INTRODUCTION:

Haemostasis refers to an important physiological process preventing blood loss and stopping bleeding in cases of vascular injury, this process can be divided into primary and secondary hemostasis. Primary hemostasis, which results in the formation of a soft platelet plug, involves vasoconstriction, platelet adhesion, platelet activation, and platelet aggregation¹. Secondary hemostasis is primarily defined as the formation of fibrinogen into fibrin, which ultimately evolves the soft platelet plug into a hard, insoluble fibrin clot. Within primary and secondary hemostasis, three coagulation pathways exist: intrinsic, extrinsic, and common.

The intrinsic pathway responds to spontaneous, internal damage of the vascular endothelium, whereas the extrinsic pathway becomes activated secondary to external trauma. Both intrinsic and extrinsic pathways meet at a shared point to continue coagulation, known as the common pathway.

The coagulation factors involved in the intrinsic pathway are factors I, II, IX, X, XI, and XII. The factors involved in the extrinsic pathway are factors X, VII, II, and I. These pathways converge at the common pathway, which consists of factors I, II, V, VIII and X. Some of the clotting factors function as serine proteases, specifically factors II, VI, IX, and X^2,3.

Prothrombin test PT testmeasures the activity of the extrinsic and common pathways. A normal PT value is between 11 and 15 seconds. Prolongation in PT refers to bleeding risk⁴.

Thrombosis refers to a condition in which a blood clot forms in a healthy blood vessel. It can occur in arteries or veins, and both cases are considered among the most important causes of death in the contemporary time, as estimates indicate that thrombotic diseases are responsible for one death out of every four deaths worldwide⁵.

The symptoms vary depending on the location of the blood clot formation. For example, a clot may form in the cerebral arteries, causing a stroke, or in the heart vessels, causing a myocardial infarction. A clot can also cause temporary blockages in blood supply, known as transient ischemic attacks (TIAs), or may form in the veins, causing deep vein thrombosis (DVT), or in the lungs, causing a pulmonary embolism (PE)⁶.

Many groups of Anti-coagulants drugs are used in the treatment of thrombotic diseases. Each of these groups has a different mechanism of action. These groups include thrombolytic drugs such as streptokinase and alteplase, vitamin K antagonists such as warfarin, drugs that inhibit the action of clotting factors II, X such as heparin and low molecular weight heparins⁷. In addition tothe previous drugs, new oral anticoagulants are used like thrombin direct inhibitors such as dabigatran, as well as factor X direct inhibitors such as rivaroxaban and apixaban⁸. Groups that inhibit platelet activation and aggregation, such as aspirin, clopidogrel, and abciximab, have also been used, with reference to the fact that every medication has a set of side effects, drug-drug or drug-food interactions, in addition to the risk of bleeding⁹.

Researchers also turn to alternative medicine, complementary and supportive therapies, in addition to monitoring dietary lifestyle, as a wide range of plants have shown anti-coagulant effects due to their content of phenolic compounds¹⁰. Phenolic compounds, in their various forms and chemical formulas, are important metabolites produced by plants and have numerous medical effects¹¹, including antioxidant effects^12,13, antimicrobial effects¹⁴, blood sugar-lowering effects^15,16, anti-inflammatory effects¹⁷, as well as blood pressure-lowering and lipid-lowering effects¹⁸. Therefore, phenolic compounds are promising therapeutic agents for individuals with predisposing factors for thrombotic events as they offer additional positive effects in addition to their anti-coagulant effect.

Ocimum Basilicum belongs toLamiaceaefamily, is a phenols-rich plant, commonly cultivated herb in Europe and Asia, known for its distinct sharp herbal aroma¹⁹. This plant is well known for its use as a flavouring agent in desserts and food, as well as being consumed as a popular infusion in Syria. O. basilicum has been extensively used in traditional medicine to treat cough, digestive disorders, helminth infestations, respiratory complaints, fever, insect bites, menstrual cramps, and sinusitis²⁰. The essential oil of O. basilicum has shown properties such as anti-flatulence, diuretic, antiseptic, and antispasmodic effects²¹. Linalool is considered the main component of basil oil, along with high levels of estragole, eugenol, caffeic acid, chlorogenic acid, gallic acid, quercetin, rosmarinic acid, camphor, catechin, and apigenin²². Based on the above-mentioned information, and due to the limited studies investigating the anticoagulant effect of basil extract, our study aimed to investigate the effect of aqueous basil extract on PT in vitro, using blood samples taken from healthy subjects and patients treated with warfarin.

MATERIALS AND METHODS:

The research was conducted at the Scientific Research Laboratory at Tishreen University, and in collaboration with the Medical Analysis Laboratory at Tishreen University Hospital, Lattakia, Syria.

Chemicals, reagents and equipment:

Gallic acid Biotech LTD, sodium carbonate BDH, England, recently distilled water, Folin-Denisreagent Sigma Aldrich, Switzerland, PT reagent from BIOREX British company (consisting of a mixture of calcium salts and a tissue-activating agent extracted from rabbits, simulating the human tissue-activating agent with an ISI ratio of 1.30 (ISI :international sensitivity index).

Sensitive balance RADWAG AS 220/C/2, electric heater, glass tubes,volumetric flasks.

Filter papers and filtration funnel, sterile containers for preservingthe extracts, Spectrophotometer-uv/vis DIAB SP UV 1100, syringes for blood sampling, citrate tubes for blood collection, scilogex d3024r high speed refrigerated micro-centrifuge c/w 24 place rotor EURO PLUG, micropipettes with different capacities 100-10/ 100-1000 microliter, electric blender and URIT coagulation device for PT test measurement.

O. Basilicum collection and extraction:

A mixture of flower tops and leaves was collected and left to dry at room temperature away from direct daylight. All extractswas prepared by infusion: 2grams of the plant mixture were extracted by 200ml of boiling water with stirring for 5 minutes, which is a simulation of traditional popular infusion use. This extract was filtered and labeled as E1 (Extract 1) then stored in sterile container. Then 2, 5, 10, 15grams of powdered plant extracted by 200ml of boiling water with stirring for 5 minutes. Filtered extracts were stored in sterile containers, and labeled as E2 to E5, respectively. The extracts used for PT test immediately after preparation.

Determination of Total Phenolic Content (TPC) in prepared extracts:

To determine the concentration of phenolic compounds in the prepared extracts, the Folin-Ciocalteu method described by Lamuela‐Raventós was followed²³. Briefly, 4ml of 2% (w/v) sodium carbonate solution was added to 0.2ml of the sample, mixed well and after 5 minutes 0.2ml of a 1:1 dilution of Folin-Ciocalteu reagent was added. The mixture was incubated in dark place for 30 minutes. The absorbance of formed blue complex was measured using a spectrophotometer at a wavelength of 750nm. A blank control solution was prepared as mentioned above using 0.2ml of distilled water instead of plant samples. A calibration curve of standard reference was established using gallic acid, solutions were prepared at concentrations of (0.1, 0.15, 0.2, 0.25, 0.3, 0.35) g/l and was treated as above. The total phenolic content was calculated based on the gallic acid standard series. The results were expressed as grams of gallic acid equivalent (GAE) to the phenolic compounds in 1 liter of extract (g GAE/l).

Blood samples collection and Platelet-poor plasma preparation:

Blood samples were collected on a sodium citrate tube from 7 healthy volunteers, with an average age of 27 years, ensuring the exclusion criteria that affect PT values (smoking, NSAID consumption, anticoagulant consumption, vitamin K deficiency, liver diseases), and a serum pool were prepared after sample centrifugation. On the other hand, blood samples were also collected from 5 male patients receiving warfarin, with an average age of 54 years. Platelet-poor Plasma was prepared by centrifugation at 4000rpm for 10min and used immediately for PT test.

In vitro (PT) assay:

For the initial Prothrombin time measurement,100µl of PT reagent were addedto 50µl of plasma, incubated at 37˚C for 70s, and the PT value appears was determined. A value of 70 s is the highest value can be read by the device. To determine the PT value after extracts addition, 50µl of each of E1to E5 extracts was added to 50µl of plasma followed by 100µl of PT reagent. Each test was performed 4 times and the mean value was calculated to indicate PT for each measurement. At the same time, a blank solution was prepared by adding 50 µl of distilled water to 50µl of plasma, incubation at 37˚C for 70 s and finally the addition of 100µl of PT reagent. The PT value of the blank was subtracted from the PT values after extract addition to obtain the prolongation in PT caused by just each extract (the studied response).

EC₅₀ calculation:

EC₅₀ (half-maximal effective concentration) refers to the concentration that achieves half of the maximum response, and is calculated depending on the response-concentration curve. The curve was drawn using Graph-Pad Prism 2009 software after converting the response to a percentage and the EC₅₀ for both healthy and warfarin-receiving subjects were calculated.

Statistical analysis:

The values were expressed as the mean and standard error of the mean SEM, and the paired samples t-test for means was used to determine the presence of a significant difference, and a significance level of 5% was adopted to interpret the results, the statistical analysis was conducted using Excel software 2016.

RESULTS:

Determination of TPC in prepared extracts:

TPC inthe filtered extract E1 was 0.693g GAE/l, while it ran 1.35, 6.10, 9.50, 13.93g GAE/l in E2 to E5 extracts respectively.

Effect of O. basilicum extracts on PT in healthy subjects:

Table 1 shows the results of PT values and the prolongation in PT after adding the extracts to healthy-subjects plasma. The mean initial PT for healthy subjects was 14.25 s, which increased to 14.82 s after adding the distilled water. The PT increased to 16.31 s after adding E1 and the prolongation of PT was 1.48 s. At the same way, E2, E3, E4 and E5 extracts caused prolongation of PT of 2.67, 8.6, 12.05 and 22.7 s, respectively. These results suggest an anticoagulant effect of basil extracts.

PT prolongation was concentration – dependent as the statistical analysis presented in table 2 refers to significant difference in PT prolongation values between each concentration and the concentration before. All PT prolongation values were significantly different from the prolongation caused by distilled water. Results are shown in figure 1. Figure 2 presents the response – concentration curve for PT prolongation according to concentrations of phenolic compounds. The EC₅₀ was 6.026g GAE/l.

Table 1: PT measurements in healthy people

GROUP	PT Mean (s)	SD	SEM	Prolongation in PT (S)
Healthy	14.25	0.478091	0.169031	...
Distilled water	14.825	0.420034	0.148504	0
Extract 1 (E1)	16.3125	0.397986	0.140709	1.4875
Extract 2 (E2)	17.5	0.9273619	0.327872	2.675
Extract 3 (E3)	23.425	1.5745748	0.556696	8.6
Extract 4 (E4)	26.85	0.4535574	0.160357	12.05
Extract 5 (E5)	37.525	0.87137	0.308076	22.7

Table 2: Paired samples t-test for means results in healthy people

Pair	Observations	T stat	P value (2 tailed)	sig
Distilled water/ E1	8	-9.73022	≤0.0001	****
E1/E2	8	-4.05239	≤0.01	**
E2/E3	8	-9.21823	≤0.0001	****
E3/E4	8	-7.39423	≤0.001	***
E4/E5	8	-28.1468	≤0.0001	****
Distilled water/ E2	8	-7.23511	≤0.001	***
Distilled water/ E3	8	-14.97067841	≤0.0001	****
Distilled water/E4	8	-48.98264243	≤0.0001	****
Distilled water/E5	8	-62.78621895	≤0.0001	****

Figure 1: Effect of different extracts on PT values in healthy people

Figure 2: Response-concentration curve in healthy people

Effect of basil extracts on PT in patients receiving warfarin:

Table 3 shows the results of PT values and the prolongation of PT after adding the extracts to the plasma from subjects receiving warfarin. The mean initial PT for subjects receiving warfarin was 28.64 s (obviously higher than PT measured in healthy subjects) which increased to 29.98 s after adding the distilled water. The PT increased to 41.04 s after adding E1 extract and the prolongation of PT was 11.06 s. At the same way, extracts E2 and E3 caused prolongation of PT of 19.64 and 34.89 s. while extracts E4 and E5 caused an increase in PT of > 70 s, which indicates that these last blood samples remain dissolved and the URIT device recorded the highest PT value can be read.

PT prolongation was concentration – dependent as the statistical analysis presented in table 4 refers to significant difference in PT prolongation values between E1 and E2 and between E2 and E3. All PT prolongation values were significantly different from the prolongation caused by Distilled water. Results are shown in figure 3. All the above-mentioned results suggest an effect of basil extract on PT in patients receiving warfarin and this effect might be synergetic. Figure 4 presents the response – concentration curve for PT prolongation according to concentrations of phenolic compounds in subjects receiving warfarin. The EC₅₀ was 1.26g GAE/lobviously lower than EC₅₀ determined in healthy subjectsreceiving the same extracts.

Table 3: PT measurements in patients

Group	PT mean (S)	SD	SEM	Prolongation in PT (S)
Patients	28.64	1.433876	0.453431	...
Distilled water	29.98	1.547615	0.489399	0
Extract 1	41.04	2.298889	0.726972	11.06
Extract 2	48.72	2.443949	0.772845	19.64
Extract 3	64.87	2.091809	0.661488	34.89

Table 4: Paired samples t-test for means results in patients

Pair	observations	T stat	P value (2 tail)	sig
Distilled water / E 1	10	-38.2011	≤0.0001	****
E 1/E 2	10	-9.2504	≤0.0001	****
E2/E3	10	-19.8284	≤0.0001	****
Distilled water / E2	10	-27.8385065	≤0.0001	****
Distilled water/ E 3	10	-123.621194	≤0.0001	****

Figure 3: Effect of different extracts on PT values in patients

Figure 4: Response-concentration curve in patients

DISCUSSION:

Clotting is a function of plasma depends upon the orderly interaction of a group of plasma clotting factors with some phospholipid and some Ca⁺⁺. It can be initiated and proceed according to two different cascading pathways, the intrinsic or the extrinsic. These pathways converge to a common pathway, through the production of thrombin by prothrombinase complex and the conversion of fibrinogen to fibrin by thrombin. In addition to the coagulation-promoting factors, there are also somefactors inhibit coagulation (e.g., an anti-thrombin factor that inactivates thrombin)²⁴. Whether or not blood coagulates depends on the balance that exists between pro-coagulants and anti-coagulants factors, the imbalance due to genetic and environmental factors could lead to the formation of unusual clots in the blood vessels. This pathological phenomenon called thrombosis, which is considered one of a cardiovascular and cerebrovascular risk². The prothrombin time test (PT) specifically evaluates the presence of the extrinsic and the common pathways factors. A prolonged PT value indicates a deficiency in any of factors V, VII, X, prothrombin, or fibrinogen.Recent studies indicate that a prolonged PT might be due to one or more coagulation factor defects in the Tissue Factor (TF) or common pathway, or might indicate the presence of inhibitors against these factors²⁵.

Plants have unlimited ability to synthesize aromatic substances, including phenolic compounds like phenolic acids, quinones, flavonoids,tannins, coumarins, these compounds have many pharmacological effects^26,27. There are also many studies that prove the existence of a wide range of plants that have anticoagulant activity, including Rosemary officinalis, Mentha piperita, Thymus vulgaris, Cinnamomum cassia, Pulmonaria officinalis, and others, and their extracts were rich in phenolic compounds^28,29.

Since O. basilicum is a phenols-rich plant, our study aimed to investigate the effect of aqueous basil extract on PT, with reference to the limited studies on the overall impact of aqueous basil extracts on the coagulation process.

As noted in the results, all extracts caused a significant prolongation in PTcompared to distilled water in healthy subjects, as shown in Figure 1, and the response was concentration-dependant. This result indicates the presence of anticoagulant activity for the basil extract, and indicates the presence of an inhibitory effect on clotting factors in extrinsic or common pathway due to the phenolic compounds content in the extracts.Actually, PT time was reported to be affected by flavonoids and tannins³⁰ which are the essential phenolic compounds in basil extracts²¹. Our results refer to the effect of phenolic compounds in preventing coagulation process by affecting one or possibly several coagulation factors in the extrinsic or common pathway, this is due to their inhibitory effects on enzymes, especially serine proteases. as demonstrated by a study by Bijaket al on the ability of a group of phenolic compounds to inhibit the activity of serine protease enzymes (thrombin), this study showed that six phenolic compounds were effective inhibitors of the enzyme, namely cyanidin, quercetin, catechin, epicatechin, silybin, and cyanin.³¹

In general, polyphenols has a complicated action on blood coagulation as it accelerated thrombin-fibrinogen interaction. This in turn retards clot retraction, enhancement of streptokinase activity on plasminogen, and inhibition of plasmin³². Moreover,The results presented in a previous study elucidated that flavonoids (types of phenolic compounds) might be potential structural bases for the design of new naturally, safely, and orally bioavailable direct Factor X inhibitors³³. Our results were consistent with a study by Taj Eldin et al, on the aqueous extracts of Zingiber officinalis, which is also a phenols-rich plant, the (25, 50, 75, 100)µml prepared solutions showed an increase in PT values, 15.8±0.12, 18.5±0.2, 20.20±0.22 and 21±0.27, respectively, depending on the phenols concentration in the extracts³⁴. Additionally, Vitisvinifera extract, which is also phenols-rich plant, showed inhibitory effects on thrombin in vitro when incubated with fresh plasma and human thrombin, leading to prolonged fibrin polymerization time³⁵. Regarding the obtained results, coagulation activity reduction may be mediated through inhibition of activity of several factors including tissue factors, thrombin, and other clotting factors.

In the present study, results in warfarin-treated subjectsshowed that the aqueous basil extracts caused a greater prolongation compared to healthy plasma, and when extracts E4 and E5 were applied the samples remained dissolved. The EC₅₀ value in healthy people was higher than the EC₅₀ value in patients. This result was consistent with a study conducted on the aqueous extract of Rosa damascene, which is also a phenols-richplant³⁶, where several concentrations of the aqueous extract were tested on plasma from patients treated with warfarin and the EC₅₀ value in healthy people (1137.354 μg/ml) was higher than the EC₅₀ value in patients (1110.374μg/ml). Our results indicate the possibility of synergy between the effects of phenolic compounds in basil and warfarin in vitro, but studies on the accompanying effect of basil extract with warfarin need further confirmation in vivo. In reference to the fact that research on interactions between drug and medicinal herbs is difficult due to the herbs’ content of various compounds that are difficult to separate and determine accurately. However, some studies have investigated the interaction between warfarin and phenols-rich Propolis which have shown a synergistic effect with warfarin in vivo³⁷.

Our research points out that consuming of O. basilicum infusion may reduce the risk of clot formation, andmay increase the risk of bleeding in patients who receive anticoagulant medications such as warfarin. We can recommend consuming popular O. basilicum infusion for people who are at risk of developing blood clots, such as smokers, patients with hypertension, atherosclerosis, dyslipidemia, and other high-risk individuals.It is apparent also that the O. basilicum infusion should be cautiously consumed with anticoagulant drugs and should be stopped before surgery. Furthermore, phytochemical and pharmacological elaborated experiments are required to purify and to characterize possible active constituents of O. basilicum in addition to the cytotoxicity evaluation.

REFERENCES:

1. Van der Meijden PEJ. Heemskerk JWM. Platelet biology and functions: new concepts and clinical perspectives. Nature Reviews Cardiology. 2019; 16(3): 166-79. https://doi.org/10.1038/s41569-018-0110-0.

2. D'Alessandro E.Posma JJN.Spronk HMH. Ten Cate H. Tissue factor (:Factor VIIa) in the heart and vasculature: More than an envelope. Thrombosis Research. 2018; 168: 130-7. https://doi.org/10.1016/j.thromres.2018.06.020.

3. Barmore W. Bajwa T. Burns B. Biochemistry, Clotting Factors: StatPearls Publishing, Treasure Island (FL); 2023 2023.

5. Paciaroni M.Palmerini F. Bogousslavsky J. Clinical presentations of cerebral vein and sinus thrombosis. Handbook on Cerebral Venous Thrombosis. 2008; 23: 77-88.https://doi.org/10.1159/000111262.

6. Wolberg AS.Rosendaal FR.Weitz JI.Jaffer IH.Agnelli G.Baglin T, et al. Venous thrombosis. Nature reviews Disease Primers. 2015; 1(1): 1-17. https://doi.org/10.1038/nrdp.2015.6.

7. Harter K.Levine M. Henderson SO. Anticoagulation drug therapy: a review. Western Journal of Emergency Medicine. 2015; 16(1): 11. https://doi.org/10.5811%2Fwestjem.2014.12.22933.

8. Mekaj YH.Mekaj AY. Duci SB. Miftari EI. New oral anticoagulants: their advantages and disadvantages compared with vitamin K antagonists in the prevention and treatment of patients with thromboembolic events. Therapeutics and Clinical Risk Management. 2015; 11: 967-77. https://doi.org/10.2147/TCRM.S84210.

9. Nilamsari WP.Yusuf M.Pertiwi DA.Dery A.Budi S. Zulkarnaen B. Study of co-prescription of drugs potentially interacting with warfarin in indonesian ambulatory patients. Research Journal of Pharmacy and Technology. 2020; 13(8): 3833-6. http://dx.doi.org/10.5958/0974-360X.2020.00678.2.

10. Kumar A. Kumar P.Shravya H. Pai A. Coumarins as potential anticoagulant agents. Research Journal of Pharmacy and Technology. 2022; 15(4): 1659-63. http://dx.doi.org/10.52711/0974-360X.2022.00277.

11. Sun W. Shahrajabian MH. Therapeutic Potential of Phenolic Compounds in Medicinal Plants and dash; Natural Health Products for Human Health. Molecules. 2023; 28(4): 1845. https://doi.org/10.3390/molecules28041845.

12. Jain N.Goyal S. Ramawat K. Radical scavenging activity and total phenolic content in selected fruits and vegetables. Research Journal of Pharmacy and Technology [Internet]. 2012; 5(1): 121-4. Available from: https://rjptonline.org/AbstractView.aspx?PID=2012-5-1-18.

13. AL-Asaad N. AL-Diab D. Determination of Phenolic Compounds Levels and Their Antioxidant Activity in Some Local Functional Juices. Tishreen University Journal for Research and Scientific Studies- Health Sciences Series 2015; 37(1). http://www.journal.tishreen.edu.sy/index.php/hlthscnc/article/view/1625.

14. Kaddar R.Hasan N. Al-diab D. Antibacterial activity of Rosa damascene petals mill extracts. Research Journal of Pharmacy and Technology. 2023; 16(11): 5074-8. https://doi.org/10.52711/0974-360X.2023.00822.

15. Anjum N.Hossain MJ.Aktar F.Haque MR.Rashid MA. Kuddus MR. Potential In vitro and In vivo bioactivities of Schleichera oleosa (lour.) oken: A traditionally important medicinal plant of Bangladesh. Research Journal of Pharmacy and Technology. 2022; 15(1): 113-21. http://dx.doi.org/10.52711/0974-360X.2022.00019.

16. Sachan AK.Rao CV. Sachan NK. Determination of antidiabetic potential in crude extract of caesalpinia bonducella wild on normal and streptozotocin induced diabetic rats. Research Journal of Pharmacy and Technology. 2020; 13(2): 857-61. http://dx.doi.org/10.5958/0974-360X.2020.00162.6.

17. Nezam A.Al Diab D. Hasan N. In-vitro Anti-inflammatory activity of Total Phenolic content of some fruit juices in Syria. Research Journal of Pharmacy and Technology. 2021; 14(7): 3685-8. http://dx.doi.org/10.52711/0974-360X.2021.00637.

18. Farkhondeh T. Samarghandian S. Pourbagher‐Shahri AM. Hypolipidemic effects of Rosmarinus officinalis L. Journal of Cellular Physiology. 2019; 234(9): 14680-8. https://doi.org/10.1002/jcp.28221.

19. Sestili P.Ismail T.Calcabrini C.Guescini M.Catanzaro E.Turrini E, et al. The potential effects of Ocimum basilicum on health: a review of pharmacological and toxicological studies. Expert Opinion on Drug Metabolism and Toxicology. 2018; 14(7): 679-92. https://doi.org/10.1080/17425255.2018.1484450.

20. Shagana S.Navenaa S.Vijay J.Vishali S. Rajendran V. Investigation of in vitro Anthelmintic activity of Ocimum basilicum Linn.(Lamiaceae). Research Journal of Pharmacy and Technology. 2021; 14(1): 52-4. http://dx.doi.org/10.5958/0974-360X.2021.00010.X.

21. Purushothaman B.Prasanna Srinivasan R.Suganthi P.Ranganathan B.Gimbun J. Shanmugam K. A comprehensive review on Ocimum basilicum. Journal of Natural Remedies. 2018; 18(3): 71-85. https://doi.org/10.18311/jnr/2018/21324.

22. Pandey AK. Tiwari SP. Biswas D.Patel Y.Jajda HM. Dave GS. Evaluation of Phytochemicals, Antioxidant and Anti-inflammatory properties of leaves of Ocimum basilicum L. Research Journal of Pharmacy and Technology. 2023; 16(4): 1981-6. https://doi.org/10.52711/0974-360X.2023.00325.

23. Lamuela‐Raventós RM. Folin–Ciocalteu method for the measurement of total phenolic content and antioxidant capacity. Measurement of antioxidant activity and capacity: recent trends and applications;https://doi.org/10.1002/9781119135388.ch62018. p. 107-15.

24. Smith SA.Travers RJ. Morrissey JH. How it all starts: Initiation of the clotting cascade. Critical Reviews in Biochemistry and Molecular Biology. 2015; 50(4): 326-36. https://doi.org/10.3109/10409238.2015.1050550.

25. Dorgalaleh A. Favaloro EJ.Bahraini M. Rad F. Standardization of prothrombin time/international normalized ratio (PT/INR). International Journal of Laboratory Hematology. 2021; 43(1): 21-8. https://doi.org/10.1111/ijlh.13349.

26. Ebraheem M. Al Diab D. Determination of some components of citrus fruits wastes and monitoring bioactivities of these wastes in experimental animals. Tishreen University Journal -Medical Sciences Series. 2023; 45(5): 579-94. https://journal.tishreen.edu.sy/index.php/hlthscnc/article/view/15891.

27. Ahmad A. AL-diab D. Antimicrobial effect of Rosmary extract to improve the shelf life of chicken meat. Tishreen University Journal -Medical Sciences Series. 2023; 45(3): 477-88. https://journal.tishreen.edu.sy/index.php/hlthscnc/article/view/15256.

28. Akram M. Rashid A. Anti-coagulant activity of plants: mini review. Journal of Thrombosis and Thrombolysis. 2017; 44: 406-11. https://doi.org/10.1007/s11239-017-1546-5.

29. Mohamed AA.Ali SI.Sameeh MY. El-Razik TMA. Effect of solvents extraction on HPLC profile of phenolic compounds, antioxidant and anticoagulant properties of Origanum vulgare. Research Journal of Pharmacy and Technology. 2016; 9(11): 2009-16. http://dx.doi.org/10.5958/0974-360X.2016.00410.8.

30. Adli N.Abu MN.Mastuki MF.Wan Yusoff W. Kamarudin E. In vitro haemostatic activity of rhodomyrtus tomentosa (Aiton) hassk. Aqueous leaf extract. Jurnal Teknologi. 2016; 78(5-6): 15-9. http://dx.doi.org/10.11113/jt.v78.8632.

31. Bijak M.Ziewiecki R.Saluk J.Ponczek M.Pawlaczyk I.Krotkiewski H, et al. Thrombin inhibitory activity of some polyphenolic compounds. Medicinal Chemistry Research. 2014; 23: 2324-37. https://doi.org/10.1007/s00044-013-0829-4.

32. Yang W-K.Sung Y-Y. Kim H-K. Antithrombotic effect and antiplatelet activity of Polygonum cuspidatum extract. Journal of the Korean Society of Food Science and Nutrition. 2012; 41(2): 168-73. https://doi.org/10.3746/jkfn.2012.41.2.168.

33. Bijak M. Ponczek MB. Nowak P. Polyphenol compounds belonging to flavonoids inhibit activity of coagulation factor X. International Journal of Biological Macromolecules. 2014; 65: 129-35. https://doi.org/10.1016/j.ijbiomac.2014.01.023.

34. IM TE.Elmutalib M.Hiba A.Hiba F.Thowiba S. Elnazeer I. An in vitro anticoagulant effect of aqueous extract of ginger (Zingiber officinale) rhizomes in blood samples of normal individuals. American Journal of Research Communication. 2016; 4(1): 113-21. http://dx.doi.org/10.13140/RG.2.1.4348.5201.

35. Bijak M.Bobrowski M.Borowiecka M.Podsędek A.Golański J. Nowak P. Anticoagulant effect of polyphenols-rich extracts from black chokeberry and grape seeds. Fitoterapia. 2011; 82(6): 811-7. https://doi.org/10.1016/j.fitote.2011.04.017.

36. Ahmad A. Hasan N. Al-Diab D. Study of the anticoagulant activity of Rosa Damascena extract in vitro. Tishreen University Journal -Medical Sciences Series. 2023; 45(3): 319-35. https://journal.tishreen.edu.sy/index.php/hlthscnc/article/view/15008.

37. Kolaylı S.Sahin H. Malkoc M.Cakir H.Kara Y. Bektas O, et al. Does Ethanolic Propolis Extract Affect Blood Clotting Parameters? Progress in Nutrition. 2022; 24(1). http://doi.org/10.23751/pn.v24i1.12017.

Received on 29.11.2023 Modified on 08.01.2024

Research J. Pharm. and Tech 2024; 17(7):3339-3345.

DOI: 10.52711/0974-360X.2024.00522