Phytochemical and Anticoagulant Activity of Mahogany(Swietenia macrophylla) King Fruit Extract on Rat Blood

Journal of Science, Engineering and Technology 5:44-55 (2017) Southern Leyte State University, Sogod, Southern Leyte, Philippines Phytochemical and Anticoagulant Activity of Mahogany(Swietenia macrophylla) King Fruit Extract on Rat Blood Adel Grace P. Gaudicos 1 * Edgardo E. Tulin 2 1 College of Criminal Justice Southern Leyte State University Sogod, Southern Leyte, Philippines 2 Philippine Rootcrops Research and Training Center Visayas State University Visca, Baybay City, Leyte, Philippines Abstract The study was conducted to investigate the anticoagulant property of mahogany Swietenia macrophylla king fruit extracts using its pulp and seeds. Extracts were evaluated for its anticoagulant effect on rat blood using blood clotting time. Blood was tested with different seed extracts; ethanol, hexane, and water with 20%, 40%, 60%, and 80% concentrations. Study revealed that ethanolic seed and pulp extracts increased the clotting time significantly at 80% concentration which confirmed the use of Swietenia macrophylla in arresting stroke or high blood pressure traditionally. Keywords: Blood thinner; blood clot; coagulation Introduction Blood clot formation is a natural and essential process. Whenever one has a wound, a blood clot is formed to stop the bleeding. This function protects a person from excessive blood loss. Without the ability to form blood clots, even a minor cut could cause profound consequences or even death. Clotting, also known as coagulation is a process resulting in blood clot formation. The process of coagulation happens when particles known as blood platelets combine in a process known as platelet aggregation. Although blood clotting is an essential natural process, platelet aggregation can have serious consequences when it happens within the circulatory system. This can cause blood flow to be reduced or even block arteries that can cause heart attack or stroke. Blood clots are dangerous when they block arteries and stop blood and oxygen from flowing to vital organs leading to tissue damage (Marieb & Haelin, 2010). Hypercoagulation or excessive blood clotting results in thick blood. This could lead to hindered circulation of nutrients, oxygen, and hormones, whenever they are not directed successfully to tissues and cells in the body. If this gets serious, it could cause widespread nutritional and hormonal deficiencies. Anticoagulants or blood thinners can slow the process of coagulation. They work by reducing the aggregation of blood platelets which lessens coagulation and hypercoagulation. Thinning the blood can improve circulation and may reduce the risk posed by the formation of blood clots. *Correspondence: adelgraceprimagaudicos@gmail.com ISSN 2545-9732

It has been reported that anticoagulants such as aspirin and other non-steroidal inflammatory drugs (NSAIDs) can be harmful to our health; thus, natural blood thinners present an additional attraction (Huang, et al., 2011). Researches have shown that there are a number of natural substances and blood thinning food that may inhibit platelet aggregation safely and without side effects. Natural blood thinners are readily available and most importantly may not incur the negative side effects of many drug-based blood thinners such as aspirin. The study aimed to assess the anticoagulant property of mahogany king fruit using rat blood; compare in vitro anticoagulant activity of mahogany king fruit extracts from the fruit s pulp and seeds; determine which solvent is effective in extracting the anticoagulant component present in mahogany king fruit; and evaluate some phytochemical properties present in mahogany king fruit. The result of this study will be beneficial to every individual, especially to those who are suffering from hypercoagulation or blood thickening. Methodology Collection and Preparation of Plant Extracts Fruits of mahogany Swietenia macrophylla king were bought from the Department of Environmental and Natural Resources, Bontoc, Southern Leyte. Mature fruits were selected. Plant materials were washed with tap water to remove dirt particles covering on the fruits. The collected plant materials were dried under shade and the seeds of the fruits were separated from the pulp. The samples were then powdered using a grinder. Approximately 30g of dried and powdered pulp and seeds of mahogany fruits were weighed and placed separately in glass bottles. Each was immersed in 300mL of solvent enough to cover all plant materials. The powdered dried seeds were extracted with solvents for 48 hours. The bottles were tightly capped to avoid unnecessary evaporation of solvent. The bottles were also wrapped with carbon paper to avoid photolytic reactions. The extracts were collected through gravity filtration using Whatman filter paper No. 42 to get a particulate-free filtrate. The solvents used for this study were ethanol, water, and hexane. The resulting filtrate were concentrated by removing the solvent using rotary evaporator (R110) at 50 C. Hexane extract was rotary evaporated for approximately 3 hours and ethanol for 5 hours. The water extract was also rotary evaporated, but it was stopped since after eight hours the target volume was not achieved. There was a very slow evaporation of solvent; so, evaporation was not continued. Experimental Animals Five 7-8-week-old adult male laboratory rats (Rattus novergicus), with approximately the same weight were used in the study. The animal procedures were followed as per internationally accepted ethical guidelines for the care of the laboratory animals. The animals were housed in PhilRootcrops Animal Facility, in standard polypropylene rat cages (44.5 x 28 x 20 cm). A room temperature ranges between 20-26 C was observed. Rats were provided with resting materials and in-cage shelters to enable them to regulate the micro-climate temperature, particularly for sleeping. Lighting within cages during day hours were held at lux ranges. Lighting intensity was reduced using recessed lighting consoles in the ceiling with fluorescent lights of about 25 watts. Shading was provided over top shelves of rack to protect rats in the top cages from overhead lights. Prior to the experiments, rats were fed with fiber-free diet and water ad libitum. Rats were acclimatized for at least 14 days prior to beginning any procedures. As there were indications of sex-linked determinants in response to anticoagulants in rats, only male rats were used. All experiments were performed in the 45

morning according to the current guidelines for the investigation of the experiential pain in the conscious animals. Collection of Blood Samples Blood samples were obtained from rats through careful cardiac puncture under chloroform anesthesia with supervision by a licensed veterinarian. Clotting Time Determination Clotting time was determined using the Lee-White method as described by Ochei and Kolhatkar (2000). In determining the clotting time, three numbered clean test tubes were placed in 37 C water bath. A 3ml of blood was drawn from the experimental animal. Stopwatch begun to start when blood first appeared in the syringe. One (1) ml of blood was placed into each of the 3 test tubes, starting with test tube 1. The test tube 3 (last to receive blood) was gently tilted every 30 seconds and coagulation was observed. After coagulation was observed in test tube 3, test tube 2 was observed. When coagulation was observed in test tube 2, test tube 1 was observed. The time was stopped when no more flow of blood was observed. Coagulation was the time it took for blood to coagulate in test tube 1. The anticoagulant activity was followed using ethanolic, hexane, and water extracts at 0%, 20%, 40%, 60%, and 80% concentrations. Determination of Phytochemical Properties of the Fruit Extracts The bioactive extracts of the mahogany (Sweitenia macrophylla) king were phyto-chemically screened for the presence of secondary metabolites using the methods of Guevarra and Recio (1985). Determination of total phenolic composition was determined using the method of Spanos and Wrolstad (1990) as modified by Lister and Wilson (2001). The screening for alkaloid was tested using Culvenor-Fitzgerald analysis. In the preliminary test, a 20ml aliquot of the extract was evaporated to a syrup consistency over a steam bath. The flask was removed from the bath and 5ml of 2M HCl was added. The solution was heated with stirring for 5 minutes and 0.5g of NaCl was added. The solution was stirred and filtered. The residue was washed with enough 2M HCl to bring the filtrate to a volume of 5ml. Filtrate (1ml) was tested with 3 drops of Mayer s reagent. In the confirmatory test for alkaloids, 28% ammonia was added to the remaining 3ml of solution until the solution is alkaline to litmus paper. The solution was extracted three times with 10 ml portions of chloroform. The lower chloroform extract was combined and evaporated to dryness over a steam bath. Chloroform residue was collected and 5 ml of 2M HCl was added. It was stirred over a steam bath for about 20 minutes and then cooled. The filtrate was divided into two portions. Each was tested separately with Mayer s and Dragendoff s reagent. The flavonoidal contents were analyzed using Bate-Smith and Metcalf test. A 20ml of extract was evaporated to incipient dryness over a water bath and was cooled to room temperature. The residue was defatted by treating with 9ml of hexane and 4.5ml (2:1) until extract is almost colorless. The hexane extract was discarded. The defatted aqueous layer was diluted with 10ml of 80% ethyl alcohol. The filtrate was divided into four test tubes. Two portions were taken as control. One portion of alcohol filtrate was treated with 0.5ml concentrated HCl and color changes were observed. It was warmed for 15 minutes in water bath and was observed for one hour for further color change. The presence of anthraquinone was detected using Borntrager s and modified Borntrager s test. One (1) ml of extract was evaporated to incipient dryness over a water bath. The residue was added with 10ml of distilled water and then filtered. The residue in the filtration process was discarded. The aqueous filtrate was extracted twice with 4-5ml portions of benzene and the benzene 46

extract was combined. The benzene extract was divided into two portions. One served as control. One portion was treated with 5ml of ammonia solution, shaken and compared with the control. In the modified Borntrager s test, 1ml of extract was evaporated to incipient dryness over a water bath. A 10ml of 0.5M KOH and 1ml of 5% hydrogen peroxide were added and stirred. The mixture was heated over a steam bath for 10 minutes and filtered. The filtrate was acidified using glacial acetic acid. The acid filtrate was extracted twice with 5ml benzene and the benzene extract was combined. The benzene extract was divided into two portions. One portion was used as control while the remaining portion was basified with ammonia and compared with the control. Tannins and polyphenolic compounds were determined using gelatin test and confirmed with ferric chloride test. A 10ml of plant extracts was evaporated to incipient dryness over a water bath. The residue was extracted with 20ml of hot distilled water. Five drops of 10% NaCl were added. The filtrate was re-filtered and was divided into three test tubes. One portion was taken as control. An aqueous solution of tannic acid was taken as reference material. In the gelatin test, 3 drops of 1% gelatin salt was added to one portion of filtrate. The same procedure was done to tannic acid solution. The filtrate was compared with control and reference standard. The presence of saponins in the extract was tested using froth and capillary test. Two (2) ml of extract was placed in a test tube and 2ml of gugo extract was placed in a separate test tube to serve as control. Each of the test tubes was added with 10ml of distilled water, stoppered, and shaken vigorously for 30 seconds. It was allowed to stand for 10 minutes. Determination of total phenolic composition was determined using the method of Spanos and Wrolstad (1990) as modified by Lister and Wilson (2001). The 5mg of gallic acid was weighed, dissolved with enough water and diluted to make 100ml solution. A gallic solution of 20%, 40%, 60%, 80%, and 100% were prepared. For the sample preparation, 2ml Na 2 CO 3 (2% w/v) was added to 0.5ml of sample of plant extract solution. A 2.5ml of 10% Folin-Ciocalteu reagent was added. The mixture was incubated at 45 C with shaking for 15 minutes. The absorbance of the sample was measured using UV/Visible light at 765nm. The results were expressed in milligrams of gallic acid (0.005 mg/ml) dissolved in distilled water. Experimental Design and Statistical Analysis The experiment conducted was analyzed based on completely randomized design (CRD). The one-way analysis of variance (ANOVA) in CRD was used to determine the significant differences among treatment means and Least Significant Difference (LSD) test was used for multiple comparisons of means. Results and Discussion The Plant Extracts The seeds and pulps of mahogany fruits were infused in three different organic solvents. Sample extraction was done sequentially using different solvents of increasing polarity, namely; hexane, ethanol, and water. The increasing polarity of solvents allowed a partial extraction of the secondary metabolites from the plant sample components since each component also has different polarity. It also allows partial separation of the different components since different solvents differ in their extraction ability. The nonpolar solvent, hexane, could extract nonpolar constituents, and ethanol, which has intermediate polarity could extract the remaining slightly nonpolar and also polar constituents. The most polar solvent, the distilled water, could extract the remaining highly polar components. Solubility rule states that like dissolves like. Based on the polarities of the solvents, it is expected 47

that some of the nonpolar or less polar plant constituents would go to less polar solvent and the polar plant constituents would go to the polar solvents. Thus, hexane fractions would contain some nonpolar constituents of the plant while ethanol and water would contain most of the polar constituents. After 48 hours, the extracts were collected through gravity filtration. The color of the extracts was clear/colorless but with different intensity. The color differences of the crude extracts may be attributed to the concentration and nature of substances present, which are likely responsible for the anticoagulant property of the extracts. The crude extracts were concentrated by removing most of the solvents using rotary evaporator at 50 C. This was done at this temperature so that the extracts will not be thermally degraded. Phytochemical Properties of Mahogany Fruits Preliminary test (Table 1) for alkaloids showed that all the extracts form white and red precipitate upon the addition of Mayer s and Dragendorff s reagent, respectively. This was further verified by the confirmatory test and found that all the extracts indeed form a precipitate, which means all extracts really contain alkaloids. False positive result that have been observed in the preliminary test were minimized by the addition of NaCl before precipitating reagents were added. The NaCl would degrade the proteins present before the precipitation (Guevarra & Recio, 1985). In the case of flavonoids, the Bate-Smith method and the Metcalf method for leucoanthocyanins were used. A strong red or violet color indicates the presence of leucoanthocynins. Crude extracts of hexane and water gave negative results. Only the ethanol extract indicated positive results in both tests. Flavonoids are soluble in polar solvents. Results show the presence of flavonoids in ethanol extract. The negative result contained in water extract could be due to the rotary evaporation process. The very low concentration of flavonoids in the water extract might be beyond detection limit. There are specific tests employed for determining the saponins. They are the Froth test and Capillary test (Guevarra & Recio, 1985). The results indicate the absence of the saponins in water and its presence in hexane and ethanol. The formations of honeycomb froth or foam in the hexane and ethanol extract which persisted for 30 minutes and maintained a height of 2cm satisfy the presence of saponins in ethanol and hexane extracts. Water extract did not exhibit froth or foaming effect. This may be caused by the less concentration of saponins extracted in water which was not detected. For the capillary method, it was observed that hexane and ethanol gave positive results while the water extract gave negative result. The results coincide for the froth and capillary methods. For the capillary method, the principle lies mainly on the surface tension. Water, which served as control has strong surface tension because it can adhere on the surface of the tube for the long period of time. It is known that saponins have detergent properties and it can act as surface acting agents that tend to lower surface tension of the container through its detergent property. A positive result is achieved when the level of extract is half or less than that of the water. Despite the results, the less surface tension of the extracts may be attributed to their concentrations which might explain why water gave negative result for saponins through this method. Tannins are determined by their ability to precipitate gelatin protein and a blue-black or brownish-green color in ferric chloride test indicate a positive result. There was no precipitate formation in gelatin test as well as in ferric chloride test for all the extracts. Gelatin test was used to detect the possible occurrence of tannins in plant samples. In such test, the tannins precipitate the gelatin protein. Reaction is accomplished by the H-bonding between polyphenolic hydroxyl group of tannins and the carbonyl group of 48

Table 1. Qualitative analysis of secondary metabolites in the seeds of mahogany king fruit using different solvents NVC = no visible change CEE = crude ethanol extract CHE = crude hexane extract CWE = crude water extract Table 2. Mean gallic acid equivalence (GAE) of mahogany king seed extracts Sample Extract GAE (mg/ml) Ethanol 0.151 b Hexane 0.298 a Water 0.074 c Values with different letters differs significantly at α = 0.05, LSD protein peptide bond. The reaction is made sensitive by the addition of the sodium chloride to enhance the salting-out of the protein tannin complex (Guevarra & Recio, 1985). For the Ferric chloride test, reaction occurs by the attack of hydroxyl group to the aromatic nucleus. Negative results were also obtained for the ferric chloride test. Borntrager s test was used to detect the presence of anthraquinones in the different crude extracts. Red coloration of the solution indicates positive result. In Borntrager s test, all the extracts failed to produce red coloration in the ammoniacal layer which indicates the absence of anthraquinones (Guevarra & Recio, 1985). Hence, modified Borntrager s test was used. The test was modified to hydrolyze and oxidize such compound. However, the results of the modified Borntrager s test still gave no coloration. For anthraquinones and for tannins and polyphenols, all of the extracts did not produce any color change in the Borntrager s and modified Borntrager s tests for anthraquinones. Also, no color change was observed for ferric chloride test and no precipitate was observed in the gelatin test for the presence of tannins and polyphenols. Results imply that anthraquinones and tannins and polyphenols in the fruits of mahogany are in small concentrations to be detected by the present method used. Table 2 shows the qualitative analysis of the secondary metabolites in the pulps of mahogany using different solvents. The results of the phytochemical screening of both pulp and seed extracts suggest that the two have the same phytochemical compositions. In the Folin-Ciocalteu method of determining total phenolics, the extracts turned blue when the reagent was added due to the reduction of Folin-Ciocalteu reagent by the phenol functional group. The total phenolics of the extracts 49

Table 3. Qualitative analysis of secondary metabolites in the pulps of mahogany using different solvents NVC = no visible change CEE= crude ethanol extract CHE= crude hexane extract CWE= crude water extract expressed as gallic acid equivalents vary in the three different seed and pulp extracts used. This means that differences in solvents would greatly affect the amount of phenolics that would be obtained from the extraction. For the total phenolic content of the extracts, a significant difference between the gallic acid equivalence (GAE) in the extracts was observed. The highest value observed was with the hexane extracts followed by the ethanol extracts then the water extracts. This implies that solvent for extraction used is a major factor for the number of phenolic compounds being extracted. The phenolic contents for the extracts could explain their respective effectiveness for their anticoagulant activities (Table 3 and 4). Table 4. Mean gallic acid equivalence (GAE) of mahogany king pulp extracts Sample Extract GAE (mg/ml) Ethano 0.144 b Hexane 0.250 a Water 0.058 c Values with different letters differs significantly at α = 0.05, LSD Blood Clotting Time Blood clotting, known as coagulation, is a very complicated process, requiring a balance between factors that promote clot formation and factors that either prevent clot formation or dissolve it when it occurs. When an imbalance between the two mechanisms occurs, there will either be an increased risk of clotting or alternatively, excessive bleeding. The formation of the visible coagulum, which is the physical manifestation of the fibrin formation, represents only the result of an intricate series of reactions that involve the number of coagulation factors. For the clotting process to work, clotting factor proteins need to be present in the right amounts and work correctly. The clotting time of whole blood is a procedure which tests the composite action of all plasma factors acting simultaneously. The time required for the blood to clot is the function of the combined factors favoring coagulation in one hand, as opposed to the combined factors inhibiting coagulation on the other hand. Powdered pulp and seeds 50

of mahogany were extracted using different solvents and applied to blood samples for determining the anti-blood clotting effect. Unexpectedly, the result obtained in the present study suggests that Mahogany Swietenia macrophylla King extract could inhibit blood coagulation via a significant increase in clotting time in Albino rat blood. This index is a measure of blood coagulation. Clotting time indicates the function of clotting factors I, II, V, VII, IX, X, and XII. The significant increase in the clotting time in this study may be indicative of the fact that there was a decrease in one or more of the clotting factors involve in the intrinsic pathway. The blood clotting time of rats is presented in Tables 5 and 6. Blood was tested with different seed and pulp extracts; ethanol, hexane, and water with 0%, 20%, 40%, 60% and 80% concentrations. Tables 7 and 8 present the multiple comparisons for the treatments of seed and pulp extracts on blood clotting time of rat blood, respectively. In table 7, it is shown that the ethanolic seed extract gave significant increase in clotting time to 473.33, 539.680.66, and 2608.33 seconds when compared to 0% (268 secs) at dose levels of 20%, 40%, 60%, and 80%, respectively. Water seed extract also caused significant increase of time for the blood to clot. This was indicated by the increase of clotting time to 294.33, 447.00, 609.02, and 1436.02 seconds compared to extract at 0% concentration at dose levels of 20%, 40%, 60%, and 80%, respectively. Hexane seed extracts also affect the clotting time but showed less pronounced effects as compared to ethanol and water extracts. Hexane extracts changed the clotting time to 330.00, 395.33, 563.33 and 686.00 seconds, lowest clotting time effect compared to the water and ethanol extracts. Ethanol seed extract at 80% concentration gave the highest significant effect on blood clotting time. Ethanolic pulp extract showed significant increase in clotting time to 284. 00, 343.33, 393.33 and 421.33 seconds when compared to 0% (268 secs) at dose levels of 20%, 40%, 60%, and 80% respectively. Water pulp extracts increase also the time it took for the blood to clot but not too long compared to the ethanolic extract. Hexane showed the lowest anticlotting effect compared to water and ethanol extracts. Ethanolic pulp extracts at 80% gives the highest anti-blood clotting effect (Table 8). As can be seen in Figure 1, ethanol seed extracts gave the highest antiblood clotting activity compared to the other two extracts, water and hexane. The anticlotting effect is followed by the water extract and the hexane extract. Differences in their anticoagulation may be due to the differences in secondary metabolites and their concentrations in each extract. All the extracts gave the highest anticlotting effect at 80% concentration, giving an idea that the anticoagulation property of mahogany seeds increases with an increase in concentration. For the pulp (Fig. 2), the extract that exhibits the highest anticlotting activity was also ethanol, followed by water and hexane. Taken together, it can be inferred that ethanol extract of 80% concentration gives the highest anti-blood clotting effect in both pulp and seed extracts. Moreover, the results also suggest that the seed extract is more effective than pulp extract. Phytochemical analysis of the extracts indicated the presence of metabolites that are usually responsible for the pharmacological activities of the medicinal plants. Flavonoids, alkaloids and saponins were found to be the active ingredients of the mahogany. Many thousands of polyphenolic compounds are produced as secondary plants metabolites. When ingested by humans they may provide health benefits. Flavonoids are a major class of polyphenols. The relationship between flavonoidal intake and risk of cardiovascular disease has been investigated in many epidemiogical studies. Results of population studies suggest that dietary flavonoids provide modest protection against cardiovascular disease (Fisher & Hollenberg, 51

Table 5. The effect of mahogany Swietenia macrophylla king seed extracts on blood clotting time CEE = crude ethanol extract CHE = crude hexane extract CWE = crude water extract Table 6. The effect of mahogany Swietenia macrophylla king pulp extracts on blood clotting time EE =crude ethanol extract CHE = crude hexane extract CWE = crude water extract 2005; Arts & Hollman, 2005). There is also a growing body of evidence from controlled trials that dietary flavonoids can improve endothelial function and reduce blood pressure in humans (Vita, 2005) and inhibit the development of atherosclerosis in animal models (Waddington et al., 2004). Flavonoids is useful for blood circulation. Flavonoids are powerful antioxidants that lower cholesterol levels, reduce blood pressure, and prevent blood clotting by making it less sticky. Another secondary metabolite which is also present in mahogany fruit are the saponins which are known to have antiplatelet activity (Rao & Gurfinkel, 2000; Sparg et al., 2004). Saponins help to clean the blood stream and prevent blocked blood vessels. They help with circulation problems, such as high blood pressure, arteriosclerosis and obesity. Saponins have efficacy as a pest deterrent, could also be used to reduce the fat in the body, helps boost immune system, prevent blood clotting, and strengthen heart function and slow the blood clotting process. Preliminary phytochemical analysis of mahogany Swietenia macrophylla king fruit indicated the presence of saponins and flavonoids. The anticoagulant property of 52

Figure 1. The effect of seed extracts on rat blood Figure 2. The effect of pulp extracts on rat blood Swietenia macrophylla extract is derived probably from antiplatelet aggregation and fibrinolytic activities and decrease one or more clotting factors in intrinsic pathway. The active ingredients of mahogany fruits, saponins and flavonoids reduce clotting by naturally thinning blood. The reduction of coagulation time of whole blood by the seed and pulp extracts is an indication that the extracts affect blood coagulation pathway. The toxicity of the seed extract of various species of Swietenia were previously described in a recent study of acute oral toxicity of seed extract from Swietenia mahogany in a murine model. Suggestions of oral organ toxicity at dose 25, 200, 2000 and 5000 mg/kg were not observed (Naveen et al., 2014). In another study which the acute oral toxicity of seed extract from Swietenia macrophylla in rats at doses of 2000 mg/kg was evaluated, the results showed that seeds did not produce any significant differences in food and water intake, biochemical and hematological parameters, or macroscopic and hematological changes in the organs of the treated animals compared with the control (Balijepalli et al., 2015). According 53

Table 7. Multiple comparisons for the treatments of seed extracts on blood clotting time of rat blood Treatment Mean) Hexane control 268.00 i Ethanol control 268.00 i Water control 268.00 i Hexane 20% extract 330.00 h Ethanol 20% extract 473.33 f Water 20% extract 294.33 hi Hexane 40% extract 395.33 g Ethanol 40% extract 539.67 e Water 40% extract 447.00 f Hexane 60% extract 563.33 de Ethanol 60% extract 680.66 c Water 60% extract 609.02 d Hexane 80% extract 686.00 c Ethanol 80% extract 2608.33 a Water 80% extract 1436.02 b Values with different letters differs significantly at α = 0.05, LSD Table 8. Multiple comparisons for the treatments of pulp extracts on blood clotting time of rat blood Treatment Mean) Hexane control 268.00 i Ethanol control 268.00 i Water control 268.00 i Hexane 20% extract 273.33 i Ethanol 20% extract 284.00 h Water 20% extract 298.33 g Hexane 40% extract 294.00 g Ethanol 40% extract 343.33 d Water 40% extract 321.66 f Hexane 60% extract 333.33 e Ethanol 60% extract 394.33 b Water 60% extract 365.33 c Hexane 80% extract 395.00 b Ethanol 80% extract 421.33 a Water 80% extract 394.06 b Values with different letters differs significantly at α = 0.05, LSD to another study conducted by the method of brine shrimp lethality assay, LD50 of oral acute toxicity for S. Mahogani Jacq. seed methanolic extract (SMCM) is more than 2500 mg/kg. The oral LD 50 value in the study suggests that the SMCM extract is a relatively non-toxic plant. The results of the study concur with the use of the plant by traditional healers as traditional medicine (Geethaa et al., 2010) Conclusion Anticoagulant property of Mahogany fruit can be determined by using albino rat blood. Ethanolic seed extract showed promising result in lowering the time it takes for the blood clot at 80% concentration. It was also revealed that the seed extract is more effective in increasing the time it took for the blood to clot compared to the pulp extract. The ethanol is the best solvent extracting the anticoagulant component of seeds and pulp as indicated by the significant increase in clotting time of blood when added to the rat blood. The secondary metabolites present in the aqueous crude seed and pulp extracts of mahogany Swietenia macrophylla king fruit are the alkaloids, saponins, flavonoids and phenolics. Recommendation From the results of the study, the following suggestions are recommended: Perform test on Partial Thromboplastin Time (PPT), and Activated Partial Thromboplastin Time (APPT) to screen the coagulation mechanism for investigation of deficiencies involving factors that make up the intrinsic pathway; Perform test on clotting time of recalcified plasma and thrombin time to detect the inhibitors of coagulation; and Conduct an in vivo study using albino white rat to further assess the behavior of mahogany fruit extracts in the living system. References Cited Arts, I. C. W. & Hollman, P. C. H. (2005). Polyphenols and disease risk in epidemiologic studies. American Journal of Clinical Nutrition, 81, 317-325. 54

Balijepalli, M. K., Suppaiah V., Chin A., Buru A.S., Sagineedu S.R., Pichika M.R. (2015). Acute oral toxicity studies of Swietenia macrophylla seeds in sprague dawley rats. Journal of Pharmacognosy Research, 7(1), 38-44. Fisher, N. D. L., & Hollenberg, N. K. (2005). Flavonoids for cardiovascular health: the science behind sweetness. Journal of Hypertension, 23, 1453-1459. Geetha, S, Surash R.,Sreenivasan S.,Mohd. N.M. Sabariah I.,and M.M Sharif ( 2010). Brine shrimp lethality and acute oral toxicity studies on Sweitenia mahogani (linn) Jacq. seed methanolic extract. Journal of Pharmacognosy Research, 2(4), 215-220. Guevarra, B. G., & Recio, B. V. (1985). A guidebook to plant screening : phytochemical and biological. Manila: University of Santo Tomas Publishing House. Huang, E. S, Strate L.L, Ho W.W, Lee S.S,& Chan A.T. (2011). Long term use of aspirin and the risk of gastrointestinal bleeding. American Journal of Medicine, 124: 426-433. Lister, E., & Wilson, P. (2001). Measurement of total phenolics and abts assay for antioxidant activity. New Zealand: Crop Research Institute Marieb, E., & Haelin, K., (2010). Human anatomy and physiology (8th ed.). New York: Pearson Naveen, Y. P., Divya, R. G., Ahmed, F., & Urooj, A. (2014). Pharmacological Effects and Active Phytoconstituents of Swietenia mahogany: A review. Journal of Integrative Medicine, 12(2): 86-93. National Research Council. (2010). National Research Council Committee for the Update on the Guide for the Care and Use of Laboratory Animals (8th ed.). Washington D.C.: National Academic Press. Ochei, J., & Kolhatkar, K. (2000). Medical laboratory science: Theory and practice (2nd edition). New Delhi: Tata Mcgraw Hill Publishing Company. Rao, A. V., & Gurfinkel, D. M. (2000). The bioactivity of saponins: triterpenoid and steroidal glycosides. Drug Metabolism and Drug interactions Journal, 17, 211-235. Spanos, G. A., & Wrolstad, R. E. (1990). Influence of processing and storage on the phenolic composition of Thompson seedless grape juice. Journal of Agriculture and Food Chemistry, 38, 1565-1571. Sparg, S. G., Light, M. E.& Standen, J. V. (2004). Biological activities and distribution of plant saponins. Journal of Ethnopharmacology, 94(2-3), 219-243. Vita, J. A. (2005). Polyphenols and cardiovascular disease: effects on endothelial and platelet function. American Journal of Clinical Nutrition, 81, 292S-297S. Waddington, E., Puddey, I. B., & Croft, K. D. (2004). Red wine polyphenolic compounds inhibit atherosclerosis in apolipoprotein E-deficient mice independent of effects on lipid peroxidation. American Journal of Clinical Nutrition, 79, 54-61. 55