunits are related by four-, three-, and twofold symmetries (432 symmetry). A subunit of ferritin forms a four-helix bundle composed of helices A to D and a short fifth helix E, which is also a common structural feature of all ferritins. Many intersubunit interactions have been observed in the crystal structures of ferritin from various organisms, giving the ferritin superfamily its super thermal stability (Bryant, 1985).The ferritin superfamily can be divided into a vertebrate type, a plant type, a bacterial type, and so on. Bacterial ferritin can be further divided into heme-containing bacterioferritin (Bfr), nonheme ferritin (Ftn), and Dps (DNA binding protein from starved cells), the latter of which exceptionally forms a dodecameric protein shell with two- and threefold symmetry axes (mini-ferritin) (Cohen, 1993). Most of the ferritins possess a di-iron oxidoreductase site (ferroxidase site) responsible for iron oxidation in the center of the four-helix bundle of the monomeric subunit, except for mammalian L chain ferritin (Barnes, 1985).
Among ferritins, the plant-type ferritin has some physiologically and functionally distinct features. Most of these features can be related to the presence of two plant-specific amino acid sequences: transit peptide (TP) and extension peptide (EP) (Johnston and Edgar, 1998). The primary structure of the plant ferritin core region without TP and EP has a relatively high level of similarity to the vertebrate H-type ferritins (∼40%) (Bakker and Boyer, 1986). The TP that is responsible for targeting plant ferritin to plastid exists in the N-terminus of the plant ferritin sequence (Currie and Milner 1988). The TP is removed from the mature region of plant ferritin after entering to the plastid. On the other hand, EP is present downstream from TP, so it exists in the N-terminus of the mature region. It has been suggested that EP is cleaved from the core region in the germination process in legume seeds, and that this cleavage induces the destabilization of the plant ferritin shell. Further, recent studies suggest that the EP, a specific domain found only in plant ferritin, has some significant role in regulating the function of plant ferritin. First, it was suggested that the EP functions as a second ferroxidase site, which assists in effective iron oxidation and incorporation especially in high iron/ferritin stoichiometry. However, unlike the well-characterized ferroxidase site in the four-helix-bundle, the structural and enzymatical basis of this EP-involved second ferroxidase site has not been fully elucidated.
CHAPTER THREE
MATERIALS AND METHODS
Materials
Fresh whole plant of phyllanthus amaruscollected in august, 2014 from an uncultivated farmland in Abraka, Delta state Nigeria. Animals (Swiss Albino mice, Bio vaccines), Glucose kit Randox assayed multisera levels; 2 (CAT No. HN1530) and level 3 (CAT No. HE1532).Ferritin assay Kit (Ferritin reference standard, containing 0, 15, 80, 250, 500, 1000 ng/ml) – (NIBSC-WHO 80/602, human liver standard) liquid, 0.5ml, TMB reagent (one-step), 11 ml. Spectrophotometer (Spec 20D, Techmel and Techmel, USA), Glassware’s (Pyrex, MBL, Germany), Ethanol, chloroform (Analar grade, BDH, Chemicals, Poole, England). Centrifuge (80D, Techmel and Techmel, USA), Water bath (HH-6, GUOHUA, China), Analytical Balance (Metler Toledo, USA). Specimen (Brain, Heart, Serum), Refrigerator (HTF 319 Haier Thermocool, Japan).
CHAPTER FOUR
RESULTS
The effect of ethanoic extractsof Phyllanthusamarusat various doseson blood glucose level and serum ferritin level in mice were assessed after seven days (7) of treatment and at the end of the experiment (21 days). The results are presented in this section using histogram and comparison made between groups and the control (untreated) group. The values are expressed as mean ± Standard error of mean (S.E.M), mean differences from LSD comparison at P<0.05 level of significance are considered and designated as follow;
- All values designated (a) showed significant increase when compared to control group
- All values designated (b) showed significant decrease when compared to control group
- Values not designated at all showed no significant changes when compared to control group
CHAPTER FIVE
DISCUSSION, CONCLUSION AND RECCOMMENDATION
Discussion
Glucose is one of the most important substrates required in the body for a very necessary metabolic process called glycolysis. It is needed by every cell, tissue and organ in the body for generation of energy. As excesses or absence of it can lead to life threatening conditions, this study investigates probable effects of administration/consumption of varying doses ofPhyllanthusamarus to blood glucose level, as well as ferritin, an intracellular protein that stores iron and releases it in a controlled fashion acting, in humans, as a buffer against iron deficiency and iron overload.
In concord with the studies done by karuna(2011), this study shows no significant changes in blood glucose level after seven days of treatment with various doses (150mg/dl, 300mg/dl and 459mg/dl) of P. amarus and also at the end of the experiment.Karunaet al revealed that normal rats treated with extracts of P. amarusremained persistently euglycemic throughout the experimental period.This may suggest that P. amarus has no hypoglycemic of hyperglycemic effect in normal individuals.The same was also observed in ferritin results showing no significant changes in serum ferritin level after seven days of treatment with various doses (150mg/dl, 300mg/dl and 459mg/dl) of P. amarus and also at the end of the experiment, suggesting that the plant, Phyllanthusamarus has no effect in iron homeostasis.
Conclusion
This study has demonstrated the potentially harmless effect of Phyllanthusamarusin relatively maintaining blood glucose and serum ferritin levels significantly in animal models.
Recommendation
Other properties of P. amarus should be studied and investigated further especially in diabetes and in iron deficient conditions in other to elucidate other potentials of the plant.
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