|Year : 2013 | Volume
| Issue : 1 | Page : 10-13
Effects of methanolic seed extract of Telfairia occidentalis on blood coagulation in Albino rats
Nubila Thomas1, Ukaejiofo Okem Ernest1, Nubila Imelda Nkoyo2, Shu Neba Elvis2, Okwuosa U Chukwubuzor3, Ukaejiofo C Ayodele4, Iyare E Eghosa5, Ogbuta Osule Ifeyinwa1
1 Department of Medical Laboratory Sciences, College of Medicine, University of Nigeria, Enugu Campus, Enugu, Enugu State, Nigeria
2 Department of Pharmacology and Therapeutics, College of Medicine, University of Nigeria, Enugu Campus, Enugu, Enugu State, Nigeria
3 Department of Oral and Maxillofacial Pathology, University of Nigeria Teaching Hospital, Ituku-Ozalla, Enugu, Enugu State, Nigeria, Nigeria
4 Department of Medical Rehabilitation, College of Medicine, University of Nigeria, Enugu Campus, Enugu, Enugu State, Nigeria
5 Department of Physiology, College of Medicine, University of Nigeria, Enugu Campus, Enugu, Enugu State, Nigeria
|Date of Web Publication||30-Dec-2013|
Department of Medical Laboratory Sciences, Faculty of Health Sciences And Technology, University of Nigeria, Enugu Campus, Unec
Source of Support: None, Conflict of Interest: None
Background: Medicinal plants have played a key role in the world healthcare with about 80% of Africans depending on phytomedicine, which has shown a wide range of uses in the treatment of diseases especially priority diseases of Africa. The objective of this study was to investigate the sub-acute effects of methanolic seed extracts Telfairia Occidentalis on prothrombin time (PT), activated partial thromboplastin time (APTT), and platelet values in Albino Wistar rats. Materials and Methods: Thirty rats were equally divided into groups A (control), B, C, D, and E (test). Groups B, C, D, and E were gavaged with 20, 40, 80 and 160 mg/kg body weight, respectively, of the extract for 9 days. Four milliliters of venous blood was collected from each animal and 2 ml delivered into 0.25 ml tri-sodium citrate and K 3 ethylene diamine tetra acetic acid (EDTA) anticoagulant bottles for the determination of PT, APTT, and platelet values, respectively. Results: APTT showed a statistically significant increase in groups D (P < 0.01) and E (P < 0.05) when compared with the control on Day 3. Platelet value demonstrated a time-dependent statistically significant decrease (P < 0.05). Conclusion: In conclusion. T. occidentalis demonstrated sub-acute inhibitory effects on coagulation.
Keywords: Coagulation, seed extract, sub-acute, Telfairia occidentalis
|How to cite this article:|
Thomas N, Ernest UO, Nkoyo NI, Elvis SN, Chukwubuzor OU, Ayodele UC, Eghosa IE, Ifeyinwa OO. Effects of methanolic seed extract of Telfairia occidentalis on blood coagulation in Albino rats. Niger J Exp Clin Biosci 2013;1:10-3
|How to cite this URL:|
Thomas N, Ernest UO, Nkoyo NI, Elvis SN, Chukwubuzor OU, Ayodele UC, Eghosa IE, Ifeyinwa OO. Effects of methanolic seed extract of Telfairia occidentalis on blood coagulation in Albino rats. Niger J Exp Clin Biosci [serial online] 2013 [cited 2019 Sep 22];1:10-3. Available from: http://www.njecbonline.org/text.asp?2013/1/1/10/123943
| Introduction|| |
The developing countries mostly rely on medicinal plants even though its use is still poorly understood by the general public. These medicinal plants are of great importance to health of individuals and the general communities. The medicinal values of these plants lie in some complex chemical substances that produce a definite physiological action on the human body. Fluted pumpkin also known as Telfairia occidentalis is a species of Cucurbitaceae family in the tropics and largely consumed in Nigeria, Ghana and Sierra Leone,  and Cameroon. The seed is widely consumed in Nigeria, especially in the southeastern part of Nigeria where it is used as a condiment in soup  and as food. The fermented seed of fluted pumpkin is used in the production of "Ogiri ugu," a locally made custard. The seeds of fluted pumpkin could also be used in cookie formulations and marmalade manufacturing. The seed is also a good source of edible oil. The seed contains oil, which is used for cooking. , The oil has saponification values that are beyond the range of most oil of plants origin and less than that of palm oil. Meaning that, the oil has larger molecular weight than the common oil.  It also has high iodine value compared with palm oil, indicating that it has high content of unsaturated fatty acids relative to palm oil. It may be used for cooking or manufacturing of margarine and other related products. It has high specific gravity compared with commonly known vegetable oil. It has low acid value also indicating that the oil is edible.  The oily seeds have lactating properties and are widely consumed by the nursing mothers.  Fluted pumpkin seed oil (FPSO) has been reported to ameliorate the effect of quinine induced testicular damage.  Due to its high phosphorus content, it is said to be a potential agent in reducing kidney bladder stone disease.  The antioxidative property of FPSO could also enhance fertility.  Fluted pumpkin is commonly grown and consumed in Nigeria because of its high nutritive value  and medicinal potentials.
Coagulation is a complex process by which blood forms clot. It is an important part of homeostasis, the cessation of blood loss from a damaged vessel, wherein a damaged blood vessel wall is covered by a platelet and fibrin-containing clot to stop bleeding and begin repair of the damaged vessel. Disorders of coagulation can lead to an increased risk of bleeding (hemorrhage) or obstructive clotting (thrombosis). Coagulation is highly conserved throughout biology; in all mammals, coagulation involves both a cellular (platelet) and a protein (coagulation factor) component. The system in humans has been the most extensively researched and is therefore the best understood.
Coagulation begins almost instantly after an injury to the blood vessel has damaged the endothelium lining the vessel. Exposure of the blood to proteins such as tissue factor initiates changes to blood platelets and the plasma protein fibrinogen, a clotting factor. Platelets immediately form a plug at the site of injury; this is called primary hemostasis. Secondary hemostasis occurs simultaneously: Proteins in the blood plasma, called coagulation factors or clotting factors, respond in a complex cascade to form fibrin strands, which strengthen the platelet plug. 
Despite the wide and large consumption of T. occidentalis leaf and seed either nutritional or medicinal purposes in the sub-Sahara countries, its influence on coagulation is still looking in scientific literature. The aim of this study therefore was to determine the sub-acute effects of crude methanolic seed extract of T. occidentalis on prothrombin time (PT) and activated partial thromboplastin time (APTT) and platelet values in Albino Wistar rats.
| Materials and Methods|| |
The seed was identified by a taxonomist, Prof J.C. Okafor, in the Department of Botany, Coal City University Enugu State, Nigeria. A voucher specimen was deposited for future reference (UNH No 1 a ). The seed was later purchased from a local market in Enugu, Enugu State, Nigeria. The seed was allowed to air-dry under shade and later grinded into fine powder.
Extraction of Plant Materials
Eight hundred (800) grams of the fine powder was weighed and dissolved in 3000 ml of 98% methanol. This was allowed for 48 hours with intermittent vigorous shaking. The mixture was strained using fine cheese cloth and filtered using Whatman paper no.1. The filtrate was then allowed to air-dry at room temperature in an open stainless silver basin. The semi-solid oily brown residual substance was stored in the refrigerator at 4°C till used.
Thirty (30) male Albino Wistar rats weighing 130-200 g obtained from the Animal House College of Medicine, University of Nigeria, Enugu Campus were used for the study. The rats were divided into five groups A, B, C, D, and E, with six rats per cage. The rats were housed in well stainless steel washed cages under standard rats' pelleted diet (starter top feed) and water throughout the study period. Ethical approval was obtained from the University Nigeria Nsukka, Enugu State, Nigeria, Animal Study Review Board and the approved guidelines for the care and use of laboratory animals was followed.
From the LD 50 value which was greater than 5000 mg/kg body weight  the test groups (B-E) were orally fed with graded doses (20, 40, 80, and 160 mg/kg body weight), respectively of the plant extract, once daily for 9 days. Group A served as control and received no treatment. Four milliliters of venous blood was painlessly and aseptically collected from each animal. Two animals were bled from each experimental group at Days 3, 6, and 9 from the retrobulbular plexus of the median cantus of the eye under very mild anesthesia. Two milliliters was delivered into a tube containing 0.25 ml of tri-sodium citrate anticoagulant and mixed gently by inversion. This was used for PT and APTT determination, while the remaining 2 ml was dispensed into a tube containing tri-potassium ethylene diamine tetra acetic acid (K 3 EDTA) anticoagulant for platelet count determination. The supernatant citrated plasma after spinning for 15 min at 3000 r.p.m. was removed and placed in a clean glass tube. All samples were analyzed within 2 hours of collection. PT, APTT, and platelets were determined by standard method as described by Dacie and Lewis,  while the platelet count was determined using an automated hematology analyzer-sysmex Kx-21N.
Data was analyzed with the Graph-Pad Prism computer Software. Students''t' test and two-way analysis of variance (ANOVA) were used to compare data. P < 0.05 was considered significant.
| Results|| |
There was no statistically significant difference (P > 0.05) when the pre- and postweights of the experimental animals were compared [Figure 1].
|Figure 1: Represents the mean weights of the rats before and after administration of methanolic seed extract of T.occidentalis (P > 0.05 between pre- and postweight in all the test groups)|
Click here to view
APTT mean value showed a statistically significant increase (P < 0.01) in group D (80 mg/kg) and (P > 0.05) in group E (160 mg/kg) when compared with the control group A, on Day 3 [Table 1].
|Table 1: Comparison of the mean ± SE of the PT, APTT, and platelet values after 3 days of oral crude extract administration with the control group|
Click here to view
However, there was no statistically significance difference (P > 0.05) in all parameters evaluated in the test groups when compared with the control group on Days 6 and 9 [Table 2] and [Table 3].
|Table 2: Comparison of the mean ± SE of the PT, APTT, and platelet values after 6 days of oral crude extract administration with the control group|
Click here to view
|Table 3: Comparison of the mean ± SE of the PT, APTT, and platelet values after 9 days of oral crude extract administration with the control group|
Click here to view
In addition, PT mean value recorded a dose-dependent statistically significant decrease (P < 0.05) on Day 3 when groups B, C, D, and E were compared. Furthermore, platelet mean value demonstrated a time-dependent statistically significant decrease (P < 0.05) when Days 3, 6, and 9 were compared.
| Discussion|| |
Several medicinal uses of fluted pumpkin in traditional medicine have not been documented, and many of these claims are yet to be validated by scientific research. Similarly, despite its widely consumption, information on its effects on coagulation has not being documented. Hence, this study was designed to investigate the sub-acute effect of T. occidentalis on PT, APTT, and platelet mean values in Albino Wistar rats. T. occidentalis commonly known as "fluted pumpkin" as a herb, has several medicinal properties; the seed extract is useful in the management of liver problems and impaired defense immune system.  There was no statistically significant difference (P > 0.05) when the pre- and postweights of the experimental animals were compared at the end of the study period. This indicates that the methanolic seed extract, most likely had no effect on weight. However, this result should be treated with caution since the extract was only administered for 9 days. Also, one could have expected a dramatic increase since earlier scientific report have claimed that the seeds are highly nutritious ,,, and a good source of four minerals required in human nutrition.  Whether there is a relationship between weight changes and blood coagulation is subject to further investigation in a chronic study.
APTT showed a statistically significant increase (P < 0.01) and (P < 0.05) in groups D (80 mg/kg) and E (160 mg/kg), respectively, when compared with the control group on day 3. This could be attributed to either the inhibitory effect of the crude seed extract indirectly on the liver where the coagulation factors are being synthesized or directly in the peripheral system where they function. It could also be due to the inhibition of the coagulation proteins by the methanolic seed extract, which invariably is most likely to prolong coagulation, more especially the intrinsic coagulation pathway. In addition, the most probable reason could be due to the phytochemical composition of T. occidentalis which flavanoid is one and has being shown to be an effective inhibitor of platelet aggregation and also possesses antithrombotic activities  because they directly scavenge free radicals, thereby maintaining proper concentration of endothelial prostacyclin and nitric oxide.
Furthermore, one could also suggest that since flavanoids are powerful antithrombotic agent because of their inhibition of the activities of cyclo-oxygenase and lipo-oxygenase pathways as earlier reported by Tzeng et al.,  is most likely to have also contributed to the prolonged APTT in particular and maybe coagulation in general. There was no statistically significant difference (P > 0.05) in all parameters investigated in the test groups when compared with the control group on days 6 and 9. This could be attributed to the fact that, the test groups might have by normal physiological phenomenon adjusted/adapted to the adverse effect on day 3. However, this was disproved by a dose-dependent statistically significant decrease (P < 0.05) in PT on day 3 in the one-way ANOVA. This was further supported by a time-dependent statistically significant decrease (P < 0.05) in platelet count in the one-way ANOVA. However, this alteration could be due to increase in arachidonic acid by platelets to form prostaglandins, endoperoxides, and thromboxane A 2 , which invariably stimulate platelet activation and aggregation. 
Finally, platelet mean values generally recorded a marginal, nonstatistically significant decrease (P > 0.05) in the test groups when compared with control group throughout the study period. This suggests that T. occidentalis is most likely to possess some antithrombopoietic activity. Hence, its consumption among subjects with bleeding disorders should be evaluated.
| Conclusion|| |
T. occidentalis demonstrated sub-acute inhibitory effects on coagulation in healthy Albino Wistar rats. Hence, patients with bleeding disorders or who are prone to bleeding disorders should consume T. occidentalis with caution and constant evaluation of their coagulation status is hereby recommended.
| References|| |
|1.||Nkang A, Omokaro A, Egbe A, Amanke G. Variation of fatty acid proportion during dessication of telfairia occidentalis seeds harvested at physiological and agronomic maturity. Afr J Biotechnol 2003;2:33-9. |
|2.||Agatemor C. Studies of Selected Phytochemical Properties of Fluted Pumpkin (Telfairia Occidentalis Hook F.) Seed Oil and Tropical Almond (Terminalis Catappia) Seed Oil. Pak J Nutr 2006;5:306-30. |
|3.||Horsfall M Jr, Spiff IA. Equilibrium Sorption Study of Al, Co 3+ and Ag 2+ In Aqueous Solution of Fluted Pumpkin (Telfairia Occidentalis Hook F) Waste Biomass. Acta Chim Slov 2005;52:174-81. |
|4.||Pearson D. The Chemical Analysis of Foods. 7 th ed. London: Churchill Living Stone; 1976. p. 105 |
|5.||Ajibade SR, Balogun MO, Afolabi OO, Kupolati MD. Sex differences in the Biochemical contents of telfairia occidentalis Hook F. J Food Agric Environ 2006;4:155-6. |
|6.||Nwangwa EK, Mordi J, Ebeye OA, Ojieh AE. Testicular regenerative effects induced by the extracts of telfairia occidentalis in rats. Caderno De Pesquisa, Serie Biologi 2006;19:27. |
|7.||Suphakam S, Yarnon C, Ngunboonsri P. The effect of pumpkin seeds on oxalcrystalluria and urinary composition of children in hyperendemic area. Am J Clin Nutr 1987;45:115-21. |
|8.||Murkovic M, Hillebrand A, Winker J. Variability of Fatty Acid Content in Pumpkin Seeds (Cucurbita Pepo L.). Z Lebensm Unters Forsch 1996;203:216-9. |
|9.||Orhue ER, Ekhomun A, Dafikpaku M, Eze L. The influence of Zinc on early growth and Nutrient uptake by Pumpkin (Telfairia Occidentalis Hook F) In Albino wistar rats. Ultisol J Appl Nat Sci 2010;2:8-12. |
|10.||Furie B, Furie BC. Thrombus Formation In Vivo. J Clin Invest 2005;115:12. |
|11.||Dacie, Lewis. Practical Haematology. 10 th ed. London: Churchill Livingstone; 2006. p. 398-400. |
|12.||Ojo NA, Adawaren EO, Tijani MB, Chiroma M, Simon J, Afisu B, et al. Acute toxicity and effect of ethanolic extract of tefairia occidentalis leaves on blood glucose level in normal rats. Vom J Vet Sci 2012;9:25-31. |
|13.||Eseyin OA, Igboasoiyi AC, Oforah E, Ching P, Okoli BC. Effects of leaf extract of Telfairia occidentalis on some biochemical parameters in rats. Pak J Biol Sci 2007;15:3240-2. |
|14.||Akubue PI, Kar A, Nncheita FN. Toxicity of extracts of roots and leaves of Telfairia Occidentalis. Planta Med 1980;38:339-43. |
|15.||Odoemena CS, Onyeneke EC. Lipids of Fluted Pumpkin (Telfairia Occidentalis) Seeds. Proceedings of the 1 st African Conference on biochemistry of lipids, (Acbl'98). Benin City, Nigeria: Ambik Press; 1998. p. 147-51. |
|16.||Okoli BE, Mgbeogu CM. Fluted pumpkin, telfairia occidentalis: West African vegetable crop. Econ Bot 1983;37:145-9. |
|17.||Okoli BE, Nyanayo BL. Polynology of Telfairia L. (Cucurbitacae). Folia Geobotanica Phytotaxonomica 1988;23:281-6. |
|18.||Taylor OO, Fetuga BL, Oyenuga VA. Accumulation of Mineral Elements in Five Tropical Leafy Vegetables As Influenced By Nitrogen Fertilization and Age. Sci Hortic 1983;18:313-22. |
|19.||Gryglewski Rj, Korbut R, Robak J, Swies J. On mechanism of antithrombotic action Of flavanoids. Biochem Pharmacol 1987;36:317-22. |
|20.||Tzeng SH, Ko WC, Ko FN, Teng CM. Inhibition of platelet aggregation by some flavanoids. Thromb Res 1991;64:91-100. |
|21.||Wang HK, Xia Y, Yang ZY, Natschke SL, Lee KH. Recent advances in the discovery and development of flavanoids and their analogues as Antitumour and Anti-Hiv Agents. Adv Exp Med Biol 1998;439:191-225. |
[Table 1], [Table 2], [Table 3]