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Research Article | DOI: https://doi.org/10.31579/2690-8816/050
1 Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Federal University of Oye-Ekiti, Ekiti State, Nigeria.
2 Department of Public and Community Health, College of Medicine, Novena University Ogume, Delta State, Nigeria.
3 Department of Biological Sciences, University of Delta, Agbor, Delta State, Nigeria
4 Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria.
*Corresponding Author: O. M. Aziakpono, Department Of Physiology, Faculty of Basic Medical Sciences, College Of Health Sciences, Delta State University, Delta State, Nigeria
Citation: O. M. Aziakpono. (2022). Hematological Changes in Administration of Garcina Kola Seed Extract to Wistar rats. Clinical Research Notes. 3(1); DOI: 10.31579/2690-8816/050
Copyright: © 2022 O. M. Aziakpono. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, providedthe original work is properly cited.
Received: 04 December 2021 | Accepted: 22 December 2021 | Published: 06 January 2022
Keywords: Garcenia kola; hematopoiesis; Heckel Gultiferae; blood
In a number of ailments, hematological tests are useful in diagnosing and determining the extent of blood production. The impact of Garcina kola seed extract (G. kola) on blood parameters was investigated in this study. Twenty-five (25) adult male wistar rats (weighing 130–180 g on average) were acclimatized for two weeks before being divided into five (5) groups of five (5) rats each (n = 5). They were subsequently fed G. kola seed extract in various quantities (doses) for 21 days (3 weeks). While normal control (Group I) mice were fed standard rat chow and water ad libitum, group 2 animals were fed 100 mg/kg of test extract, while groups 3 - 5 were fed 200 mg/kg, 300 mg/kg, and 400 mg/kg of the same G.kola seed extract, respectively. Rats were venipunced, blood samples were obtained, and haematological alterations were analyzed in the laboratory after a period of administration of test chemicals. During this time, student's t-test was used to compare the connection in means between each group and the control group. P-values less than 0.05 were considered statistically significant. Finally, hematological measures such as PCV, RBC, HGB, LYM, MON, BAS, EOS, and NEU counts showed a significant effect. Oral administration of aqueous extract of G. kola seed to the mice decreased RBC and HGB levels insignificantly compared to control, while higher dose administration significantly increased PCV and HGB counts (p 0.05). In comparison to the control, lymphocyte counts were considerably (p 0.05) higher in group 4, but not in the other extract-fed groups. The counts of basophils and eosinophils, as well as neutrophils, were significantly higher than the control group, especially as the dose was raised. In rats, an extracts of G. kola raised the immune system while lowering RBC production, causing anemia and having a negligible effect on PCV and HGB. Further research into the likely molecular mechanism for this could be useful in clinical treatment, thus it is recommended.
Garcinia kola (Heckel Gultiferae) is an extensively grown forest tree native to Sub-Saharan Africa. It's been termed a "magic plant" because nearly every part of it has been shown to have medicinal properties [1]. In traditional hospitality, cultural, and social traditions, the masticatory seed (also known as bitter kola, male kola, or fake kola) is used.
In African folkloric medicine, Garcinia kola seed is frequently advised for the treatment of diabetes and its consequences. In STZ-induced diabetic rats, garcinia kola seed administration dramatically reduced hyperglycemia-mediated damage by lowering blood glucose levels, enhancing the antioxidant system, inhibiting lipid peroxidation, and improving the architecture of the kidney, liver, and testes. Furthermore, in STZ-induced diabetic rats, G. kola seed intervention was observed to restore kidney and liver function markers, sperm qualities, and plasma concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone, triiodothyronine (T3), and thyroxine (T4) to normal [2]. Laryngitis, liver disorders, and cough have all been treated with extracts from the plant in the past. The seeds are used to ease coughs, prevent or relieve colic, and heal head or chest colds. Anti-inflammatory, antibacterial, antidiabetic, antiviral, and antiulcer activities are also found in the seed. Garcinia kola extracts have been shown to have a variety of bioactivities, which include hepatoprotection, antidiabetic properties, and antigenotoxic attributes [3, 4].
Bioassay-guided separation of Garcinia kola seed generated diverse combinations of phenolic chemicals, triterpenes, and benzophenones, according to research. Moisture content, crude protein, crude fibre, ash, crude fat, and carbohydrate make up the approximate composition of Garcinia kola (in g/100g). Garcinia kola has a lot of tannin, oxalate, phytate, and trypsin inhibitor, according to qualitative anti-nutrient screening. It also contains tannin, oxalate, phytate, and trypsin inhibitor, as well as magnesium, zinc, iron, manganese, copper, lead, phosphorus, sodium, potassium, and calcium, according to quantitative anti-nutrient screening [4]. The well-known health implications of G. kola seed in traditional medicine for liver and reproductive disorders, as well as its ability to suppress oxidative stress in various experimental models of organ toxicity, has piqued interest in its effect on the hypothalamic-pituitary-gonadal axis-hypothalamus (HPGA) axis in wistar rats [5]. These findings support the idea that it may affect hormones in the hypothalamic-pituitary-gonadal axis. Given G. Kola's anti-oxidant properties, it's important to look into its effect on the female reproductive system.
G. Kola has also been implicated in not only sleep deprivation, but also as a physiologic stressor in the altering of homeostatic balance, including changes in hematological variables [6, 7]. The main element in G. Kola, Kolaviron, has been demonstrated to interfere with hormone release via the hypothalamic-pituitary-Gonadal axis and the autonomous nervous system, lowering serum levels of circulating hormones and altering blood variables. This report, together with the increased frequency of blood illnesses (such as amenia) around the world, has remained a concern that the research community has yet to completely investigate [8, 9].
Hematological health indicators, that are good indicators of an animal's physiology, have recently been connected to blood organ creation [10, 11], which serves as a pathological reflector of the status of toxicants and other situations to which animals are exposed. Animals with a normal blood composition, as per Isaac et al. (2013) [12], are more likely to perform successfully. According to Afolabi et al. [13], knowing variations in hematological parameters may be relevant in identifying stresses produced by environmental, infectious, and/or nutritional factors. As a result, this research was required.
Study Area
The study was carried out in Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka, Anambra State.
Animal Procurement
Twenty five (25) adult female wistar rats of an average weight of between 160g - 200g were procured and acclimatized for two (2) weeks. The animals which were housed in the study institution's animal house (AAU), were provided with regular rat chow and clean water delivered to them at liberty.
Study Design
Following acclimatization period, animals were divided into five (5) groups of five (5) rats each (n = 5). They were then fed with aqueous seed extract of Gacinia Kola for 21 days (3 weeks) as follows;
Group 1 (Control): Received standard rat diet and water ad libitum
Group 2: Received 100 mg/kg body weight of aqueous seed extract of Gacinia Kola
Group 3: Received 200 mg/kg body weight of aqueous seed extract of Gacinia Kola (Olaleyeet al., 2006, 2014)
Group 4: Received 300 mg/kg body weight of aqueous seed extract of Gacinia Kola (Obi andNwoha, 2014)
Group 5: Received 400 mg/kg body weight of aqueous seed extract of Gacinia Kola (Onasanwo and Rotu, 2014)
Preparation of g. Kola extract
Nearly 8.2kg of crushed Garcinia kola seed was measured into a glass container, then mixed with 5 liters of solvent (pure n-hexane), swirled every 2 hours, and allowed to sit for 72 hours. The defatting procedure was repeated for another 72 hours by injecting another 2 liters of pure n-hexane to the plant shaft. This was done to ensure that the fat in the Garcinia kola was properly removed. The crude fat-containing solvent (n-hexane) was collected. After being filtered, the solvent (n-hexane) containing the crude fat collected after 72 hours was combined and concentrated using a rotary evaporator. It was then set at 40°C and concentrated further in a vacuum oven at 40°C and 600mm Hg.
To eliminate the traces of n-hexane utilized, the Garcinia kola shaft (that is, defatted seeds) was distributed and air-dried for 5 hours. The defatted, dried marc was repacked into a glass container with 5 liters of solvent (water), which was added in two-hour intervals and let to stand for 72 hours. After 72 hours, the process was repeated by adding another 5 liters of distilled water to the plant shaft. After 72 hours, the solvent (pure water) containing the crude aqueous extract was collected and concentrated using a rotary evaporator after being filtered. It was then set at 40 degrees Celsius and further concentrated in a vacuum oven at 40 degrees Celsius and 600mm Hg.
With methanol and an equivalent volume of water, the crude extract was converted into a solution. It was carried out in batches. 200ml of this combination (methanol/water) was added to 200ml of chloroform and transferred to a 500ml separating funnel, which was properly shaken and allowed to sit for 30 minutes for good chloroform and mixture (methanol/water) partitioning. With the help of chloroform, this process was repeated four times for effective extraction of kolaviron (active component of G. Kola). A rotary evaporator was used to collect and concentrate the chloroform fraction at a temperature of 40°C. To completely eliminate any trace of solvent, the crude chloroform fraction was further concentrated in a vacuum oven at 40°C and 600mmHg (chloroform). The following is how the percentage yield was calculated:
Preparation of stock solutions from g. Kola extract
After weighing 2 grams of G. Kola with an electronic scale, the powder was homogenized in a pestle and mortar with 10 milliliters of distilled water, then filtered with Wattmann filter paper. This yielded a stock solution with a concentration of 200 mg/mL. G. Kola doses were graded [high, medium, low, and very low] based on a previously determined lethal dose (192mg/kg). To make the aforementioned stock solutions, 1g, 2g, 3g, and 0.4g were dissolved in 100 ml, 200 ml, 300 ml, and 400 ml of distilled water, respectively. The animals' body weights were then measured, and the millilitre dose of test medications to be supplied was computed.
Administration of g. Kola
For two weeks, the rats in the treatment groups were given estimated doses of G. kola stocks (as specified) per kilogram body weight each day. This was accomplished using an orogastric tube, while the control rats were given an identical volume of distilled water via the same method and for the same amount of time.
Blood Sample Collection
The animals were terminated by cervical decapitation at the conclusion of the twenty-first (21) day, and blood samples were taken from the superior vena cava. The sample was centrifuged for 15 minutes at 3000rpm, and the sera obtained were frozen. Blood was drawn from the animals once a week for three weeks and placed in an anti-coagulant (EDTA) vial for hematological analysis. Hematological characteristics The Mind ray Hematology Analyzer was used to determine hematological parameters (Mind ray BC-2800, Guangzhou Shihai Medical Equipment Co. Ltd, China).
Ethical Consent
Ethical approval was granted by the research and ethics committee of the Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka, Anambra State. global best practices for use of laboratory animals were also strictly adhered to.
Statistical Analysis
After statistical analysis, the obtained data were reported as Mean SEM (Standard Error of Mean), with one-way analysis of variance (ANOVA) used to analyze mean differences across various groups, with p-values less than 0.05 (p 0.05) being statistically significant. For all statistical procedures, the graph pad prism was utilized (version 8.1).
Key: PCV = Packed Cell Volume, HGB = Hemoglobin, RBC = Red Blood Cell count
Values are presented as Means ± SEM, n = 5. Values in each column with different alphabets superscripts are statistically differ significantly (p ˂ 0.05).
\Key: WBC = White Blood Cell Count, NEU = Neutrophil Count
Values are presented as Means ± SEM, n = 5. Values in each column with different alphabets superscripts are statistically differ significantly (p ˂ 0.05).
Key: LYM = Lymphocyte Count, EOS = Eosinophil Count, BAS = Basophil Count, MON = Monocyte Count
Values are presented as Means ± SEM, n = 5. Values in each column with different alphabets superscripts are statistically differ significantly (p ˂ 0.05).
Many plant-derived bioactive compounds have been shown to benefit both plants and animals [14]. Some of these useful compounds have been industrially manufactured and employed to imitate the effects of the compounds in their natural state in living beings in a number of ways. Blood compositions are fairly consistent under typical settings, and fluctuations in specific species are kept to a minimum. The biological effects of dietary components on animals are indicated by blood indicators. Typically, each change in an animal's physiological state leads to changes in its hematological parameters [15].
In this study, the observed declines in PCV and HGB numbers (table 1) indicates that G. kola has the capacity to lower RBCs. This significant drop suggests that the seed of G. kola may have RBC-lowering properties and, as a result, may be an anemia-promoting agent. Given the clinical relevance of PCV and HGB, unrestricted use of G. kola may have negative health consequences. Although insignificant, the current study's findings on the effect of G. kola extract on RBC count are in contrast to those of Uko et al (2001) [16], who reported a significant reduction in HGB (g/100ml), Haematocrite (percent), and RBC (106 ml) counts, but are in agreement with those of Ahumibe and Braide (2009) [17], who reported PCV, HGB, and RBC counts to increase G. kola has a favorable effect on haematological indices, according to Dada and Ikuerowo (2009), who found no significant change in RBC levels of fish given various concentrations of G. kola. Intriguingly, Esomonu et al. (2005) [18] observed a substantial reduction in PCV, RBC, and HGB in rats treated with 2 g/kg of G. kola in the first week but thereafter became insignificant compared to control in the second through fifth weeks. G. kola has been shown to have the ability to regulate aberrant RBC indices linked to diseases such polycytemia [19]. G. kola seed extract possesses erythropoietic properties, according to Oluyemi et al. [20], it is possible that it lowers RBC production by reactive oxygen species (ROS). The antioxidant capacity of G. kola is responsible for its ability to effectively scavenge ROS that destroys RBC and reduce lipid peroxidation in the membranous tissues of erythrocytes [19].
The observed changes in WBC count (table 2) as a result of ingesting G. kola extract were considerable, implying that G. kola has a significant impact on the immune system. This finding supports the findings of Uko et al., (2001) and Dada and Ikuerowo (2009) [16, 21], which found that total WBC counts increased with increasing G. Kola administration, but contradicts the findings of Ahumibe and Braide (2009), who found that total WBC counts decreased with increasing G. Kola administration. The effects of G. kola on Neutrophil and WBC counts, given the ever - increasing dose administration (table 2), supports numerous claims about its antimicrobial potentials, given the important role that these haematological parameters are said to play in the body's immunity defense mechanisms, both in man and animals, as disclosed by Dada and Ikuerowo (2009). Furthermore, results on differential WBC counts demonstrated a substantial rise (p 0.05) in basophil and eosinophil counts, as well as lymphocyte and monocyte counts, when compared to control animals (table 2 and 3).
G. kola extract administration at 300 mg/kg body weight resulted in a substantial rise in lymphocytes, implying an immunological impact. Okoko and Oruambo (2008) [22] and Dada and Ikuerowo (2008) [23] also support this conclusion (2009). Other studies have found that it has antibacterial, antiviral, antifungal, antimicrobial, anti-inflammatory, and anti-parasitic properties when compared to a control group [3, 4]. However, the fact that G. Kola seed had no significant effect on platelet and monocyte counts in this investigation demonstrates that the test extract had no negative impact on the thrombopoietic system's functioning.
G. kola's stimulatory effect on HGB and PCV levels may be clinically useful; RBC counts, hemoglobin, and hematocrit have all been documented to be changed in some way by G. kola extract. Because reticulocytes are immature red blood cells, the decreasing effect of increasing test extract dose on RBC could indicate a decrease in erythropoiesis (table 1), even while boosting HGB and reticulocyte numbers, which could induce (or exacerbate) hemolytic anemia, as previously indicated.
Functionally, RBCs operate as simple containers (the membrane) for one key cytoplasmic protein, but their membranes, along with cell geometry and cytoplasmic viscosity, are thought to be major drivers of RBC deformability in disease situations. Understanding of the various components of a membrane (lipids - 50% molecular weight, proteins - 40% molecular weight, and carbohydrates - 10% molecular weight) has recently been used to explain how the deformability method is altered in hemolytic anemia, whether directly by bacteria or indirectly by enzymes and/or reactive oxygen species (ROS) produced by WBCs and/or platelets.
There seem to be various theories that could account for the differences in RBCs between treatment groups seen in this study. First, low flow rates owing to anemia induction may have resulted in the depletion of arteriolar O2 and reduction of arteriolar blood PO2, a similar effect seen in the venular circulation as a result of reduced flow speed after interchange with rigid RBCs to low PO2. As a result of the low count, RBC residence time within the vessel is crucially influenced, affecting the amount of O2 that diffuses into the surrounding tissue and, in turn, O2 supply to the capillary network. The changed RBC is linked to a higher surface area-to-volume ratio, which supports the idea of stiff RBCs in the lungs loading less oxygen. This impact, together with the decreased arteriolar circulation and effective capillary permeability, may explain why RBC and PCV values were not significantly reduced despite high dosage extract administration in the current investigation. These possibilities, however, remain theoretical, particularly under low RBC circumstances, where decreased ATP release by changed RBCs has never been investigated.
In this study, low dose administration of G.kola seed extract to rats caused an insignificant drop in RBC, with substantial increase in WBC and HGB counts at high dose. The extract’ increase capability for WBC count is a pointer to the fact that it may contain some biologically active components of immune system improving values; a feat it likely achieve by raising the population of protective white blood cells. The lack of a significant effect of kola seed on HGB, PCV, and RBC count suggests that the herb had no negative impact on the erythropoietic system's functioning. As it stands, the significant drop in RBC levels shows that the seed of G. kola may have killer activity and, as a result, pro-anemia. As a result, uncontrolled ingestion of G. kola may have negative health and wellness consequences, as evidenced by clinical symptoms of increased PCV and HGB levels. Furthermore, an increase in WBC numbers indicates that the test extract has anti-infection properties.
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