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ORIGINAL ARTICLE
Year : 2014  |  Volume : 2  |  Issue : 2  |  Page : 75-78

Sub-acute effects of crude methanolic leaf extract of Irvingia gabonensis (Aubry-Lecomte et O'Rorke Baill) on activated partial thromboplastin time, prothrombin time and platelet values in albino wistar rats


1 Department of Medical Laboratory Sciences, College of Medicine, University of Nigeria, Enugu, Nigeria
2 Departments of Pharmacology and Therapeutics, College of Medicine, University of Nigeria, Enugu, Nigeria
3 Department of Oromaxillofacial Pathology, University of Nigeria Teaching Hospital, Ituku-Ozalla, Nigeria
4 Department of Mathematics, Faculty of Physical Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria

Date of Web Publication17-Nov-2014

Correspondence Address:
Thomas Nubila
Department of Medical Laboratory Sciences, College of Medicine, University of Nigeria, Enugu Campus, Enugu State
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2348-0149.144840

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  Abstract 

Background: The use of herbs such as Irvingia gabonensis (Aubry-Lecomte et O'Rorke Baill) for nutrition and to treat diseases is almost universal among nonindustrialized societies, as it is often more affordable than modern pharmaceuticals. Therefore, the objective of this study was to screen the coagulation potentials of the crude methanolic leaf extract of I. gabonensis in albino wistar rats. Materials and Methods: Thirty albino wistar rats were used for the study. They were grouped into: A, B, C, D and E, with Group A as the control. The test Groups (B-E) were orally administered with graded concentrations (50, 100, 150 and 200 mg/kg body weight) respectively once daily for 9 days. Four ml of venous blood was collected from the ocular plexus on days 3, 6 and 9. Two ml was delivered into a test tube containing 0.25 ml of trisodium citrate anticoagulant for the determination of prothrombin time (PT) and activated partial thromboplastin time (APTT). The remaining 2 ml was emptied into tripotassium ethylene diamine tetra-acetic acid anticoagulant bottle for the determination of platelet count. Results: All investigations were analyzed using standard hematological methods. There was no statistically significant difference (P > 0.05) when all the parameters evaluated in the test groups were compared with the control group on day 3. However, there was a statistically significant decrease in Groups B and C (P < 0.05 each) in APTT when compared with the control group on day 6. In addition, there was no statistically significant difference (P > 0.05) recorded in all the parameters investigated in all the test groups when compared with the control group on day 9. There was neither duration of exposure nor dose-related statistically significant difference (P > 0.05) in all the parameters evaluated in all the test groups when days 3, 6 and 9 were compared. Conclusion: From the result of the present study, it can be concluded that I. gabonensis crude methanolic leaf extract has no adverse effects on PT, APTT and Platelet mean values at low dose and short duration.

Keywords: Coagulation, Irvingia gabonensis, sub-acute


How to cite this article:
Nubila T, Ukaejiofo EO, Nubila NI, Shu EN, Okwuosa CN, Okofu MB, Ogbe J. Sub-acute effects of crude methanolic leaf extract of Irvingia gabonensis (Aubry-Lecomte et O'Rorke Baill) on activated partial thromboplastin time, prothrombin time and platelet values in albino wistar rats . Niger J Exp Clin Biosci 2014;2:75-8

How to cite this URL:
Nubila T, Ukaejiofo EO, Nubila NI, Shu EN, Okwuosa CN, Okofu MB, Ogbe J. Sub-acute effects of crude methanolic leaf extract of Irvingia gabonensis (Aubry-Lecomte et O'Rorke Baill) on activated partial thromboplastin time, prothrombin time and platelet values in albino wistar rats . Niger J Exp Clin Biosci [serial online] 2014 [cited 2019 Sep 22];2:75-8. Available from: http://www.njecbonline.org/text.asp?2014/2/2/75/144840


  Introduction Top


The consumption of plant materials has been on the increase all over the World with Africa and some other developing countries top on the list (Nubila et al., 2013). [1] More than 80% of the world's population depends primarily on traditional medicine for the treatment of ailments and other related health care. [2] This is more predominant in developing countries, where modern Western medicine is often unavailable or is simply too expensive. Hence, medicinal plants do form a major part of indigenous or traditional medicine practice. The medicinal values of these plants lie in some complex chemical substances that produce a definite physiological action on the human body. [3]

Irvingia gabonensis (Aubry-Lecomte et O'Rorke Baill) is a tropical forest tree mostly found in Southern and Eastern Nigeria, Sierra Leone and Equatorial Africa. It is specie from the family Irvingiaceae. The inedible fruit pulp is bitter and acrid although it can be eaten and has a turpentine flavor. [4] It acts mainly as a source of human food and is commonly known as "ogbono" in Igbo land in Nigeria where the seed is used as a food thickener. The seeds are rich in oil (54-67%) calculated on dry kernel. This is known as "dika" fat, which has become evaluated and used now as tablet lubricant. [4]

It has been reported that the seed reduces fasting blood sugar in obese subjects. [5] The leaf extract has also been reported to increase urine output and electrolytes in adult wistar rats. [6] A decoction of the leaves of I. gabonensis is used to treat spleen infection. [7] The aqueous leaf extract of I. gabonensis has been found to cause a significant dose-dependent decrease of gastrointestinal motility in mice. [8] The stem bark has been reported to have analgesic properties. [9] Preliminary phytochemical screening of aqueous leaf extract of I. gabonensis revealed the presence of saponins, tannins, phenols and alkaloids and this leaf is largely used in both traditional and modern medicine for the treatment of several illnesses.[10] The ethanol extract of I. gabonensis may also contain a biologically active properties, which can exhibit agonistic activity on uterine smooth muscles. [6]

The ability of the body to control the flow of blood following vascular injury is paramount to continued survival. The process of blood clotting and then the subsequent dissolution of the clot following repair of the injured tissues is termed hemostasis. Hemostasis is composed of four major events that occur in a set order following the loss of vascular integrity:

  1. The initial phase of the process is vascular constriction. This limits the flow of blood to the area of injury.
  2. Next, platelets become activated by thrombin and aggregates at the site of injury, forming a temporary, loose platelet plug. The protein fibrinogen is primarily responsible for stimulating platelet clumping. Platelets clump by binding to collagen that becomes exposed following rupture of the endothelial lining of vessels. Upon activation, platelets release nucleotides, adenosine diphosphate and eicosanoid, thromboxane A2 (both of which activates additional platelets), serotonins, phospholipids, lipoproteins and other proteins important for the coagulation cascade. In addition to induced secretion, activated platelets change their shape to accommodate the formation of the plug.
  3. To ensure stability of the initially loose platelet plug, a fibrin mesh (also called the clot) forms and entraps the plug. If the plug contains only platelets it is termed white thrombus, if red blood cells are present, it is called red thrombus, and
  4. Finally, the clot must be dissolved in other for normal blood flow to resume following tissue repair. The dissolution of this clot occurs through the action of plasmin.


The objective of the present study was to determine the sub-acute effects of crude methanolic leaf extract of I. gabonensis on prothrombin time (PT), activated partial thromboplastin Time (APTT) and platelet values in Albino wistar rats.


  Materials and methods Top


A sample of the leaf of the plant was authenticated at the Herbarium Unit of the Department of Botany, University of Nigeria Nsukka by a taxonomist. A voucher specimen was deposited at the Herbarium Unit for future references with UNH No 221a. The fresh leaves were collected from their natural habitat from Obeagu village in Enugu South Local Government Area of Enugu state, Nigeria. The leaves were carefully separated from the stem and air-dried under shade for 6 days. The dried leaves were grinded into fine powder with a grinding machine.

Extraction of Leaf Material

Eight hundred grams of the fine powder was weighed and dissolved in 2000 ml of 98% methanol in a 10 l container with daily intermittent vigorous shaking for 2 days. The mixture was strained using fine cheese cloth and the filtrate was further filtered with Whatman filter paper number 1. This was allowed to air-dry at room temperature in an open metallic basin for 5 days.

Forty grams of the semi-solid green residue was stored in the refrigerator (4°C) until use.

Experimental Animals

Thirty male 2-3-month-old Albino wistar rats weighing 2500-280 g were purchased from an Animal Farm in Enugu state and grouped into five cages (A, B, C, D and E) of six rats per cage according to their body weights. These animals were kept in the Animal Research Unit of the University of Nigeria Enugu campus under standard conditions. They were fed with standard rat pelleted diet (super starter feed) for 2 weeks to acclimatize. Good hygiene was maintained by constant cleaning and removal of their feces and leftover feed from the cages daily. The animals were handled according to the Institutional and National guidelines for the care and use of laboratory animals.

Experimental Design

The test Groups (B-E) were orally administered with graded doses (50, 100, 150, 200 mg/kg body weight respectively) of the crude leaf extract once daily for 9 days. Group A served as the control group and received no leaf extract. Two animals were bled from each group after days 3, 6 and 9 of the oral extract administration through the retro bulbar plexus of the median cantus of the eye. [11]

Two ml of venous blood was painlessly collected under thiopentone sodium anesthesia and was delivered into 0.25 ml trisodium citrate anticoagulant bottle using a capillary tube. By gentle inversion, the blood was immediately mixed with the anticoagulant to avoid clothing. Another 2 ml of sample was collected and delivered into tripotassium ethylene diamine tetra-acetic acid anticoagulant bottle which was used for platelet count determination.

The samples were analyzed within 2 h of collection as described by Dacie and Lewis, [12] while the platelet count was analyzed using hematology auto-analyzer (Sysmex KX-21 N, Sysmex America Inc, One Nelson (White Pkwy), Mundelein, IL 60060, USA) following the manufacturers' operational guidelines.

Statistical Analysis

Statistical analysis was carried out using computer statistical software GraphPad-Prism (GraphPad Software Inc, 7825 Fay Avenue, Suite 230 La Jolla, CA 92037, USA). One-way analysis of variance (ANOVA) with post-hoc test for linear trend and Student's t-test were adopted for comparison. The data were expressed as mean ± standard error. P < 0.05 was considered significant.


  Results Top


There was no statistically significant difference (P > 0.05) when all the parameters evaluated in the test groups were compared with the control group on day 3 [Table 1]. However, there was a statistically significant decrease in Groups B and C (P < 0.05) in APTT when compared with the control group on day 6 [Table 2].
Table 1: Comparison of the PT, APTT and platelet values after 3 days of oral crude extract administration with the control group

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Table 2: Comparison of the PT, APTT and platelet values after 6 days of oral crude extract administration with the control group

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In addition, there was no statistically significant difference (P > 0.05) recorded in all the parameters investigated in all the test groups when compared with the control group on day 9 [Table 3].
Table 3: Comparison of the PT, APTT and platelet values after 9 days of oral crude extract administration with the control group

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  Discussion Top


The use of herbs to treat diseases is almost universal among non-industrialized societies. Many of the herbs and spices like I. gabonensis (Aubry-Lecomte et O'Rorke Baill) used by humans to season food also yield useful medicinal compounds. [13],[14]

The result of this present study revealed no statistically significant difference (P > 0.05) when all the parameters evaluated in the test groups were compared with the control group on day 3. This could possibly indicate that the crude methanolic leaf extract did not have any effect on the coagulation factors or any of the coagulation pathways, neither did it have any thrombopoietic activity indirectly on the liver that produces thrombopoietin which is an important hormone that stimulates the bone marrow to produce thrombocyte, which plays a major role in the initiation and regulation of coagulation and other hemostatic activities. Furthermore, one might be tempted to suggest that this leaf may have a high safety margin and hence is not likely to have altered any of the normal physiological coagulation mechanisms, instead one is expecting it to have or possess a stimulatory coagulation activity due to its high nutritional and medicinal potentials. However, this interpretation should be viewed with caution since neither a toxicity study nor mineral and the Vitamin contents (especially the K and B-Vitamins) of the methanolic leaf extract was investigated in the present study. However, from its long-term and high rates of consumption in this community, it could be considered safe even though this might not be enough evidence for its safety.

Interestingly, the statistically significant decrease in Groups B and C (P < 0.05) in APTT mean values when compared with the control group on day 6. This suggests that the methanolic crude leaf extract of I. gabonensis had both dose and duration-related stimulatory effects on the coagulation factors, more especially on the intrinsic pathway of coagulation, hence a reduction in APTT. However, this was contradicted by a nonstatistically significant dose or time dependent difference (P > 0.05) in all the parameters evaluated in all the test groups in the one-way ANOVA. This could further be attributable to the inhibition of the plasmas protease inhibitors such as antithrombin, which can act by dampening or neutralizing the activity of proteolytic enzymes generated in the coagulation, fibrinolytic, and kinin gemerating system, which invariably will stimulate the coagulation factors, hence coagulation and some other hemostatic processes. The crude leaf extract could also have stimulated the production of some of the coagulation factors in the liver. However, there was no previous scientific literature for comparison.

Surprisingly, there was no statistically significant difference (P > 0.05) recorded in all the parameters investigated in all the test groups when compared with the control group on day 9. This tends to confirm that the crude methanolic leaf extract seems not to have any adverse effects on coagulation and hemostasis at low dose and short duration and also point to the fact that its consumption could be relatively safe by subjects prone to developing coagulation and other related bleeding disorders and on the other hand could be used to ameliorate bleeding disorders.


  Conclusion Top


From the result of the study, it can be concluded that I. gabonensis crude methanolic leaf extract has no adverse effects on APTT, PT and platelet mean values at low dose and short duration. However, a chronic study at higher dose is advocated.



 
  References Top

1.
Nubila T, Ukaejiofo EO, Nubila NI, Okwuosa CN, Chijioke CP, Shu EN, et al. Subacute effects of crude methanolic leaf extract of Mucuna flagellipes (Vogel ex Hook) on haematological profile and haematinic activities in albino wistar rats. Int J Med Aromat Plants 2013;3:452-8.  Back to cited text no. 1
    
2.
Nubila T, Ukaejiofo EO, Nubila NI, Iyare EE, Chijioke CP, Ukaejiofo AC, et al. Methanolic crude leaf extract of Occimum gratissimum reverses phenylhydrazine-induced anaemia in albino wistar rats. Niger J Exp Clin Biosci 2013;1:23-7.  Back to cited text no. 2
    
3.
Nubila T, Ukaejiofo OE, Nubila IN, Shu NE, Okwuosa CU, Ukaejiofo CA, et al. Effects of methanolic seed extract of Telfaira occidentalis on blood coagulation in albino wistar rats. Niger J Exp Clin Biosci 2013;1:10-3.  Back to cited text no. 3
    
4.
Udeala OK, Onyechi J, Agu SI. Sourcing pharmaceutical raw materials from indigenous medicinal plants. J pharm pharmacol 1980;32:6.  Back to cited text no. 4
    
5.
Ngondi JL, Oben JE, Minka SR. The effect of Irvingia gabonensis seeds on body weight and blood lipids of obese subjects in Cameroon. Lipids Health Dis 2005;4:12.  Back to cited text no. 5
    
6.
Nosiri CI, Abdu-Aguye I, Abdulrahman E, Hussaini MI. Leaf extract of Irvingia gabonensis increase urine output and electrolytes in rats. Internet J Altern Med 2011;8:2.  Back to cited text no. 6
    
7.
Sofowora A. The State of Medicinal Plant Research in Nigeria. Vol. 3. Chichester: John Wiley and sons Ltd.; 1986. p. 251-4.  Back to cited text no. 7
    
8.
Abdulrahman F, Inyang IS, Abbah J, Binda L, Amos S, Gamaniel K. Effect of aqueous leaf extract of Irvingia gabonensis on gastrointestinal tract in rodents. Indian J Exp Biol 2004;42:787-91.  Back to cited text no. 8
    
9.
Okolo CO, Johnson PB, Abdurahman EM, Abdu-Aguye I, Hussaini IM. Analgesic effect of Irvingia gabonensis stem bark extract. J Ethnopharmacol 1995;45:125-9.  Back to cited text no. 9
    
10.
Lowe AJ, Gillies ACM, Wilson J, Dawson IK. Conservation genetics of bush mango from central/west Africa, implications from RAPD analysis. Mol Ecol 2000;9:831-41.  Back to cited text no. 10
    
11.
Parasuraman S, Raveendran R, Kesavan R. Blood sample collection in small laboratory animals. J Pharmacol Pharmacother 2010;1:87-93.  Back to cited text no. 11
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12.
Dacie JV, Lewis SM. Practical Hematology. 10 th ed. Belfast: University Press; 2006. p. 110-5.  Back to cited text no. 12
    
13.
Lai PK, Roy J. Antimicrobial and chemopreventive properties of herbs and spices. Curr Med Chem 2004;11:1451-60.  Back to cited text no. 13
    
14.
Tapsell LC, Hemphill I, Cobiac L, Patch CS, Sullivan DR, Fenech M, et al. Health benefits of herbs and spices: the past, the present, the future. Med J Aust 2006;185:S4-24.  Back to cited text no. 14
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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