1. Introduction

Pentaclethra macrophylla (African oil bean) produces its seeds used for ugba production from a perennial legume tree ^[1]. The trees are often planted along the sides of roads as shade trees and around communities as cash crops. African oil bean is a tropical tree crop found mostly in the Southern rain forest zone of West Africa and has been cultivated since 1937. It belongs to the leguminosae family and sub-family Mimosoideae. African oil bean tree grows to about 21 meters in height and to about six meters in girth and it is well branched, forming crown-like canopy. The leaves have stout angular petiole. The compound leaves are usually about 20 centimeters large and covered with rusty hairs. The flowers are creamy, yellowish or pinkish-white and sweet smelling. The main flowering season is between March-April after which the pods (brown and woody when matured) open by explosive mechanism, dispersing the seeds and curls up, releasing about eight flat, glossy brown seeds measuring about 5-7 cm in diameter and weighing between 15-20g ^[2].

In Nigeria it thrives in the eastern and western parts. Ugba is the Igbo name for the fermented African oil bean seeds. It is called Apara by the Yoruba’s and Ukana by the Efiks ^[2]. The African oil bean seed is fermented and consumed especially in eastern states of Nigeria. The fermented product Ugba (old Imo State) or Ukpaka (Old Anambra State) is eaten alone or with other ingredients like stockfish, garden egg, sliced tapioca or can be mixed with vegetable popularly known as Africa salad in the Eastern part of Nigeria ^{[3, 4]}.

The oil bean seeds contain 8-10% carbohydrate and 47-48% of fatty acid ^[5]. About 75% of the total oil in the seed is made up of unsaturated fatty acids, predominantly oleic and linoleic acid. The pharmacological importance derivable from its seeds, roots, bark and leaves are of benefit in the treatment and management of obesity, itching, heart problems, high blood pressure ^[6]. The seed is a source of oil used for candle making and soaps. The seed shells are decorative and often used as craft beads, which are worn as necklaces and sometimes as local dance apparels.

Anticoagulants are substances or agents which prevent or retard clotting of blood ^[7], by removing calcium ^[8]. It could be used in vivo (inside the body) or in vitro (outside the body). This work therefore is an exploratory research effort towards identifying and characterizing new anticoagulants. Extract of Pentaclethra macrophylla was subjected to anticoagulation activity and compared to EDTA and sodium citrate.

2. Materials and Methods

The seeds of Pentaclethra macrophylla were procured from a local market in Elele, Rivers State. Then washed clean with tap water before they were manually cracked to extract the edible cotyledons, which were sliced and dried to constant weight at 75oC in an air draught oven (Astell-Hearson, London, UK). The dried samples were pulverized, mixed thoroughly and stored in polythene bags in a refrigerator. The dried sample was weighed out (0.1g, 0.2g and 0.5g) and introduced into plain containers, 2ml of blood was equally added to each container and observed for clotting.

Verbal consent was obtained from ten (10) human volunteers. 4ml of blood sample was collected from ante cubital part of the arm and dispensed 2ml each into 0.1g of the extract and EDTA/sodium citrate container. The blood samples were analyzed within six hours of collection. Packed cell volume (PCV), Platelet count, total white blood cell count, activated partial thromboplastin time (APTT), prothrombin time (PT) and haemoglobin concentration were analyzed using standard methods ^{[8, 9]}.

3. Results

Table 1. Clotting time for different concentrations of the extract

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When 2ml of blood was introduced into plain bottles containing 0.2g and 0.5g of the extract, clotting was achieved at 2520 sec and 105 sec respectively while at 0.1g/2ml of blood clotting was unattained.

Table 2. Mean ± SD of extract compared to EDTA/sodium citrate

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4. Discussion

Some plants have been attributed to possess anticoagulation property ^{[10, 11, 12]} but have not been properly utilized. Anticoagulant from plant source should definitely have better safety margin and eliminate monitoring of therapy. Such anticoagulant may present with little or no side effects both for laboratory and clinical use ^[13]. In this present study, the extract of Pentaclethra macrophylla clotted 2ml of human blood sample at concentration of 0.2g and 0.5g in 2520sec and 105sec respectively while at 0.1g/2ml of blood, coagulation was not achieved. This indicates that the extract of Pentaclethra macrophylla exhibits anticoagulation activity. Medicinal plants show good therapeutic effect comparable to orthodox drugs and yet exhibit minimal unwanted side effects ^[14]. Salvia multiorrhiza and D. chrysamthum have also been shown to possess anticoagulation activity ^{[12, 15]}.

The extract-anticoagulated blood was compared with other anticoagulants, EDTA and sodium citrate using some routine haematological parameters. The PCV of the extract was 39.2 ± 3.83% while that of EDTA was 40.0 ± 4.00% which is statistically not significant (p>0.05). Also, at haemoglobin concentration of 13.0 ± 1.69g/dl and 13.3 ± 2.19g/dl for the extract and EDTA respectively, the difference was not significant (p>0.05). The extract is rich in potassium/calcium content ^[16], vitamins and iron ^[17]. This could possibly be the reason for its anticoagulation activity.

White blood cell count (WBC) of the extract-anticoagulated blood gave 9.4 ± 1.55 x109/l while EDTA gave 8.7 ± 2.26 x109/l. The difference was insignificant (p>0.05) when compared. Platelet count which is used to assess coagulation was also analyzed. The platelet count was 307.4 ± 86.30 x109/l and 305.2 ± 84.54 x109/l for extract-anticoagulated blood and EDTA respectively. Though much literature is unavailable to compare with previous works, the difference between the extract of Pentaclethra macrophylla and EDTA showed little or no difference. This shows a promising prospect for its use for in vitro studies.

The extract-anticoagulated blood recorded a reduced APTT value of 9.0 ± 1.41sec compared to that of sodium citrate which was 44.2 ± 5.63sec. The prothrombin time was 20.8 ± 1.30sec and 14.8 ±2.16sec for the extract and sodium citrate respectively. The APTT and PT were both statistically significant (p<0.05). Based on this significant difference, the extract might not be of importance in coagulation studies. In laboratory practice, some anticoagulants are reported to cause deleterious effects on blood cellular elements, while others confer unwanted bizarre colouration on cells ^[18]. This makes the choice of anticoagulant to be selective depending on the type of investigation so desired.

In this work, in vitro anticoagulation activity of the extract of Pentaclethra macrophylla have been established. The extract has potential which could be harnessed and produced into anticoagulant for in vitro/laboratory use.

Statement of Conflicting Interest

We declare that we have no conflict of interest.

Abbreviations

PCV　　  Packed Cell Volume;

Hb　　　 Haemoglobin concentration;

WBC　　 White Blood Cell count;

APTT　　Activated Partial Thromboplastin time;

PT　　　 Prothrombin time.

EDTA　  Ethylene diamine tetra acetic acid.

Sec　　  seconds

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