Rutin Exhibits In-Vitro Anticoagulant Activity in Human Blood Samples by Prolonging Coagulation Pathway Times

Abstract

Background

Rutin is a plant-derived flavonoid with reported biological activities, but its effect on blood coagulation parameters has not been clearly characterized under in-vitro conditions.

Objective

This study aimed to evaluate the in-vitro anticoagulant activity of rutin in human blood by measuring changes in clotting time (CT), prothrombin time (PT), and activated partial thromboplastin time (aPTT) at increasing concentrations, compared with baseline control values.

Materials and Methods

Rutin (1-6 mg/mL) was added to human blood samples, and standard CT, PT, and aPTT assays were used to measure coagulation parameters. Heparin and Phosphate-buffered saline (PBS) were used as controls.

Results

In a concentration-dependent manner, rutin significantly increased activated partial thromboplastin time (aPTT), prothrombin time (PT), and clotting time (CT) compared with the PBS control, with the most pronounced effects observed at higher concentrations (4 and 6 mg/mL; P < 0.05 vs control). At 6 mg/mL, CT increased to 11.42 ± 1.11 minutes, PT to 21.33 ± 3.28 seconds, and aPTT to 57.20 ± 9.31 seconds.

Conclusion

Rutin exhibits significant in vitro anticoagulant activity, supporting its potential as a natural anticoagulant candidate.

Introduction

Flavonoids are the most common secondary metabolites found at high concentrations in fruits and vegetables. It has immense pharmacological activity (anti-oxidant, anti-inflammatory, immune-stimulant, anti-diabetic, etc.) that can help maintain the body’s immunological response and prevent disease progression.^1-4 People from developing countries suffer from haematological complications (blood coagulation diseases, including deep vein thrombosis, cerebral haemorrhage, and vitamin K toxicity) due to insufficient intake of dietary foods.^5,6 Anti-coagulants are essential for preventing haematological disease. Thus, they depend on synthetic medicines to manage complications. There are no significant alternative anti-coagulants available to reduce the haematological complications with tolerable adverse effects. Few recombinant anticoagulants, like hirudin, are available, but they also have adverse effects. ⁷ The long-term and frequent use of these medications shows severe adverse effects like thrombocytopenia, irreversible effects on coagulation factors, hypersensitivity, etc. ⁸

Numerous medicinal plants possess anti-coagulant properties and can serve as alternatives to most current anti-coagulants. ⁹ Rutin, also known as rutoside, quercetin-3-rutinoside, and sophorin, is a flavonoid glucoside that is present in buckwheat, leaves of petioles of Rheum species, and fruits of the Dimorphandra mollis, as well as other sources. Its name ‘rutin’ comes from Ruta graveolens, containing rutin. ¹⁰ It is a glycoside consisting of flavonoid aglycone quercetin along with disaccharide rutinose. Rutin is a flavonoid glycoside composed of the flavonol aglycone quercetin and the disaccharide rutinose. Quercetin has been previously reported to exhibit anticoagulant and antiplatelet activities, including inhibition of thrombin generation and factor Xa activity, suggesting that rutin may share similar anticoagulant mechanisms after partial hydrolysis or direct interaction with coagulation proteins. Incorporating this mechanistic link strengthens the rationale for investing in rutin as a potential anticoagulant agent. ¹¹

Rutin exhibits several pharmacological activities, including antioxidant, cytoprotective, vasoprotective, anticarcinogenic, neuroprotective, and cardioprotective effects. ¹² Only a few numbers of plants, Fagopyrum esculentum and Cappris species, particularly Capparis spinosa L., are recognised as the biggest sources of rutin (or rutoside). ¹³ Buckwheat is considered the third most abundant source of rutin among plants. Gardenia may rank fourth in rutin accumulation.^14,15 An aromatic plant, Capparis spinosa L. (Capparidaceae), is widely distributed in the coastal areas of the Mediterranean Basin and has been found to contain a 2.8% rutin content in its leaf tissue. ¹³ This study aimed to determine the effect of rutin as an anti-coagulant.

Material and Methods

The Institutional Review Committee (IRC) investigated the research proposal. The IRC approved the proposal with registration number UCMS/IRC/006/23 after carefully reviewing it and giving helpful feedback. After receiving IRC approval, the study started and was carried out over 4 months, from April 2023 to July 2023 in Department of Pharmacy and Pharmacology at Universal College of Medical Sciences, Bhairahawa, Rupandehi, Nepal.

Rutin was investigated for in-vitro anticoagulant activity through coagulation tests: clotting time (CT), prothrombin time (PT), activated partial thromboplastin time (aPTT).

The concentration range of rutin (1-6 mg/mL) was selected based on previous in vitro anticoagulant and flavonoid screening studies, which commonly employ higher concentrations to detect direct effects on coagulation pathways in plasma-based assays. The concentrations represent levels achievable in experimental settings using concentrated plant extracts rather than physiological plasma concentrations obtained through dietary intake. The present study was designed as a preliminary in vitro investigation to evaluate direct anticoagulant potential rather than clinical relevance.

Drugs and Chemicals

Rutin (GLR INNOVATIONS), heparin, thromboplastin (Tulip Diagnostic Pvt. Ltd), partial thromboplastin, sodium chloride (Thermo Fischer Scientific India Pvt. Ltd), potassium chloride (Thermo Fischer Scientific India Pvt. Ltd), sodium phosphate dibasic (SD fiNE-cHEM limited), potassium phosphate monobasic (SD fiNE-cHEM limited), calcium chloride (Thermo Fischer Scientific India Pvt. Ltd), hydrochloric acid (ACS grade).

Rutin was prepared as a freshly mixed suspension in phosphate-buffered saline (PBS) and vortexed thoroughly prior to use. No organic co-solvent such as DMSO was employed. All samples were mixed immediately before incubation to ensure uniform exposure during the assay period.

Blood Collection and Plasma Sample Preparation

After obtaining approval from the Institutional Review Committee, blood samples were collected by venipuncture from 15 healthy volunteers (9 males and 6 females; aged 18-35 years) with their consent. Donors were non-smokers, had no known bleeding or cardiovascular disorders, and were not taking anticoagulant, antiplatelet, anti-inflammatory, or hormonal medications for at least 1 week prior to sample collection. The blood was placed in a blood collection tube without any anticoagulant. After blood collection, coagulation was recorded immediately, and the remaining blood was centrifuged at 2500 g for 10 minutes to separate plasma from blood cells for the PT and aPTT tests. The plasma was separated and stored at 4°C until further use.^16,17

Clotting Time Measurement

This study employed a modified method, as described by Choi SS et al. 2021 and Ayodele et al. 2019. The clotting tube containing rutin was suspended in phosphate-buffered saline (PBS) at concentrations of 1-6 mg/mL. PBS (negative control) and heparin as an anti-coagulant (positive control) were incubated using a water bath at 37°C. A total of 0.5 mL of freshly drawn blood was transferred into each tube containing either the standard or test drugs, respectively. After that, all the tubes were incubated, and the stopwatch started simultaneously. At 30 s intervals, tubes were slanted at 45°C to check clot formation. The time required for the formation of the first clot was recorded, and slanting at intervals continued until they turned out without blood flowing. As soon as the stopwatch stopped, the time was recorded as the final clotting time.^16,18 After determining the clotting time for various concentrations of rutin, PT and aPTT were subsequently measured using fresh blood samples from the same volunteers.

Prothrombin Time (PT) Measurement

The PT was determined using the standard procedure by Quick AJ et al. and Choi JH et al. 2015, after minor modification.^11,19 The calcified thromboplastin reagent was incubated in a water bath at 37°C for 10 minutes. ¹⁶ The 100 µL of plasma was incubated with various concentrations of rutin at 1-6 mg/mL and heparin 1000 U (standard drug). After that incubation, a calcified thromboplastin (uniplastin) reagent was added, and the time required for clot formation was recorded. ⁹

Activated Partial Thromboplastin Time (aPTT) Measurement

The RGT2 reagent (CaCl₂, sodium azide, salts and stabilisers) and test tubes were pre-warmed in a water bath at 37°C for 10 min based on kit instructions. The patient’s plasma (100 µL), control (PBS), heparin 1000 U (standard), and various rutin concentrations (1-6 mg/mL) were added to a pre-warmed test tube. The RGT1 reagent (rabbit brain cephalin, ellagic acid, and sodium azide) was prepared by mixing the components before use and then added to the test tubes. The reagents in the test tube were mixed and incubated for 3 min at 37°C. The pre-warmed RGT2 reagent was added. The stopwatch started simultaneously, and the tube was slanted at regular intervals until a clot formed, and the time was recorded to form a clot. ¹⁶

Statistical Analysis

The data were statistically evaluated, and the results were expressed as Mean ± SD. One-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test.

Results

Effect of Rutin on Whole Blood Clotting Time (CT)

At doses between 1 and 6 mg/mL, the physiological effect of rutin on whole blood clotting time was assessed (Table 1). The heparin-treated group exhibited total inhibition of clot formation, whereas the PBS (pH 7.4) control group exhibited a baseline clotting time of 4.5 min. Treatment with rutin induced the clotting time to increase in a concentration-dependent manner. Rutin considerably improved the clotting time to 7.30 ± 0.012 min at 1 mg/mL (P = 0.0250). At 2 mg/mL (8.15 ± 0.027 min), 4 mg/mL (9.05 ± 1.186 min), and 6 mg/mL (11.42 ± 1.114 min), additional increases in clotting time were observed with increasing concentration.

Table 1.

Determination of Coagulation Time of Rutin at Different Concentrations

S.N.	Group	Concentration	Average clotting time (min ± SD)	P value
1	PBS (pH 7.4) control		4.5
2	Heparin	1000 U	-
3	Rutin	1 mg/mL	7.30 ± 0.012	0.0033
4		2 mg/mL	8.15 ± 0.027	0.0011
5		4 mg/mL	9.05 ± 1.186	0.0031
6		6 mg/mL	11.42 ± 1.114	0.0134

The results are presented as a mean standard deviation (SD). Statistical significance was considered at P < 0.05.

Effect of Rutin on Prothrombin Time (PT)

Prothrombin time (PT) and INR values were used to measure the impact of rutin on the extrinsic coagulation pathway (Table 2). The heparin-treated group possessed total coagulation inhibition, however the PBS control group showed a PT of 11 s with an INR of 0.848 ± 0.045. PT was markedly and dose-dependently prolonged by rutin administration. PT increased to 13.6 ± 1.019 s at 1 mg/mL, with an INR of 1.00 ± 0.086 (P = 0.0002). At 2 mg/mL (14.733 ± 1.123 s; INR 1.096 ± 0.094), 4 mg/mL (18.466 ± 2.156 s; INR 1.406 ± 1.183), 6 mg/mL (21.333 ± 3.279 s; INR 1.657 ± 0.276), and further prolongation was noted.

Table 2.

Determination of Rutin Prothrombin Time (PT) at Different Concentrations

S.N.	Group	Concentration	PT time (s)	INR (mean ± SD)	P value
1	PBS (pH 7.4) control		11	0.848 ± 0.045
2	Heparin	1000 U	-	-
3	Rutin	1 mg/mL	13.6 ± 1.019	1.00 ± 0.086	0.0134
4		2 mg/mL	14.733 ± 1.123	1.096 ± 0.094	0.0048
5		4 mg/mL	18.466 ± 2.156	1.406 ± 1.183	0.0177
6		6 mg/mL	21.333 ± 3.279	1.657 ± 0.276	0.0004

The results are presented as a mean standard deviation (SD). Statistical significance was considered at P < 0.05.

Effect of Rutin on Activated Partial Thromboplastin Time (aPTT)

The activated partial thromboplastin time (aPTT) assay was used to evaluate the intrinsic coagulation pathway (Table 3). Heparin totally prevented clot formation, while the PBS (pH 7.4) control showed an aPTT of 25 ± 4 s. APTT was significantly prolonged by rutin in a concentration-dependent manner (P < 0.005). At 1 mg/mL, aPTT increased to 38.67 ± 6.53 s; further prolongation was observed at 2 mg/mL (42.73 ± 7.93 s), 4 mg/mL (50.67 ± 7.94 s), and 6 mg/mL (57.20 ± 9.31 s).

Table 3.

Determination of Activated Partial Thromboplastin Time (aPTT) of Rutin at Different Concentrations

S.N.	Group	Concentration	aPTT time (s)	P value
1	PBS (pH 7.4) control		25 ± 4
2	Heparin	1000 U	-
3	Rutin	1 mg/mL	38.666 ± 6.531	0.0313
4		2 mg/mL	42.733 ± 7.932	0.0018
5		4 mg/mL	50.666 ± 7.943	0.0002
6		6 mg/mL	57.2 ± 9.305	0.0011

The results are presented as a mean standard deviation (SD). Statistical significance was considered at P < 0.05.

Discussion

The coagulation process occurs due to the complex interaction of cellular and molecular components. Initially, both intrinsic and extrinsic pathways are involved in clotting; however, it has recently been found to be due to a balance between pro-coagulants and anti-coagulants. ²⁰ In both healthy and diseased conditions, such as cardiovascular disease, diabetes mellitus, and bleeding disorders, anti-coagulants and pro-coagulants are frequently used to control blood coagulation. Although several drugs have been developed over the years, most are usually accompanied by undesirable side effects. Therefore, there remains a need to develop novel anti-coagulants and pro-coagulants with fewer side effects. ¹⁶ Plant-based products are effective therapeutic tools to treat diseases and injuries. Increased demand for natural products and their derivatives in developing and developed countries has increased interest in medicinal plants as an alternative medicine. ²¹ Therefore, according to WHO, approximately 80% of the world’s population uses traditional medicine to cure various illnesses including hemolytic diseases, cardiovascular diseases, etc. ²² Most plants contain flavonoids, a ubiquitous polyphenolic compound in seeds, fruit skin or peel, bark, and flowers. Rutin is flavonoid, consisting of the Flavonol quercetin and the disaccharide rutinose. Rutin are commonly used to treat chronic venous insufficiency because it helps prevent haemorrhages and ruptures in the capillary and connective tissues. ²³ The present study investigated the in vitro effect of rutin on the blood coagulation of human blood samples. A similar study was reported by (Asha S. et al. 2017) whose findings indicated that the in vitro anticoagulant effect of Nelumbo nucifera leaf extracts on normal healthy blood plasma increased the PT time. ²⁴ Mahajan and More 2006; also reported increased PT time of human blood in an anti-coagulant activity study with aqueous and methanol extracts of Encheostema litorrale, Acherenthaus aspera, Abutilon indicum, and Tridax procumbens. ²⁵ In contrast, AL-Farawachi and AL-Badranii 2014; reported increased PT and aPTT in anticoagulant studies using Eminium spiculatum aqueous leaf extract in rabbits. ²⁶ The methanol extract of Crassocephalum crepoidiodes also showed prolonged prothrombin and activated partial thromboplastin times in blood obtained from volunteers. ¹⁶

The simultaneous prolongation of PT and aPTT suggests that rutin may interfere with both intrinsic and extrinsic coagulation pathways. Based on previous studies involving flavonoids, this effect may be mediated through inhibition of key coagulation enzymes such as factor Xa or thrombin, which play central roles in the common pathway. Molecular docking and in vitro enzyme inhibition studies on quercetin and related flavonoids have demonstrated binding affinity toward factor Xa and thrombin, supporting this hypothesis. However, direct enzyme-specific assays are required to confirm these interactions for rutin. ¹¹ As previously noted for flavonoid drugs, the dual inhibitory impact may be determined by the indirect reduction of thrombin production and regulation of important coagulation factors, such as factor Xa. Additionally, rutin’s anticoagulant potential is further supported by the concentration-dependent increase observed across clotting time (CT), PT, and aPTT assays, which shows a consistent inhibitory effect on the coagulation cascade.

The current investigation showed that rutin inhibited several coagulation pathways by dose-dependently prolonging both PT and aPTT. Guan et al. 2011 reported similar results, showing prolonged aPTT after ethanolic rutin preparation administration. All of these investigations provide credence to rutin’s anticoagulant properties in vitro. ²⁷ another study showed that an ethanolic rutin solution increased aPTT, similar to the result obtained. ²³ As the concentration increases, the ethanolic solution of rutin inhibits coagulation time and increases the prothrombin time (PT). The present study showed that all concentrations (1-6 mg/mL) of rutin have prolonged PT time compared to normal control values. The reported data on rutin at concentrations of 1-6 mg/mL for PT are presented in Table 2. Rutin exhibits the highest PT time (21.33 ± 3.27 s) at a concentration of 6 mg/mL. Rutin may influence elements of the common coagulation cascade, possibly involving thrombin or factor Xa, as indicated by the simultaneous lengthening of PT and aPTT; however, this needs direct mechanistic validation. ¹¹

Similarly, the results showed that all concentrations of rutin prolonged the aPTT time compared to normal control values. The recorded data on the effect of rutin at different concentrations (1-6 mg/mL) on aPTT is presented in Table 3. Rutin shows the highest aPTT time (57.2 ± 9.305 s) at a concentration of 6 mg/mL. The in-vitro study used a limited number of blood samples, emphasizing specific parameters. Future research will include molecular techniques, pharmacokinetic analysis, and in vivo studies to comprehensively elucidate the anticoagulant mechanism of rutin.

Conclusion

The present study demonstrates that rutin exhibits significant, concentration-dependent anticoagulant activity in vitro by prolonging clotting time, prothrombin time, and activated partial thromboplastin time, indicating inhibition of both intrinsic and extrinsic coagulation pathways. These findings identify rutin as a promising in vitro anticoagulant candidate that requires further validation through in vivo studies to assess its safety, pharmacokinetics, and bioavailability before clinical relevance can be established.