1. INTRODUCTION:

The coronary thrombosis developed in circulatory system, is the result of failure of hemostasis and it may be because of atherosclerosis, leads to vascular blockage and in turn show severe upshots of atherothrombotic diseases like acute myocardial or cerebral infarction commonly called as stroke which may eventually leads to sudden death¹. Thrombogenesis, closely linked to atherogenesis, is a crucial condition that requires addressing at various levels because relying solely on anticoagulation seems insufficient. The agents utilized in clinical practice to reduce cardiovascular events either target the risk factors of atherosclerosis or directly address thrombosis².It is projected that by 2030, thrombosis of blood arteries will cause acute myocardial infarction, accounting for about 23 million annual deaths from cardiovascular disease³.Thrombolytic therapy is a breakthrough treatment has saved untold lives when governed endogenously lyses the thrombus by prompting plasminogen to serine protease plasmin. A natural fibrinolytic substance called plasmin breaks down the crosslinks between fibrin molecules in clots to weaken their structural integrity⁴. Thrombolytic treatment should be given with the intention of minimize the risk of bleeding and magnify the chances of smoothly thrombolysis of clot. Although the thrombolytics that are now licensed and, on the market, alteplase, anistreplase, streptokinase, urokinase, and reteplase—are unquestionably safe and efficient, they also have some significant side effects.^5,6. These include the requirement of higher doses, restricted fibrin specificity, haemorrhage, hypersensitive reactions, and the fact that some thrombi have been shown to be resistant to intravenous t-PA⁷. It is necessary to acknowledge the possibility of unfavourable interactions, such as bleeding risk, haemorrhage, limited therapeutic window, antigenicity, and hypertensive crisis. Therefore, the goal of these extraordinary pharmacological efforts is to develop natural antithrombotic medicines with the fewest possible side effects and effective therapy⁸. Many studies have shown that natural phytochemicals have a significant natural source of antioxidants and has potential therapeutic value in the management of cancer, coronary heart diseases and several inflammatory diseases⁹. Natural products from higher plants contains amazing secondary metabolites like phenolic compounds, steroids, flavonoids, alkaloids and glycosides responsible for therapeutic potential. Thus, the researchers are now focusing on natural resources to identify safer, more effective alternatives for thrombolytic therapy without any side effects¹⁰.

Cordia macleodii Hook. (Boraginaceae) a folklore medicinal plant, native to India, has historically been used for mouth sores, jaundice and aphrodisiac purposes ¹¹. About 300 species of this deciduous blooming trees and shrubs are found in tropical regions and are commonly known as Phankhi/Shikari. Locally it is referred as "Dahiman" or "Dahipalas,"¹². It is a prominent drug distributed over India's Deccan, Carnatic region and in the forests of Madhya Pradesh, Chhattisgarh, Karnataka, and Bihar¹³. The leaves, fruit, bark, and seeds of Cordia macleodii were discovered to have rich ethnomedicinal value; nevertheless, leaves were most frequently employed to treat a wide range of illnesses including respiratory conditions, stomach pain, wounds, inflammation, myalgia, cough, dysentery, and diarrhoea¹⁴. A wide range of pharmacological activities like anti-inflammatory, antioxidant, larvicidal, hepatoprotective, analgesic, antibacterial, and antidiabetic are documented for several Cordia species extracts and their isolated components¹⁵. The various organs of Cordia macleodii species has been utilized ethnobotanically for many purposes like healing malaria, tuberculosis, menstrual cramps, headaches, illness, fever, colds, snakebite¹⁶. Preliminary phytochemical examination of targeted C. macleodii bark revealed a presence of terpenoids, saponins, tannins, glycosides, alkaloids, carbohydrates, resins, reducing sugar, phenolics, flavonoids and steroids¹⁷. Soni and Bodakhe (2014) evaluated the anti-snake venom potential of an ethanolic extract of Cordia macleodii bark in relation to the pharmacological effects produced by Naja venom at dosages of 400 and 800mg/kg¹⁸.

Cissus repanda Vahl. is a succulent plant belonging to family-Vitaceae, generally called as Panivel in Hindi.In India approximately 8 genera and 13 species have been distributed and identified scientifically¹⁹. Kumaun, Tripura, Arunachal Pradesh, Bihar, Assam, Madhya Pradesh, Orissa, and the Western Ghats area of Maharashtra are the prominent place to the ethnobotanically significant Cissus repanda herb, which may reach elevations of up to 1350 meters above sea level²⁰. However, this plant’s distribution in Maharashtra specially in Vidarbha is found rare or uncommon. It is a huge perennial climber with a smooth bark and silky tendrils. The wood is extremely porous and has corky bark²¹. The root of Cissus repanda was discovered to be long, smooth-surfaced, elongated, and 15-20cm in diameter. Its color was determined to be highly brown with an interior yellowish orange hue. Most of the species of genus Cissus listed in Ayurveda system have been used for the treatment of diarrhoea, dysentery and blood cancer. Commonly it is used for the treatment of piles, asthma, digestive troubles, cough and loss of appetite²². The powdered roots and leaves of Cissus repanda are widely utilized to heal wounds, boils, cuts, and fractures²³. Hydroalcoholic extract of roots of species reveals the presence of polyphenols, alkaloids, carbohydrates, flavonoids, steroids, tannins, proteins and these phytochemicals might be responsible for numerous therapeutic potential²⁴. It is reported that the roots and stem of its allied species Cissus ripens Lamk is being employed for treating snake bites, rheumatic pain, nephritis, long term cough, diarrhoea and many diseases ^25-26.Cissus repanda leaves are widely utilized as traditional vegetable and has strong hepatoprotective properties. Because of its high nutritional content, it helps to maintain excellent health and shield the body from many dietary disorders²⁷.

Celosia argentea Linn. belongs to Amaranthacea family is a herbaceous, erect and branching plant commonly known as plumed cockscomb, Sitivara and Safed murga in Hindi^28-29. It is usually consumed as a leafy vegetable as it is found to contain very high nutritive value³⁰. Of the 17 species of Celosia known to exist in India, 11 are known to be found growing in Maharashtra. The major constituents found in the roots of Celosia argentea are carbohydrates, proteins, alkaloids, steroids, flavonoids, saponins, phenolic compounds and fixed oils³¹. Conventionally, it is used to treat fever, wounds, gonorrhoea, calculi, burning urination, dysuria, and jaundice. The plants' leaves are used to treat fever, inflammation, and itching³². The seeds as well as roots of Celosia argentea used to treat diarrhoea, mouth sores, blood disorders, eczema, gonorrhoea, colic infections and whole plant is used for counteracting the effects of snake poison³³. Based on ethnobotanical profile and its traditional application, plant was explored for its numerous therapeutic potentials like anti-inflammatory, antipyretic, antidiabetic, antibacterial and diuretic properties^34-38. A review of the literature revealed that herbal remedies rich in flavonoids and phenolic compounds have strong antioxidant qualities and are thus used to treat a variety of age-related illnesses, including depression, anxiety, and Parkinson's disease³⁹. As per the scientific study, it is found that consuming antioxidants, phytochemicals with anticoagulant qualities or natural dietary anticoagulants can ultimately lower the incidence of thrombotic diseases^40-41. In view of this and alleged usefulness of these herbal crude drugs in traditional treatment, the considerable efforts have been made towards the evaluation of their anti-thrombotic potential.

2. MATERIALS AND METHODS:

2.1 Collection of plant materials:

Fresh bark of Cordia macleodii Hook was collected from its natural habit, from the forest of village Pimpalgaon, Tahsil- Lakhandur Dist-Bhandara (Maharashtra). Roots of Cissus rependa Vahl was collected from the campus, area of Department of Physics, RTM Nagpur University Campus Nagpur. While roots of Celosia argentea Linn. was collected from village Adasa Tah- Kalmeshwar, District-Nagpur. All these collected crude drugs were identified and authenticated by Dr. S. M. Bhuskute, Taxonomist and former Principal, Bhawbhuti Mahavidyalaya Amgaon-441902 District- Gondia (Maharashtra). The verified specimens have been preserved in herbarium museum of Department of Botany with vide no. BMV704, BMV703, BMV702 for Cordia macleodii, Cissus rependa and Celosia argentea respectively for future references. Before the extraction procedure, the materials underwent a two-week period of shade drying after being completely cleansed with water.

2.2 Material and standard drugs:

Blood from healthy volunteers (n=5), electronic balance, syringe, eppendorf tubes, eppendorf tubes stand, active fractions of herbal drugs, microcentrifuge, distilled water, streptrokinase. Streptrokinase (SK) was purchased from Cadila Pharmaceuticals Limited, Ahmedabad.

2.3 Preparation of plant extract:

Dried crude drugs material was extracted by maceration process using Ethanol: Water (40:60) as a solvent. About 1.5kg of each coarse powdered material was soaked in10 litres of hydro-alcoholic mixture for 24 hours. Soaked crude drug mixture then heated at 800C for 4 hours in reflux condenser. Extraction was repeated twice and combined extract was evaporated to dryness by rotary evaporation at 40°C. The extract was then stored in refrigerator for further studies^42,43.

2.4 Fractionation of hydroalcoholic extract:

The hydroalcoholic crude extract (mother extract) was further partitioned into different fragments on the basis of increasing polarity of solvents ranges from petroleum ether, chloroform, ethyl acetate to ethanol in separating funnel. Most productive fraction (active fraction) was obtained in ethanol solvent.

2.5 Specimen:

Venous blood was obtained (utilizing a procedure that Institutional Ethics Committee has authorized) from five healthy human volunteers who had not taken an oral contraceptive or an anticoagulant medication. 500μl of blood was added to each micro-centrifuge tube that had been weighed earlier in order to form clots⁴⁴.

2.6 Sample preparation:

Ten millilitres of distilled water were mixed with 100 milligrams of each active component of the three herbal extracts, and the mixture was vortexed. After an overnight storage period, the suspension was decanted to extract the soluble supernatant, which was then filtered using filter paper. To assess the clot lysis activity, 100 μl of this transparent solution was introduced to microcentrifuge tubes holding clots⁴⁵.

2.7 Preparation of streptokinase:

The commercially available lyophilized SK vial containing 15,00,000 I.U. was filled with about 5 ml of sterile distilled water and thoroughly mixed. For the in-vitro thrombolysis experiment, 100μl (30,000 I.U.) of this solution was employed as a stock⁴⁵.

2.8 In-vitro thrombolytic activity:

About 15ml of venous blood from healthy participants were placed into 27 separate, pre-weighed, sterile Eppendorf tubes (0.5ml/tube), and the tubes were incubated for 45 minutes at 37°C^48. After the serum was completely removed without disturbing the clot, each tube holding a clot was weighed once again to determine the clot weight (W₂). Each eppendorf tube holding a pre-weighed clot had 100μl of these active extracts added to it, along with various ratios of mixes containing active fraction⁴⁶. 100μl of sterile distilled water was added as a negative control and 100μl of SK was added as a positive control to the control tubes that contained clots. After that, each tube was incubated for 90 minutes at 37 °C. Following incubation, all of the fluid that had been discharged was collected, and the tubes were weighed again as (W₃) to see if the weight had changed following clot disruption. A percentage of clot lysis was used to illustrate the weight variations between pre- and post-clot lysis⁴⁷.

% clot lysis = (dissolved clot wt. / clot wt.) × 100

= (WR/WC) × 100

Where,

W₁ = Wt. of empty eppendorf tube

W₂ = Eppendorf tube wt. with clot

W₃ = Eppendorf wt. following clot lysis

WC = Wt. of clot before lysis (W₂ - W₁)

WR = Wt. of clot after lysis (W₂–W₃)

2.9 Statistical analysis:

All the experimental data were carried out in triplicates and resulting evidences is represented as mean±SEM. One-way ANOVA analysis and the Newman-Keul Multiple Comparison Test were used to determine the significance of the weight difference between the percentage clot lysis of streptokinase and the herbal extract.

3. RESULTS AND DISCUSSION:

Coronary atherothrombotic disorders (CADs) are connected to progressive atherosclerosis that leads to arterial thrombosis. The pathogenesis process encompassed persistent, progressive atherosclerosis interspersed with platelet thrombus development and plaque rupture⁴⁸. While treating thrombosis, the basic difficulty associated with thrombolytic therapy includes slow and incomplete thrombolysis. Moreover, thrombolytic treatment generates most typical adverse conditions such recurrent intestinal bleeding and stomach ulcers and sometimes the patients may die owing to bleeding and embolism^49,50. Almost all thrombolytic medicines work by changing plasminogen activator into plasmin, which dissolves fibrin and fibrinogen to lyse the clot.^51-52. By taking into consideration these severe complications, we attempt to explore active fraction of CMB, CRR and CAR root extract for thrombolytic effect. Several research evidences confirmed the powerful relationship between antioxidant properties and thrombolytic potential for a variety of natural products⁵³. Eventually it was found that phenolic compounds consisting phenolic acids and flavonoids showing strong antioxidant properties, thereby may reduce the risk of thrombosis⁵⁴.

Cordia macleodii, Cissus rependa and Celosia argentea are different species but they possibly contain similar biological active components like phenolics, flavonoids, alkaloids, saponins, carbohydrates and triterpenoids^62-63, that’s why attempt has made for evaluation of antithrombotic activities. Experimental data shows streptokinase, positive control was able to dissolve 55.61 % of the clot and sterile distilled water, negative control shows 2.12% clot lysis. Encouraged by the result of positive control, we compared active fractions of CMB, CRR and CAR and their combinations in various ratio in same way with the negative control and noticed moderate thrombolytic activity. The active ethanolic fraction of each drug extract on an average were able to dissolve 22.63%, 22.78% and 26.63% of the clot independently. Herbal combination containing CMB, CRR and CAR fraction in a proportion such as M1 (1:1:1), M2 (2:1:1), M3 (1:2:1) and M4 (1:1:2) were able to lyse 35.84%, 38.42%, 51.49% and 39.96% clots respectively. From the experimental findings, it was observed that herbal mixture in (1:2:1) ratio shows more significant clot lysis effect as compared to other combinations used during the study.

3.1 Extraction of plant materials:

About 2kg fresh material of each of Cordia macleodii bark, Cissus rependa roots and Celosia argentea roots was dried under shade for a two week and grind to coarse powder and it was extracted by maceration process using Ethanol: Water (40:60) mixture. 1.5kg coarse powder of each crude drug was soaked in 10 litres of hydro-alcoholic mixture for 24hours. Soaked crude drug mixture then heated at 80⁰C for 4hours in reflux condenser. Extraction was repeated twice and combined extract were evaporated to dryness by rotary evaporation at 40⁰C. Percentage yield of Cordia macleodii bark, Cissus rependa root and Celosia argentea root extract was found to be 8.70%, 8.25% and 7.90% respectively.

3.2 Fractionation of hydro-alcoholic crude extract:

The hydroalcoholic crude extract of each herbal, Cordia macleodii bark, Cissus rependa and Celosia argentea root (approximately 15 g) was partitioned into different fractions based on varying polarity of solvents like petroleum ether, chloroform, ethyl acetate and ethanol in separating funnel. When the separating funnel is agitated, the solute from the mixture get migrated to the solvent in which it is more soluble. (Table:1)

3.3 Yield of Active fraction:

After partitioning of hydroalcoholic crude extract of Cordia macleodii bark, Cissus rependa and Celosia argentea root into respective solvent fraction, it was then evaporated to dryness by rotary evaporation at 40 0C. Every non-polar solvent fraction obtained through separating funnel in dry form was in very negligible amount except ethanolic fraction called as productive active fraction. The % yield of ethanolic active fraction of Cordia macleodii bark, Cissus rependa and Celosia argentea root hydroalcoholic extract was found to be 82.2%, 85.1% and 83.5% respectively.

4. DECLARATION OF INTEREST:

The authors declare that they have no conflicts of interest with regard to their co-authorship and/or publishing. The authors alone are accountable for the writing and content of the paper.

5. ACKNOWLEDGEMENTS:

The authors would like to thank to Prof. Dr. S. M. Bhuskute, Taxonomist and Former Principal, Bhawbhuti Mahavidyalaya, Amgaon-441902 District- Gondia (Maharashtra) for the identification and authentication of herbal drugs used in research project. I extend my deep gratitude to the HOD, for letting me avail their infrastructure facility and extending their cooperation and support while carrying out this research work. Further, I would like to record my gratitude to Dr. Satender Prasad, Assistant Professor, Department of Pharmaceutical Sciences, Nagpur for their constant source of guidance, encouragement and support for my research project.

6. LIST OF SYMBOLS AND ABBREVIATIONS:

CMB- Cordia macleodii bark

CRR- Cissus rependa root

CAR- Celosia argentea root

μl - Micro-litre

t-PA- tissue plasminogen activator

SK- Streptokinase

I.U.- International unit

ANOVA- Analysis of variance

CAD- Coronary atherothrombotic diseases

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Received on 01.09.2024 Revised on 21.12.2024

Accepted on 27.02.2025 Published on 12.06.2025

Available online from June 14, 2025

Research J. Pharmacy and Technology. 2025;18(6):2447-2453.

DOI: 10.52711/0974-360X.2025.00349

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