Evaluation of Hemostatic and Antihemolytic Effects of Aqueous Extract of Garcinia kola (Clusiaceae) Fresh Seeds

Objectives: Garcinia kola seeds (EAgk) (Clusiaceae) contain coagulant components use to pretreat aquaculture wastewater in Nigeria. This work was undertaken to assess the effects of EAgk on blood coagulation because of its contents. Methods: Five groups of four rats of both sexes (200 ± 5 g) received orally distilled water, phytomenadione (15 mg/kg b.w.) and garcinia kola seeds extract (250, 500 and 1000 mg/kg b.w.) for four days and after this period, bleeding time was measured by tail hemorrhage model. Activated partial thromboplastin time (aPTT) and platelet count were determined by coagulometer and hematological analyzer respectively. Blood red cells protection of EAgk was measured by the methods of 2,2’-Azobis(2-methylpropionamidine) dihydrochloride (AAPH)-induced hemolysis and inhibition of 2,2-Diphenyl-1-picryl hydrazyl (DPPH) radical and hydrogen peroxide. Results: EAgk at 1000 mg/kg b.w. induced a significant decrease in bleeding time from 420 ± 27.3 s to 157.5 ± 18.9 s. The platelet count of group of rat treated with EAgk (1000 mg/kg b.w.) increased significantly (p < 0.01, n = 4). EAgk, decreased aPPT in a concentration dependent manner (p > 0.05, n = 4). EAgk inhibited hemolysis, DPPH radical and hydrogen peroxide with IC 50 values of 1.02 ± 0.2; 3.2 × 10 −2 ± 0.12 and 1.01 ± 0.5 mg/mg respectively. Conclusions: EAgk exhibits hemostatic effects, which could justify the presence of coagulant components in the extract. Erythrocytes’ protective activity of Garcinia kola seeds may be due to free radicals and hydrogen peroxide inhibition.


Introduction
Hemostasis is a great protective mechanism that depends on an important balance of procoagulant and anticoagulant processes [1]. When a blood vessel is injured, platelets and fibrin play an essential role to build blood clot and stopping blood loss from the damaged vessel [2]. The clotting cascade (secondary hemostasis) is broken up into two pathways which are intrinsic and extrinsic.
The term intrinsic refers to a series of sequential reactions involving the plasma proteins factors VIII, IX, XI and XII; and prekallikrein and high molecular weight kininogen that are required for initiation of coagulation in the activated partial thromboplastin time (aPTT) assay, while the extrinsic pathway consists of the transmembrane receptor tissue factor (TF) and plasma factor VII/VIIa (FVII/FVIIa) [3] [4].
Uncontrolled bleeding remains the leading cause of preventable deaths in many cases of hemorrhage. Over the past 15 years, pharmaceutical industries have developed news hemostatic agents [5]. Also, several studies, worldwide have focused on the hemostatic activity of many medicinal plants with the hope of finding new and more effective agents [6] [7].
Garcinia kola is widely known as Bitter kola, belongs to the family of plants called Clusiaceae. The seeds of Garcinia kola play an important role in traditional herbal medicine worldwide [8]. Garcinia kola seeds are chewed as an aphrodisiac or used to cure cough, dysentery, chest colds, liver disorders, diarrhoea, laryngitis, bronchitis, and gonorrhea [9]. Bitter kola is used to prevent and relieve colic; it can also be used to treat headache, stomachache and gastritis [10] [11].
Garcinia kola seeds possess several pharmacological properties such as antioxidant, hepatoprotective, antidiabetic, antimicrobial and antipyretic activities [12] [13] [14]. The phytochemical compounds isolated from Garcinia kola include alkaloids, cardiac glycosides and biflavonoids [15] [16]. Seeds contain polysaccharides and can form a gel in a solution, so they can act as a coagulant to pre-treat aquaculture wastewater [17]. However, the effects of Garcinia kola seeds on blood coagulation still remain unknown.
The aim of the present work was to investigate in vitro and in vivo hemostatic effects of Garcinia kola.

Animals and Ethics
Rats (Ratus norvegicus) and rabbits (Oryctologus cuniculus) weighing 200 ± 5 g and 2 ± 0.4 kg respectively were used in our experiments. These animals were obtained from the Animal House of the Laboratory of Biology and Health of UFR Biosciences at Cocody University in Abidjan (Côte d'Ivoire). They were housed in constant temperature rooms with a light/dark cycle of 14/10 hours. All animals were fed and given water ad libitum until use. Experimental procedures and protocols used in this study were approved by ethical committee of Health Sciences, University Felix Houphouet-Boigny of Cocody-Abidjan. These guidelines were in accordance with the internationally accepted principles for laboratory use and care [18].

Preparation of Rabbit Erythrocytes
Five rabbits were anesthetized with ketamine (100 mg/kg b.w.) and the blood was collected from saphena vein. Erythrocytes were isolated and stored according to the method described by [21].with slight modifications. The blood samples collected were centrifuged at 3000 rpm for 10 min. The erythrocytes were separated from the plasma and buffy coat and were washed three times by centrifugation (3000 rpm, 5 min). The supernatant and buffy coats of white cells were carefully removed with each wash. The washed erythrocytes were stored at 4˚C and used within 6 h for further studies.

Hemolytic Assay
The method described by [22] was used to evaluate the percentage of hemolysis of red blood cells. Two (2) concentrations of Garcinia kola extract (10 −3 and 10 −1 mg/mL), were added to 20% red blood cell solution. The saline solution NaCl

Antihemolytic Activity
In vitro study of the antihemolytic effect of the extract of Garcinia kola was carried out according to the AAPH (2-2'-Azobis(2-methylproprionamidine) dihydrochloride) method used by [23]. The standard control used for the study is ascorbic acid. Conveniently it was added to (200 µL) extracts or ascorbic acid at different concentrations (10 −1 -1 mg/mL), 200 µL of 20% red blood cells.
The mixture was incubated for 30 min at 37˚C. 400 µL of AAPH (200 mM) was added to the mixture and was incubated again at 37˚C for 2 h. Before centrifugation of the mixture at 1200 rpm for 10 min, 3 mL of PBS was added.
The percentage of inhibition of hemolysis was determined at the longwave of 540 nm, as follows: with AC: Absorbance of the positive control and AE: Absorbance of the sample.

DPPH Free Radical Scavenging Activity
The radical scavenging activities of the plant extracts against 2,2-Diphenyl-1picryl hydrazyl radical were determined by spectrophotometry at a longwave of 517 nm. Radical scavenging activity was measured by a slightly modified method previously described by [24]. The following concentrations of the extracts were prepared, 0.001, 0.01, 0.1 and 1 mg/mL in ethanol. Ascorbic acid was used as the antioxidant standard at the same concentrations. 1 mL of the extract was placed in a test tube, and 2 mL of ethanol solution of DPPH (0.1 mM) was added. A blank solution was prepared to contain the same amount of ethanol and DPPH. The radical scavenging activity was calculated using the following formula: ( ) with A 0 : Absorbance of control at a longwave of 517 nm and A 1 : Absorbance of tested substance at a longwave of 517 nm.

Hydrogen Peroxide (H2O2) Scavenging Activity
In this study, we used a prior method of [25] with A 0 : Absorbance of the control and A 1 : Absorbance in the presence of the sample.

Preparation of Platelet-Poor Plasma
Platelet-poor plasma preparation was realized according to the method described by the professional order of medical technologists of Quebec [26]. Platelet-poor plasma (PPP) was separated from citrated rabbit whole blood. At first, the whole blood was centrifuged at 2500 rpm for 15 min. The supernatant obtained, was removed without disturbing the pellet. To be sure that the plasma was devoided of platelet, second centrifugation was operated at 2500 rpm for 10 min. The new plasma was taken without cellular debris and was stored at −20˚C until used.

Activated Partial Thromboplastin Time (aPPT) Assay
Intrinsic and extrinsic pathway of coagulation was determined according to the method described by [27], with slight modifications. Plasma (43 µL) was pipetted into clotting tubes and warmed for 2 -3 min at 37˚C. Then, 7 µL of distilled water (for control), and of plant extracts (10 −1 -1 mg/mL) for the test were added. Cephalin-koalin reagent (50 µL) and calcium chloride (50 µL) were added to the mixture respectively. Cephalin-koalin reagent and calcium chloride were pre-warmed at 37˚C for 2 -3 min. Phytomenadione (10 −1 -1 mg/mL) was used as reference drug. The coagulation time was recorded with a coagulometer (CyanCoag, Belgium).

Prothrombin Time (PT) Assay
To explore the extrinsic pathway of coagulation, 43 µL of plasma was pipetted into clotting tubes and incubated for 2 -3 min at 37˚C. Then, 7 µL of distilled water (for control), and of plant extracts (10 −1 -1 mg/mL) for the test, were added to clotting tubes. Prothrombin reagent (100 µL) pre-warmed at 37˚C for 2 -3 min was added to the mixture. Phytomenadione (10 −1 -1 mg/mL) was used as reference drug. The coagulation time was recorded with a coagulometer (CyanCoag, Belgium).

Acute Toxicity
The acute oral toxicity of Garcinia kola seeds extract was performed according to the OECD 423 [28], guidelines. Nine female Rats were divided in three groups of three animals. The first group received distilled water used as reference, and the two other groups were treated with 2000 and 5000 mg/kg b.w. of the extract.
To study the behavioral changes, the treated rats were observed every 30 min for a period of two (2) hours [29]. The mortality of animals was appreciated after 24 hours and the toxicity of the extract was determined.

Bleeding Time Activity
Rat tail hemorrhage model is one of the most commonly used animal models for preclinical efficacy studies of hemostatic agents [7] [30]. In vivo hemostatic activity of Garcinia kola seeds extract was investigated as described by [30]. Twenty rats of both sexes, were divided in five groups of four animals. Distilled water was administered per os to the control group. The second group received phytomenadione (15 mg/kg b.w., positive control) . The three other groups were pretreated orally with 250, 500 and 1000 mg/kg b.w. of the extract for four days.
The treated rats were anesthetized with ketamine (100 mg/kg b.w.) and the tip of their tails was cut to induce bleeding. The site of bleeding was gently blotted with filter paper every 30 s, till cessation of bleeding. The observation of time was limited to 20 min. Care was taken that no pressure was exerted on the tail tips that could affect homeostasis.

Hematological Assay
Blood samples were collected from rats used to achieve bleeding time assay, into heparinized tubes. Hematology Analyzer (Sysmex XN-1000, Japan) was used to determine platelet counts. This automate was able to measure several others parameters such as white blood cell counts, red blood cell count. Blood is sampled and diluted, and moves through a tube thin enough that cells pass by one at a time. Characteristics about the cell are measured using lasers (fluorescence flow cytometry) or electrical impedance.

Statistical Analysis
All statistical analyses were performed using Graph-pad Prism 5 (Graph-pad Software Inc., USA). The results were expressed as mean ± sem of four independent measurements. Statistical analysis was determined by using One-way Analysis of Variance (ANOVA), and Turkey's multiple comparison test was also applied. Results were indicated as significant at p < 0.05.

Phytochemical Screening of Garcinia kola Seeds Extract
The phytochemical study of the aqueous extract of Garcinia kola seeds revealed the presence of sterols and polyterpins, polyphenols and alkaloids (Table 1).

Peroxide Scavenging Activity of Garcinia kola Seeds Extract
The extract scavenged hydrogen peroxide in a concentration-dependent manner with the IC 50 value of 1.01 ± 0.5 mg/mL compared with IC 50 value of 0.52 × 10 −1 ± 0.1 mg/mL for ascorbic acid. EAgk and ascorbic acid at the unique concentration of 10 −1 mg/mL, induced peroxide scavenging activities of 26.85% ± 2.41% and 32.57% ± 2.3% respectively (Figure 4).

Hemostatic Activity of Garcinia kola Seeds Extract
The plasma poor platelet was used to study the hemostatic effects of Garcinia kola seeds and Phytomenadione (vitamin K 1 ).

Effects of Garcinia kola Seeds Extract on Activated Partial Thromboplastin Time (aPPT)
The aqueous extract of garcinia kola seeds caused the decrease of activated partial thromboplastin time in a concentration dependent manner. The most important decrease of this time, induced by EAgk and phytomenadione (Vitamin K 1 ) used as positive control, were operated at the concentration of 1 mg/mL. But these decreases were not significant compared to the negative control which was distilled water (p > 0.05, n = 4). The outcomes are summarized in Figure 5.

Effects of Garcinia kola Seeds Extract on Prothrombin Time (PT)
The prothrombin time was not affected by the aqueous extract of Garcinia kola seeds and vitamin K 1 . Garcinia kola seeds extract and vitamin K 1 effects are shown in Figure 6.

Acute Oral Toxicity of Garcinia kola Seeds Extract
EAgk at the dose of 5000 mg/kg b.w. did not cause any mortality in rat. However

Effects of Garcinia kola Seeds Extract on Bleeding Time
Bleeding

Effects of Garcinia kola Seeds Extract on Platelet Count
Platelet

Discussion
The hemostatic effects of the aqueous extract of Garcinia kola seeds and phytomenadione were assessed under different conditions. Phytomenadione or vitamin K1 (coagulant) was considered a reference drug. In vivo, ex vivo and in vitro tests were used in this study. Garcinia kola seeds extract as well as phytomenadione shortened significantly bleeding time in rat. To further confirm the seeds extract bleeding time shortened action, we evaluated its effects on platelet count, on prothrombin time and on activated partial thromboplastin time.
In ex vivo experiments, Garcinia kola extract and vitamin K 1 increased platelet count. The correlation between platelet counts and bleeding times has been reported by several authors. Platelet count was significantly lower and bleeding time significantly prolonged in patients with preeclampsia compared with the control group [31]. Also, high platelet counts were associated with 1.8-fold risk of arterial thrombosis in the brain [32]. Thus, platelet count increase induced by garcinia kola seeds extract could justify bleeding time shortened effects. Garcinia kola extract and phytomenadione shortened activated partial thromboplastin time in vitro whereas prothrombin time was not affected. However, the decrease of activated partial thromboplastin time was not significant. The extract could act by intrinsic pathway of the coagulation. This way involves the plasma proteins factors VIII, IX, XI and XII; and prekallikrein and high molecular weight kininogen [4].
It is well documented in the literature that phytomenadione strengthens coagulation as measured by prothrombin time decrease and increases in the activity of vitamin K-dependent clotting factors and thrombin generation [33]. Vitamin K 1 effects on prothrombin time in this study are different from those obtained in our experiments. This difference may be due to experimental conditions which are not the same.
According to professional order of medical technologists of Quebec, it is strongly advised not to carry out hemostasis analysis on a hemolyzed sample [26]. Hemolytic samples are unsuitable for coagulation assays because of the release of hemoglobin, intracellular components, and thromboplastic substances from damaged blood cells [34].
Hemolytic and antihemolytic activities of Garcinia kola seeds extract were undertaken to appreciate the ability of the extract to protect red blood cells against hemolysis.
Preincubation of red blood cells with Garcinia kola seeds extract, did not show any harmful effects on cells. In addition, the extract inhibited 2,2'-Azobis(2methylpropionamidine) dihydrochloride (AAPH)-induced hemolysis. At physiological temperature, AAPH can decompose to generate alkyl radicals to initiate lipid peroxidation and hemolysis [35]. Protective action of Garcinia kola seeds extract against hemolysis may be due to the ability of the extract to scavenge 2,2-Diphenyl-1-picryl hydrazyl radical and peroxide radical assessed in our study.
The radical scavenging properties of Garcinia kola seeds extract could be attri-

Conclusion
The present study indicates that the aqueous extract of Garcinia kola seeds shortens bleeding time, activated partial thromboplastin time and increases platelet counts. The extract protected blood red cell against hemolysis and could accelerate blood coagulation through intrinsic pathway. Garcinia kola seeds possess hemostatic properties. This property is justified by the presence of coagulant components in the extract. More studies are needed to isolate the coagulant component and to provide a better insight into the mechanism of these pharmacological actions.