Medicinal plants have long played significant roles in maintaining human health and have also served as sources of food for humans. World health organization estimated that over 80% of the earth’s inhabitants rely on traditional medicine for their primary health care needs and most of this therapy involves the use of plant extracts or their active components [1]. Plants evolved the ability to synthesize chemical compounds that help them defend against attack from a wide variety of predators. By chance, some of these compounds, while being toxic to plant predators, turn out to have beneficial effects when used to treat human diseases [2]. Medicinal plants are the oldest known health-care products, where renewed interest is growing based on the ethnological, medical and historical background of each country [3]. The use of traditional medicine has increased in developed countries also, mainly due to the failure of modern medicine to provide effective treatment for chronic diseases and emergence of multi-drug resistant bacteria and parasites. The current emphasis of new drug discovery processes from plants is the development of products with new pharmacological modes of actions [4].

Blood coagulation is one of the haemostatic processes of humans, which consists of a complex, physiological cascade. When the blood vessel is damaged, the substances released from the destroyed endothelium into blood induce formation of a platelet aggregation [5]. After activation, platelets tend to adhere to the damaged vessel wall and finally, an aggregated platelet plug is formed to prevent the loss of blood. During this process, plasma clotting also happens. When the cells that are placed in endothelium is exposed to blood, the plasma clotting, known as blood coagulation, is activated. Blood coagulation process has more complex cascade, which consists of enzymatic reactions in blood plasma [6,7]. As a result of complicated process, polymerized fibrin is formed from fibrinogen to prevent the loss of blood cells. The studies on the haemostatic activity of these plants are rather limited. Despite its wide range of folk medicinal use, there is insufficient scientific data on the efficacy of this plant in arresting wound bleeding or hemorrhage. Most of the reports are anecdotal. Hence, the need to investigate the claims.

Experimental Section

Plant Material: Fresh leaves of Murraya exotica were collected from the front of Agram Pharmacy, Ilishan-Remo, Ogun State and ientified by Professor F.D. Onajobi of the Department of Biochemistry, Babcock University.

Extraction Procedures of M. Exotica

The M. exotica leaves were washed with distilled water and oven dried at 40 0C for 72 hours. The dried leaves were pulverized using a blender. 50 g of M. exotica leaves were extracted using 70% methanol. The leaves were extracted with methanol three times at intervals. The mixture was stirred continuously over a period of five hours. The mixture was filtered with a filter paper and funnel. The extract filtrate was concentrated using rotary evaporator and was dried using an air pump. The concentrated sample was kept in the evaporator until further use.

Preparation of M. Exotica Stock Extract

About 3.5 mL of distilled water was added to 2.1 g of M. exotica extract and it was mixed thoroughly. After the addition of distilled water, 2.4 mL of methanol was then added using a micropipette and mixed until all the extract was dissolved.

Preparation of Solvent

About 4 mL of distilled water and 1mL of methanol was measured into a beaker.

Preparation of the Dilute Extracts

• 0.05 mL of the stock extract prepared initially was diluted with 4 mL of water and 1 mL of methanol in a beaker

• 1 mL of the stock extract prepared initially was diluted with 4 mL of water and 1 mL of methanol in a beaker

• 1 mL of the stock extract prepared initially was diluted with 2 mL of solvent in a beaker

Preparation of Acid Citrate Dextrose

1.3 g of sodium citrate was weighed into a 100 mL volumetric flask and dissolved with 85 mL of distilled water. 0.28 g of acid was also weighed and dissolved in the solution. 1.47 g of dextrose was also weighed and dissolved in the solution. Distilled water was added to make up100 mL and the solution was labeled.

Assay for Blood Clotting Time

A modified method of Lee and White as reported by Wintrobe [8], was used for the assay of blood clotting time. Clean labeled glass test tubes containing different solutions was placed in the water bath at 37 0C. The contents of the test tube are as follows; Test tube A Clean test tube. Test tube C1 containing 0.05 mL of extract. Test tube D1 containing 0.05 mL of solvent. Test tube C2 containing 0.1 mL of extract. Test tube D2 containing 0.1 mL of solvent. Test tube C3 containing 0.2 mL of extract. Test tube D3 containing 0.2 mL of solvent. Test tube C4 containing 0.3 mL of extract. Test tube D4 containing 0.3 mL of solvent. Blood was collected from healthy adult volunteers by venipuncture into sterile disposable 10 mL syringes. Starting a stopwatch. 1 mL of blood was immediately transferred into each of the equilibrated test tubes by carefully allowing the blood to run down the side of the tube and swirling it gently to mix content in the tubes. At intervals of 30 seconds, the tubes still in the water bath were gently tilted to an angle of 45 0C to check for blood clot formation. When there was a noticeable clot, the time of the first clot was recorded. This was continued until the tubes could be inverted without the blood flowing. The stopwatch was immediately stopped and the final blood clotting time was recorded. The blood clotting time was obtained by deducting the starting time from the time of the final clot.

Effect of Acid Citrate Dextrose on Human Blood Clotting Time of Health Donor

The clotting time of blood samples from volunteers treated with acid citrate dextrose is shown in Table 1. Acid citrate dextrose is a standard anticoagulant used in this experiment for the purpose of comparison.

Table 1: Effect of Acid Citrate Dextrose on Blood Clotting Time of Healthy Donors

Data were expressed as mean±SD, SD: Standard Deviation, * indicate p˂0.05

Effect of Murray Exotica Methanol Extract on Human Blood Clotting Time

The clotting time of first volunteer used in the experiment treated with different concentration of the methanol extract of M. exotica shown in Table 2. 

Table 2: Differences in Blood Clotting Time of Healthy Donor’s Blood Treated with 0.03 g/ml M. Exotica Concentration

Data were expressed as mean±SD, SD: Standard Deviation, * indicate p˂0.05

The blood samples treated with 0.003 g of the plant extract and 0.006 g of the extract significantly p˂0.05 decreased blood clotting time 5.50±1.0412* and 5.68±0.8536* min respectively compared to the control test tube clotting time 6.11±0.6377 min. The test tubes containing blood samples treated with solvent 0.1 mL  and 0.2 mL of the solvent reduced the blood clotting time 5.55±1.1112 and 4.96±0.6697 min respectively compared to the Control test tube clotting time 6.11±0.6377 min.

The clotting time of the volunteers, used in the experiment treated with different concentrations of the methanol extract of M. exotica is shown in Table 3. 

Table 3: Differences in Blood Clotting Time of Healthy Donor’s Blood Treated with 0.06 g/mL M. Exotica Concentration

Data were expressed as mean±SD, SD: Standard Deviation, * indicate p˂0.05

The blood samples treated with 0.003 g and 0.006 g of M. exotica significantly p˂0.05 decreased blood clotting time 6.29±2.3721* and 6.35±1.6980* min respectively compared to the control 7.35±2.6720 mins, while the blood samples treated with 0.012 g and 0.018 g of M. exotica increased blood clotting time 7.86±2.3432 and 7.87±2.1873 mins compared to Control. 

Table 4 shows the clotting time means of the ten subjects used in the experiment treated with different concentrations of the methanol extract of M. exotica. The blood samples treated with 0.006 g and 0.012 g of M. exotica (of the extract significantly (p˂0.05) decreased blood clotting time (6.16±1.8829 and 7.32±1.1459) respectively compared to the control (7.82±1.279) while the blood samples treated with 0.024 g of plant extract (8.53±1.246) and 0.036 g of the plant extract (10.89±1.276) increased blood clotting time compared to Control. This shows that an increase in the amount of extract increased blood clotting time.

Table 4: Differences in Blood Clotting Time of Healthy Donor’s Blood Treated with 0.12 g/mL M. Exotica Concentration

Data were expressed as mean±SD, SD: Standard Deviation, * indicate p˂0.05

The effect of Murraya exotica leaves extracts (methanol) on human blood clotting time was observed in vitro by a modified Lee and White method. Primarily, blood was drawn from ten volunteers. Hemostatic effects were measured by determination of blood clotting time using glass tubes noted in minutes. After testing on the blood samples of volunteer, it was observed that the extracts increased the human blood clotting time of volunteers.

The extrinsic pathway usually produces clot in as little as 15 seconds, while the intrinsic pathway requires 2-6 minutes. Clotting time is a qualitative measurement of factors involved in intrinsic pathway [7]. Since the effect M. exotica leaves extract are shown in minute range, it is likely that the intrinsic pathway is affected due to deficiency in factors I, II, V, VIII, IX, X, XII, XIII.

Presence of coumarins in M. exotica has been detected by Negi et al., [9], some of these coumarins include isomurralonginolsenecioate, isomurralonginoic acid, murrangatin 2’-formate and meranzin hydrate 2’-palmitate. Coumarins are vitamin K antagonists that produce their anticoagulant effect by interfering with the cyclic interconversion of vitamin K and its 2, 3 epoxides (vitamin K epoxide). Vitamin K is a cofactor for the posttranslational carboxylation of glutamate residues to g-carboxyglutamates on the N-terminal regions of vitamin K-dependent proteins. These coagulation factors (factors II, VII, IX and X) require g-carboxylation for their biological activity [10]. Coumarins produce their anticoagulant effect by inhibiting the vitamin K conversion cycle, thereby causing hepatic production of partially carboxylated and decarboxylated proteins with reduced procoagulant activity. In addition to their anticoagulant effect, the vitamin K antagonists inhibit carboxylation of the regulatory anticoagulant proteins C and S and therefore have the potential to exert a procoagulant effect.

This study showed that M. exotica could have anticoagulant property; with this the plant extract could be used to treat blood circulation related diseases such as a deep vein thrombosis. The increase in blood clotting times observed after the administration of methanol extracts of M. exotica attests to the anti-haemostatic and fibrinolytic effect of the extract. However, further researches may be carried out to buttress these findings and work need to be done on toxic studies to eliminate any hazardous consequences.

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