INTRODUCTION:

Surgery in dentistry generally includes exodontia, odontectomy, preprosthetic surgery, cleft lip and palate surgery, endodontic surgery and so on. These surgeries can result in a high risk of complications. If not immediately controlled and handled properly, it can lead to complications in the form of bleeding. Bleeding that is difficult to stop triggers disruption of the stability of the body’s haemostasis function by blood fluid^1,2.

Haemostasis itself has an important role in stopping bleeding spontaneously through three mechanisms: blood contraction, platelet plug formation and blood coagulation³. Control of bleeding should be done immediately either by applying tampons to suppress bleeding, suturing the surgical wound or by administering haemostatic drugs^4,5,6. One form of haemostatic medicine is tranexamic acid, which functions to increase platelet aggregation by inhibiting the activation of plasminogen into plasmin⁷. However, tranexamic acid can cause mild to severe side effects in patients such as hypersensitivity reactions, pulmonary embolism and deep vein thrombosis, so materials with haemostatic properties that have minimal side effects are needed as an alternative treatment in stopping bleeding ⁵.

Natural ingredients can be a substitute option due to their minimal side effects. Natural ingredients such as soybeans can be used as an option because they have been widely consumed by the public in fresh and processed forms. Edamame as a type of soybean with various benefits that have a higher selling value than ordinary soybeans. Edamame seeds as a natural ingredient have the results of phytochemical screening of flavonoids, saponins and tannins which are components with good probability in shortening bleeding time in the haemostasis mechanism^8,9. Based on the explanation above, researchers are interested in conducting research on effectiveness of edamame seeds (Glycine max l. Merril) extract on fibrinolytic activity test in strain BALB/c mice, including bleeding time, clotting time, and platelet count tests ^10,11. Before the test was carried out, a phytochemical screening test was carried out on the active ingredients suspected to be present.

MATERIALS AND METHODS:

The type of research used is laboratory experimental research with the posttest only control group design). The research sample used was BALB/c strain mice with inclusion criteria of male sex, weight 20-30grams, age 3-4 months, body and tail in good health while exclusion criteria include mice that die during the study, diarrhea, tail wounds or lesions. Edamame seeds (Glycine max L. Merril) in this study came from PT Mitra Tani Dua Tujuh Jember and plan identification was carried out at the Integrated Agricultural Development Laboratory, Jember State Polytechnic (No. 16/PL17.8/PG/2023).

Ethical clearance for experimental animal research subjects is done at The Ethical Committee of Medical Research, Faculty of Dentistry University of Jember (No. 1824/UN25.8/KEPK/DL/2022).

Edamame Seed Extraction Procedure:

Preparation of edamame seed extract in Pharmaceutical Chemistry Laboratory, Faculty of Pharmacy, University of Jember, using the Hasanah et.al¹²method with modifications . Fresh edamame seeds as much as +5.6 kg were cleaned from impurity contamination using water, cut into small parts and dried using an oven at a temperature of +50^oC, then blended to obtain crude plant powder and sieved using 60 mesh size. After that, edamame seed powder was extracted with 70% ethanol solvent using a powder and solvent ratio of 1:5. The filtrate obtained was evaporated using a vacuum rotary evaporator at +50⁰ C until a thick extract was obtained.

Thin-Layer Chromatography (TLC) Assay of Edamame Seed Extract (ESE):

TLC examination aims to conduct an initial screening test of the active ingredients of frangipani extract in both groups. The active ingredients detected were flavonoids, saponin, and tannin.

Flavonoid Compound:

The extract (0.1g) was put in 1ml n-hexane and repeatedly stirred until the extract was colourless. The residue was dissolved in some drops of ethanol, and then it was dropped on thin-layer chromatography (silica gel 60 F254). After that, it was eluted by the mobile phase of butanol-glacial acetic acid-water (4:1:5). It was subsequently sprayed stain viewer of borate citric. The intensity yellow colour indicated a flavonoid presence.

Saponin Compound:

1gram of extract is dissolved in 5ml of distilled water and put into a test tube. Then add 1ml of distilled water 1:1 and shake the solution for 1minute. If there is foam, add 2N HCl, if the foam can last for 10minutes so that the height is 1-10cm, then the extract is positive for containing saponins

Tannin Compound:

Extract 1mg, put into a test tube and add 2-3 drops of 1% FeCl3 solution. The solution changes color to blackish green or dark blue, then the extract contains tannin.

Sample Grouping and Treatment:

The research sample of 20 mice strain BALB/c was divided into 3 groups, namely the negative control group (C-), the positive control group (C+), and the treatment group (P) which was divided into 3 subgroups, namely treatment group 1(ESE1), treatment group 2(ESE2) and treatment group 3(ESE3). All groups were adapted for 1 week. On day 8, all sample groups were fasted for 7 hours.

Na-CMC, tranexamic acid and edamame seed extract were only given once before the tail bleeding time test. Group (C-) was given Na-CMC 0.5%, group (C+) was given a dose of tranexamic acid 0.065mg/gr BW mice, group (ESE1) was given a dose of edamame seed extract 0.112mg/gr mice, (ESE2) a dose of 0.14mg/gr BW mice and (ESE3) a dose of 0.168mg/gr BW mice in Na-CMC 0.5% according to the capacity of the mice's stomach using a gastric tube. The treatment was given for 3 days at 7-8am. After the 3rd day of treatment (day 4), all groups were tested.

Fibrinolytic Activity Test:

All treatment groups were performed bleeding time, clotting time, and platelet count tests.

Bleeding Time Test:

Bleeding time was measured using the Duke method, namely by cutting the tip of the rat's tail 0.5cm long which had previously been cleaned with 70% alcohol. The blood that came out was absorbed using filter paper at certain time intervals. The time from the first blood flow until the blood stopped dripping and could not be absorbed by the filter paper was calculated as the length of the bleeding time.

Clotting Time Test:

Clotting time was tested using the glass object method, namely by cutting the tip of the rat's tail 0.5cm long which had previously been cleaned with 70% alcohol. The blood that came out was first removed using filter paper, the blood that came out was then dripped onto the glass object. The tip of the lancet was moved upwards on the blood drop until fibrin threads were visible. Clotting time was seen when fibrin threads were visible on the blood drop.

Platelet Count Tests:

Platelet count examination is done automatically using the Automatic Hematology Analyzer. Blood is taken through the orbital vein of the eye using microhematocrit and collected in a tube that has been given EDTA (ethylenediamine tetraacetic acid). The blood sample is placed in the sample tube on the Automatic Hematology Analyzer, then the sample is sucked by the tube and the device begins to calculate. The results will be displayed on the monitor screen and printed in the form of a print out.

RESULT:

Result of Thin-Layer Chromatography (TLC) Assay:

The presence of active ingredients detected is obtained from comparison of the results obtained with the reference values

Table 1. Result of Thin-Layer Chromatography (TLC) Assay on Edamame Seed Extract (ESE)

Phytochemicals Assay	Reference Value of Positive Result	Result
Flavonoid	Yellow color is formed	+
Saponin	The foam can last for 10 minutes so that the height is 1-10 cm	+
Tannin	a blackish green or dark blue color is formed	+

Notes: +: indicates the presence of the active ingredient being tested

The table above shows that ESE contains 3 active ingredients that are suspected to be present previously, namely flavonoids, saponins and tannins.

Bleeding Time Test:

The results of the Bleeding Time Test are shown in Figure 1.

Figure 1. Result of Bleeding Time Test in BALB/c Strain Mic

Notes: C-: negative control group (Na-CMC 0.5%), C+: Positive control group (tranexamic acid dose 0.065 mg/gr BW), ESE1: dose 0.112 mg/gr BW, ESE2: dose 0.14 mg/gr BW, ESE3: dose 0.168 mg/gr BW.

Figure 1 shows that in the treatment group there is a tendency that the higher the concentration of ESE, the smaller the test result value. This shows that ESE has the potential to accelerate bleeding time, where the smallest value or closest to the C+ group is the ESE3 group.

Clotting Time Test:

The results of the Clotting Time Test are shown in Figure 2.

Figure 2. Result of Clotting Time Test in BALB/c Strain Mice

Notes: C-: negative control group (Na-CMC 0.5%), C+: Positive control group (tranexamic acid dose 0.065mg/gr BW), ESE1: dose 0.112mg/gr BW, ESE2: dose 0.14mg/gr BW, ESE3: dose 0.168mg/gr BW

Figure 2 shows that in the treatment group there is a tendency that the higher the concentration of ESE, the smaller the test result value. This shows that ESE has the potential to accelerate clotting time, where the smallest value or closest to the C+ group is the ESE3 group.

Platelet Count Tests:

The results of Platelet Count Test are shown in Figure 2.

Figure 3. Result of Platelet Count Tests in BALB/c Strain Mice

Notes: C: negative control group (Na-CMC 0.5%), C+: Positive control group (tranexamic acid dose 0.065mg/gr BW), ESE1: dose 0.112mg/gr BW, ESE2: dose 0.14mg/gr BW, ESE3: dose 0.168 mg/gr BW

Figure 3 shows that in the treatment group there is a tendency that the higher the concentration of ESE, the greater the test result value. This shows that ESE has the potential to increase the number of platelets, where the largest value or closest to the C+group is the ESE3 group.

DISCUSSION:

Based on the results of the analysis showing the average value of tail bleeding time of mice group C- which was given a dose of 0.5% Na-CMC with group C+ which was given a dose of tranexamic acid 0.065mg/gr BB mice there was a significant difference. The difference when viewed in the calculation of the length of tail bleeding time and clotting time is that the group of mice given tranexamic acid has a shorter tail bleeding time compared to the group of mice given Na-CMC. This is in accordance with the expected results, because in theory Na-CMC only acts as a thickener to make it easier for mice to digest the extract given without affecting hemostasis activity while tranexamic acid is a patent drug that has been tested for its effectiveness in shortening bleeding time.^13,14

Tranexamic acid was chosen as the positive control as it acts as an inhibitor of the proenzyme plasminogen to the active enzyme plasmin.¹⁵ The inhibition caused by the presence of tranexamic acid is able to trigger plasminogen to prevent plasmin from destroying the GPIb and GPII/GPIIIa glycoprotein receptor bonds resulting in increased platelet adhesion and aggregation which will significantly continue the blood clotting process and overcome bleeding.¹⁶ On a broad spectrum, tranexamic acid plays a role in reducing the amount of blood loss in surgery and health conditions associated with increased bleeding.¹³

The treatment groups have the ability to shorten the bleeding time when compared to the C- group. This can occur because Na-CMC has no hemostasis effect, while edamame seed extract is thought to have hemostasis activity in shortening bleeding time. This activity is shown through its active compounds, namely flavonoids, saponins and tannins.

Flavonoids have been shown to exhibit significant fibrinolytic activity, which is the process of dissolving blood clots. Flavonoids can interact with fibrinogen, affecting its activity or concentration. For example, studies have shown that flavonoids like epicatechin and epigallocatechin gallate can bind to fibrinogen, influencing its function and potentially its role in clot formation and dissolution.c Beside that, Flavonoids from various plant sources have demonstrated thrombolytic activity in vitro. For instance, extracts from plants like Madras Nelli (Phyllanthus maderaspatensis) and Homalomena aromatica have shown significant clot lysis effects, comparable to or even surpassing that of standard thrombolytic drugs like streptokinase.¹⁷ The other researcher states the thrombolytic activity of flavonoids is attributed to their ability to inhibit the formation of blood clots or enhance the breakdown of existing clots. This is often linked to their antioxidant properties, which help in reducing oxidative stress and inflammation, both of which are crucial in the pathogenesis of thrombosis.¹⁸

Flavonoid compounds have an influence on hemostasis by inhibiting prostacyclin production. Prostacyclin inhibition triggers an increase in Plasminogen Activator Inhibitor-1 (PAI-1).⁹High amounts of PAI-1 result in inhibition of the formation of plasminogen into plasmin.¹⁹ Inhibition of plasmin is known to increase the distribution of GPIb and GPIIb/ GPIIIa receptor binding to fibrinogen resulting in increased platelet adhesion and aggregation, thus accelerating wound healing.^7,16

In summary, flavonoids play a significant role in fibrinolytic activity by either directly interacting with fibrinogen or enhancing the body's natural fibrinolytic processes, thereby aiding in the prevention and treatment of thrombotic disorders.

Saponins, particularly those derived from various plant sources, have been studied for their effects on fibrinolytic activity, which is the process of dissolving fibrin clots. Studies on Paris saponins, such as those from P. fargesii var. brevipetala, have shown that these compounds can enhance thrombin activity, which is a key enzyme in the coagulation cascade. However, the direct fibrinolytic activity of Paris saponins is not explicitly mentioned in the provided sources. Instead, they are noted for their hemostatic effects, which include enhancing thrombin activity and potentially influencing blood coagulation pathways.²⁰ The other study states Fermented ginseng using Bacillus subtilis has been found to increase fibrinolytic activity. The fermentation process enhances the content of ginsenosides, which are known to exhibit fibrinolytic activity. The fibrinolytic activity of the ginseng medium was measured using a method that involves creating a fibrin plate and assessing the area of transparency generated by fibrinolytic enzymes.²¹ Beside that, Diosgenyl saponins, particularly diosgenin and its derivatives, have been shown to have anti-thrombotic activity. These compounds inhibit platelet aggregation and prolong bleeding time, indicating a potential role in fibrinolysis by preventing clot formation and promoting the dissolution of existing clots.²²

Saponins play a role in the process of hemostasis by increasing the synthesis of PAI-1. PAI-1 is a single-chain glycoprotein member of serpins that functions as the main inhibitor of tissue-type plasminogen activator (t-PA) and urokinase type (u-PA) so that it plays an important role in the mechanism of inhibiting the formation of plasminogen into plasmin.²³ Inhibition of plasmin stimulates platelet binding to endothelium with GPIb receptors and enhances platelet aggregation in tissues with GPIIb/ GPIIIa receptors to fibrinogen.^7,16 This will accelerate the formation of temporary platelet plugs in the blood vessels in the injured area.⁵

In summary, while saponins like those from Paris and ginseng may not directly exhibit significant fibrinolytic activity, they can influence coagulation pathways and potentially contribute to hemostasis. Diosgenyl saponins, however, have been demonstrated to have anti-thrombotic effects, which include inhibiting platelet aggregation and prolonging bleeding time, suggesting a role in fibrinolysis.

Tannins have an effect in inhibiting plasmin activity. In a study conducted by Pereira et al ²⁴ revealed the ability of tannins to inhibit plasmin activity in vitro using flower Brownea grandiceps extract. Inhibition of plasmin activity is important in increasing platelet plug formation as it enhances platelet adhesion and aggregation mechanisms through mobilization of GPIb and GPIIb/GPIIIa receptors to fibrinogen in tissues, preventing further blood loss.^8,16

Tannins have been shown to have both profibrinolytic and antifibrinolytic effects on the fibrinolytic system, which is responsible for dissolving blood clots and maintaining vessel patency. This ellagitannin, isolated from Phyllanthus urinaria, has been found to increase tissue plasminogen activator (t-PA) activity and decrease plasminogen activator inhibitor 1(PAI-1) activity in plasma and in platelets stimulated by thrombin. This increase in t-PA activity enhances the fibrinolytic process, promoting the dissolution of blood clots. Besides that, in studies on diabetic rats, the extract from Potentilla erecta rhizome increased t-PA activity, which is beneficial for fibrinolysis.²⁵

On the other hand, Tannins have antifibrinolytic effects. the same extract from Potentilla erecta rhizome has been shown to inhibit clot dissolution in normoglycemic rats, leading to prolonged euglobulin clot lysis time. This indicates an antifibrinolytic effect under certain conditions. Besides that, The procyanidin fraction from Brownea grandiceps flowers inhibited plasmin activity, which is a key component of the fibrinolytic system. This inhibition suggests an antifibrinolytic effect.^25,26

In summary, tannins can modulate the fibrinolytic system in both positive and negative ways, depending on the specific tannin and the experimental conditions. Understanding these dual effects is crucial for assessing their potential therapeutic applications in managing thrombotic disorders.

The use of three different doses of edamame seed extract in this study was used to see the dose-effect relationship in each experimental result.²⁷ The successive increase in edamame seed extract dose is thought to result in a shortening of bleeding time and clotting time, increase platelet count. This occurs because the more doses used, the more the influence of flavonoids, tannins and saponins that play a role in all three tests.²⁸

Based on the results of the three texts, the ESE1 group has very different values when compared to the C+ group. This can occur because the dose given to the group of mice with the administration of edamame seed extract dose of 0.112mg/kg BW of mice is thought to be ineffective or not optimal enough compared to the dose used in the group of mice with the administration of tranexamic acid dose of 0.065mg/kg BW of mice. In addition, because edamame seed extract is a form of traditional medicine. Traditional medicine is known to have a weaker pharmacological impact and a slow absorption period when compared to the patent drug tranexamic acid.^29,30According to Dewastisari's research ³¹, the yield value (total extract) is related to the amount of active substance content in plants so that a small dose of extract has a small total active component as well. The presence of ballast substances or impurity compounds in the form of chlorophyll, resins, fats, proteins or other nonpolar compounds in traditional medicine content can also trigger a weak pharmacological impact.²⁸However, in the treatment group with the administration of three different doses of edamame seed extract, the average bleeding time closest to the C+ group or 0.065mg of tranexamic acid was the ESE1 group so that the 0.112mg dose of edamame seed extract can be recommended as an alternative to shorten bleeding time because it has an effect that is not much different from tranexamic acid in stopping the amount of blood loss.

CONCLUSION:

Based on the results of the study, it can be concluded that the edamame seed extract (Glycine max L. Merril) is effective in shortening tail bleeding time, clotting time and increase platelet count in BALB/c strain mice.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

This research is financially supported by Grant “Hibah Reworking Skripsi/Tesis”, Institute for Research and Community Service, Universitas Jember

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Received on 09.09.2024 Revised on 10.01.2025

Accepted on 01.04.2025 Published on 01.10.2025

Available online from October 04, 2025

Research J. Pharmacy and Technology. 2025;18(10):4609-4614.

DOI: 10.52711/0974-360X.2025.00662

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.