In-Vitro Anti-Coagulant Activity of Ethyl Acetate and Aqueous Extract of Syzygium cumini Leaves on Normal Blood Plasma
B. D. Prasad1, Ajey B. Gadagi2, S. Dadakhalandar3, Dyamanagouda3, Jameer4, Vikas4, Nagaraj4, Veeresh4, Sharavan Naragund5
5Principal, Dr. Gurachar Nargund College of Pharmacy, Muradi, Koppal.
*Corresponding Author E-mail: ajeygadagi@gmail.com
ABSTRACT:
The present research was undertaken to evaluate the in-vitro anticoagulant activity of aqueous and ethyl acetate leaf extracts of Syzygium cumini. The plant leaves were collected, shade-dried, powdered, and subjected to extraction using distilled water and ethyl acetate. The obtained extracts were screened for phytochemical constituents, which revealed the presence of carbohydrates, tannins, flavonoids, and glycosides, indicating the potential biological activity of the plant. The anticoagulant activity was determined by Prothrombin Time (PT) method using platelet-poor plasma isolated from healthy human volunteers. Plasma samples were treated with different concentrations of plant extracts ranging from 0.125 g/ml to 1 g/ml, and the clotting time was observed after the addition of calcium chloride. The findings demonstrated that both aqueous and ethyl acetate extracts significantly prolonged the prothrombin time in a concentration-dependent manner. The aqueous extract showed the highest anticoagulant activity, increasing the clotting time up to 48 minutes and 02 seconds at a concentration of 1 g/ml, whereas the ethyl acetate extract extended the clotting time to 45 minutes and 57 seconds at the same concentration. These results were notably higher compared to the normal control clotting time of 8 minutes and 16 seconds. The enhanced anticoagulant potential may be attributed to phenolic compounds and flavonoids that are known to inhibit clotting factor activation or chelate calcium ions essential for blood coagulation. Result: The normal plasma exhibited clot formation within 8 minutes and 16 seconds, indicating a normal physiological response. When plasma was treated with the standard anticoagulant (EDTA), the clotting time increased to 23 minutes and 14 seconds, confirming test accuracy. Upon treatment with plant extracts, the clotting time increased progressively with concentration. The aqueous extract produced the highest clotting time of 48 minutes and 02 seconds at 1 g/ml, while the ethyl-acetate extract prolonged clotting to 45 minutes and 57 seconds. This indicates a strong dose-dependent anticoagulant effect, with aqueous extract showing greater potency due to higher solubility and concentration of phenolic compounds in water.
KEYWORDS: Syzygium cumini, Anti-coagulant Activity, Prothrombin Time (PT), Aqueous Extract, Ethyl Acetate Extract.
INTRODUCTION:
Blood Coagulation:
Blood is a vital biological fluid composed of cellular and plasma components that collectively maintain physiological equilibrium, nutrient transport, gas exchange, immune defense, and hemostasis. The coagulation system, a critical component of hemostasis, functions through a cascade of enzymatic reactions that convert soluble fibrinogen into insoluble fibrin, leading to clot stabilization at sites of vascular injury. This process involves the intrinsic, extrinsic, and common pathways, which act synergistically to maintain the balance between hemorrhage and thrombosis. Disruption in this equilibrium results in either hypocoagulable or hypercoagulable states, both of which are associated with significant clinical morbidity and mortality.1.
Coagulation disorders include a spectrum of inherited and acquired abnormalities. Inherited conditions, such as Hemophilia A (Factor VIII deficiency) and Factor V Leiden mutation, result from genetic alterations in key clotting factors.
In contrast, acquired conditions, including Antiphospholipid syndrome, malignancy, and prolonged immobility, often arise secondary to systemic or environmental influences2. The “multiple-hit hypothesis” highlights the synergistic interplay between genetic predisposition and external risk factors in the development of thrombotic disorders.3.
Globally, venous thromboembolism (VTE) is the second most prevalent cardiovascular disorder after myocardial infarction, while Hemophilia A remains the most common X-linked hereditary bleeding disorder, affecting approximately one in 5,000 males worldwide4. Although diagnostic and therapeutic advances have markedly improved disease management, limited access to specialized care in developing regions continues to pose major public health challenges.5.
Anti-Coagulant:
Recent developments in coagulation management, such as the use of direct oral anticoagulants (DOACs) and advances in gene therapy, have improved safety and treatment outcomes. Nevertheless, limitations related to bleeding risk, high costs, and drug interactions highlight the need for safer, more affordable alternatives.6.
Emerging evidence indicates that its extracts possess anticoagulant potential, possibly by influencing intrinsic and extrinsic pathways of coagulation7.
Introduction to Plant (Syzygium Cumini Leaves):
Syzygium cumini (L.) Skeels, commonly known as Jamun or Java plum, is an evergreen tropical tree belonging to the family Myrtaceae, native to India and Southeast Asia but now widely distributed across tropical and subtropical regions8. Traditionally, its bark, leaves, seeds, and fruit have been extensively used in the Ayurvedic and Unani systems of medicine for treating diabetes, inflammation, ulcers, and microbial infections owing to its rich phytochemical profile that includes alkaloids, flavonoids, tannins, terpenoids, and triterpenoids9.
Scientific investigations have demonstrated that the biological activities of S. cumini are closely related to its high phenolic and flavonoid content, which contributes to its potent antioxidant, anti-inflammatory, and antidiabetic effects10,11. Moreover, extracts of S. cumini have shown notable antimicrobial activity against pathogenic strains of Staphylococcus aureus and Pseudomonas aeruginosa, confirming its traditional use in infectious conditions12. Recent studies have also highlighted its anticancer and cytoprotective potential through reactive oxygen species (ROS)-mediated apoptosis in cancer cells13.
Given its diverse pharmacological activities and bioactive constituents, Syzygium cumini represents a promising source of natural compounds with therapeutic potential. The present study, therefore, focuses on elucidating the plant’s anticoagulant potential, aiming to expand the scientific understanding of its pharmacological relevance and support the development of safe, effective, and affordable natural alternatives to synthetic drugs14.
Purpose:
In-vitro anticoagulant activity of aqueous and ethyl acetate extract of syzygium cumini leaves on normal blood plasma.
Extraction:
Preparation of aqueous extract of leaves of Syzygium cumini:
Fresh Syzygium cumini plant leaves were collected and washed properly with distilled water. The leaves were shade dried at room temperature for 15 days. Dried parts were uniformly grinded using mechanical grinder. The dried powder of plant material was extracted in distilled water. 10gm of ground plant material was soaked in 100ml of distilled water in a round bottom flask and loaded in the heating mantle at a temperature of 70°C for 15minutes for boiling. After 15min temperature was reduced to 40°C and kept in that temperature for 1hour. Then the mixture was filtered using what-mann filter paper number 1. Each grounded plant materials were extracted separately. The dried extracts were weighed and stored in air tight container with necessary markings for identification and kept in refrigerator (0-4°C) for future investigation.15
Ehyl Acetate Extraction Method:
The fresh leaves were shade-dried and cleansed properly to remove any extraneous material. The dried leaves were ground into coarse powder. The powdered plant material (300g) was then refluxed with ethyl acetate (900 ml) and kept in the dark for 3 to 5 days with occasional stirring and shaking. The mixture was filtered using a cotton plug followed by filtration through a Whatman filter paper no. 1. The filtrate was dried at 50˚C under reduced pressure. The extract was preserved in an air-tight flask in a refrigerator as a crude extract.16
In-vitro studies of Prothrombin Time (Pt) Determination for anticoagulant activity
By making vein puncture of healthy volunteers, 10ml of blood was drawn. Volume of blood about 9ml, volume of 1ml 3.8% tri-sodium citrate solutes added for Ignoring of coagulation process Which occurs naturally. Then centrifugation has done about for 15minutes at 3000 RPM rate. Then from plasma the blood cells get separated. From that pure platelet, plasma was obtained. PT test was used for Pure platelet plasma (PPP).
Plasma sample was divided into five groups.
Group I: Negative control group 0.2ml. Plasma + 0.1ml of saline water + 0.3ml of CaCl2(0.5g/ml).
Group II: positive control group 0.2ml of plasma + 0.1 ml of 50mg/ml of EDTA + 0.3ml of CaCl2. (0.5mg/ml)
Group III: 0.2ml of plasma + 0.1ml of. 0.125g/ml of plant extract + 0.3ml of CaCl2 (0.5g/ml).
Group IV: 0.2ml of plasma + 0.1ml of 0.25g/ml of plant extract + 0.3ml of CaCl2.(0.5g/ml).
Group V: 0.2ml of plasma + 0.1ml of 0.5g/ml of plant extract + 0.3ML of CaCl2 (0.5g/ml).
Group VI: 0.2ml plasma + 0.1ml of 1g/ml of plant extract+0.3ml of CaCl2 (0.5g/ml).
At the angle of 45° all tubes are tilted for every 30 seconds for measuring of clotting time. for clot formation stopwatch is used. The time is called Pro-thrombin time PT. This was repeated for three times then calculation of average time was done.
For determination of anticoagulant activity, aqueous extract of Syzygium-cumini skin was investigated he concentrations of preparations are 0.125, 0.25, 0.5g/ml and 1gram.17
RESULT:
The phytochemical screening of Syzygium cumini leaf extracts revealed that the aqueous extract contained carbohydrates, tannins, phenolic compounds, alkaloids, flavonoids, and glycosides, while the ethyl acetate extract lacked alkaloids and amino acids. The anticoagulant activity, assessed by the prothrombin time test, showed that both extracts significantly prolonged clotting time in a concentration-dependent manner compared to the control. The aqueous extract increased clotting time from 13 minutes (0.125 g/ml) to 48 minutes 02 seconds (1 g/ml), and the ethyl acetate extract from 10 minutes 28 seconds to 45 minutes 57 seconds, demonstrating strong anticoagulant potential.
Table No: 1 Phytochemical Screening of Syzygium Cumini Leaf Extracts
|
Constituent |
Aqueous Extract |
Ethyl Acetate Extract |
|
Carbohydrates |
Present |
Present |
|
Tannins |
Present |
Present |
|
Phenolic Compounds |
Present |
Present |
|
Alkaloids |
Present |
Absent |
|
Flavonoids |
Present |
Present |
|
Glycosides |
Present |
Present |
Amino Acids |
Absent |
Absent |
|
GROUP 01: -8 min 16 sec |
GROUP 02: -23 min 14 sec |
|
AQUEOUS EXTRACT |
ETHYL ACETATE EXTRACT |
|
GROUP 03: -13 min |
GROUP 03: -10 min 28 sec |
|
GROUP 04: -25 min 12 sec |
GROUP 04: -13 min 32 sec |
|
GROUP 05: -34 min 08 sec |
GROUP 05: -37 min 44 sec |
|
GROUP 06: -48 min 02 sec |
GROUP 06: -45 min 57 sec |
Prothrombin Time (PT) and Partial Thromboplastin Time (PTT) are important evaluation tests used to determine the functionality of the extrinsic and intrinsic pathways of coagulation, respectively.
Syzygium cumini has been reported that the inhibition of intrinsic coagulation factors is responsible for the prolongation of the Partial Thromboplastin Time (PTT) test, whereas inhibition of extrinsic factors leads to prolongation of the Prothrombin Time (PT) test.
Many researchers have reported that Syzygium cumini possesses anticoagulant properties. In the present study, different extracts of Syzygium cumini were tested on normal blood plasma. An increase in the concentration of Syzygium cumini extract resulted in an increased Prothrombin Time (PT), indicating delayed blood coagulation. Both aqueous and ethyl acetate extracts produced a mild prolongation of clotting time at lower concentrations.
At higher concentrations, a significant increase in clotting time was observed for both extracts. When comparing the two, the aqueous extract showed a greater prolongation of coagulation time than the ethyl acetate extract, likely due to higher water solubility of phytochemical components in the aqueous medium.
EDTA was used as the standard anticoagulant drug, which delays coagulation by chelating calcium ions. Finally, when comparing Syzygium cumini extracts with the standard drug, the aqueous extract showed a higher prolongation of clotting time than EDTA.
CONCLUSION:
The present investigation demonstrated that the aqueous and ethyl acetate extracts of Syzygium cumini leaves exhibit significant in-vitro anticoagulant activity. The Prothrombin Time (PT) increased with rising extract concentrations, indicating a dose-dependent inhibition of blood coagulation. This anticoagulant effect may be attributed to the presence of flavonoids, tannins, and phenolic compounds in the extract.
Therefore, Syzygium cumini has the potential to act as a natural anticoagulant agent, and may serve as a supportive herbal alternative in the management of conditions associated with hyper-coagulation and thrombosis.
However, to establish its therapeutic value, further research is recommended, including:
· Isolation and characterization of active constituents
· Pharmacological evaluation in animal models
· Toxicity and safety assessment
· Clinical validation in human subjects
This study provides a promising foundation for the development of plant-based anticoagulant formulations and contributes to the growing evidence supporting the medicinal significance of Syzygium cumini.
CONFLICT OF INTEREST:
The authors have no conflicts of interest regarding this investigation.
ACKNOWLEDGEMENTS:
Author express deepest gratitude to Dr. Prasad B, Professor and HOD & Dr. Shravan L. Nargund, Principal, Dr. Gurachar Nargund College of Pharmacy, Muradi, for thire continual support.
|
T |
Prothrombin Time |
|
PPP |
Platelet Poor Plasma |
|
EDTA |
Ethylene-di-aminetetraacetic Acid |
|
CaCl2 |
Calcium Chloride |
|
RPM |
Revolutions Per Minute |
|
g/ml |
Grams per millilitre |
|
min |
Minutes |
sec |
Seconds |
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Received on 09.12.2025 Revised on 27.12.2025 Accepted on 13.01.2026 Published on 12.02.2026 Available online from February 14, 2026 Res.J. Pharmacology and Pharmacodynamics.2026;18(1):111-114. DOI: 10.52711/2321-5836.2026.00015 ©A and V Publications All right reserved
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